Surgical PATHOLOGY UPDATE

PATHOLOGY UPDATE: SurgicalDiagnostic Pearls for the Practicing Pathologist

Sunday, October 9, 2016 Aria® Resort & Casino • Las Vegas, Nevada

Educational Symposia TABLE OF CONTENTS

Sunday, October 9, 2016 Barrett Esophagus Update: Treatment Effects on Dysplasia and Do Current Surveillance Protocols Make Sense? (Marie E. Robert, M.D.)...... 327 Common Morphologic Patterns in Soft Tissue Tumors Parts 1 and 2 (John R. Goldblum, M.D.)...... 339 Common Pitfalls in Medical Liver Disease Interpretation: How Not to Fall in a Hole (John Hart, M.D.)...... 401 The Critical Role of the Pathologist in the Management of Bladder Cancer (David J. Grignon, M.D., FRCP(C))...... 433

• Definition of clinical problem and current Barrett Esophagus: When is practice in United States Surveillance Justified? • Definition of Barrett esophagus – Impact of pathology diagnosis on surveillance Marie Robert, M.D. Yale University School of • Controversies in diagnosis of dysplasia Medicine – Crypt dysplasia, subsquamous intestinal metaplasia

Clinical Significance of Barrett: Clinical Significance of Barrett Epidemiology Definition of Barrett Esophagus: Risk of adenocarcinoma: 0.12 -0.38% per • Endoscopic finding of columnar mucosa (salmon year (Dunbar and Spechler. Mayo Clinic Proceedings, pink) above the gastroesophageal junction (GEJ) 2014) AND • Lower than previously thought • Histologic finding of columnar mucosa with • Risk may decrease with each endoscopy intestinal metaplasia (goblet cell) in the esophagus from the endoscopically abnormal with biopsies negative for dysplasia (Gaddam, et al. Gastroenterology, 2013) region • Low grade dysplasia: progression to HG American Gastroenterology Association Position Statement, or cancer is 0.5-13.4% Gastroenterology, 2011 – Studies flawed by subjectivity of diagnosis

Decision Points in Barrett Barrett Epidemiology Esophagus • High grade dysplasia: meta analysis of • First diagnosis of Barrett esophagus multiple historical studies reports overall – Places patient in surveillance category risk of progression to cancer is 6% per • First diagnosis of dysplasia in Barrett patient/per year. – Impacts interval of surveillance • Higher rates in some studies, may be due • Diagnosis of signiﬁcant neoplasia to prevalent (concurrent) cancer at time – High grade dysplasia, intramucosal or of HG diagnosis invasive carcinoma • Evidence stronger; interobserver – Triggers therapeutic intervention agreement better than for LG dysplasia

329 Surveillance Guidelines in Barrett Practice Parameters Committee, ACG, 2008 Does Surveillance Prevent Cancer?

Dysplasia Documentation Follow-Up • Data from numerous studies reveal that None Two EGDs with 3 years (?5 years) mortality from esophageal biopsy adenocarcinoma not impacted by Indefinite or Highest grade on 1 year until no surveillance Low Grade repeat; LGD expert dysplasia vs ablation confirmation (controversial) – Corley, et al. Gastroenterology, 2013 High Grade Repeat EGD to rule One focus- every 3 out cancer, expert months pathologist • At least 40% of patients with cancer confirmation High Grade Multifocal- Ablation present with malignancy, having never with RFA or Cryo been in surveillance program High Grade Mucosal irregularity- EMR f/ by ablation

Surveillance Paradox

• How to Reconcile: surveillance performed despite evidence that it is not effective Incidence of Gastroesophageal – Doesn’t prevent cancer or death well – Psychological impact, life insurance rates, Adenocarcinoma Rising procedure risk – Expense in face of shrinking health care Identiﬁcation and surveillance of dollar Barrett patients is not the answer to early prevention

Pathologist’s Play Crucial Role Deﬁning Barrett on biopsy In face of data showing lack of efﬁcacy, • Mimics of goblet cells: short/tall blue goal for pathologists is to not increase cells surveillance pool incorrectly Who is initially screened by endoscopy? • Intestinal metaplasia at GEJ • GERD plus at least one additional risk factor • Are goblet cells required for a diagnosis • Male, over 50, white, hiatal hernia, of Barrett? tobacco, elevated body/mass index

330 Clinical Scenario

• A 53 year old white male with symptoms suggestive of gastroesophageal reﬂux disease for several years undergoes upper endoscopy • The endoscopic examination reveals an irregular GEJ with a question of short segment Barrett esophagus

Normal cardia GE Junction

331 Special Issues in/near GEJ

• Carditis Diagnosis: Carditis with – Chronic inﬂammation gives rise to expected reactive changes in gastric surface epithelial reactive gastric surface cells cells • Tall Blue cells or “Short” (alcian blue) The Short Blues – Need to distinguish from goblet cells

Intestinal Metaplasia at the Gastroesophageal Junction

Endoscopically normal GEJ

Goblet cells in the Endoscopically Are Goblet Cells Still Required? Normal GEJ • Meaning of intestinal metaplasia in the GEJ or cardia • Several studies opinion papers suggest dropping controversial requirement of goblet cells for diagnosis of – Clinical signiﬁcance given to difference in location of Barrett a few millimeters – Playford, 2006, British Society of Gastroenterology (they don’t survey!) – Balance with fact that incidence of GEJ carcinoma has risen 5-6 fold in Western Countries – Riddell and Odze, 2009 – Barrett and GEJ cancer have more similarities • Distinguish academic entity of “columnar genetically and clinically than differences metaplasia” from condition associated with • Bottom line: No data to support surveillance cancer. – Spechler. Clin Gastro and Hepatol, 2012 – Practice guideline: Do not biopsy normal GEJ; do not – Increased cancer risk has only been associated with survey if intestinal metaplasia found presence of goblet cells

332 Goblet Cells

• In response, recent studies support retaining goblet – Rebay, 2011,Westerhoff, 2012, Montgomery, 2013 • Cancers developed almost exclusively in patients with goblet cells • Dropping requirement would increase surveillance pool exponentially Figure 3. Kaplan–Meier curve for progression to dysplasia. There is a statistically signiﬁcant difference between patients with GCs and patients without.

Recent Controversies in Diagnosis 2014: Goblet Cells Are Required of Dysplasia • Regulatory societies continue to require goblet cells for diagnosis of Barrett • Crypt Dysplasia esophagus American Gastroenterology Association • Post ablative therapy buried metaplasia American College of Gastroenterology

Dysplasia Grading Criteria Reid et al, Human Pathology, 19:166-178, 1988 Montgomery et al, Human Pathology, 32:368-378, 2001

• Dysplasia is graded on degree of cytologic and architectural atypia – Cytology = nucleus/cytoplasm (high mag) – Architecture = relation between glands and lamina propria (low mag) • Surface changes vs deep gland changes – Presence or absence of maturation • Background inﬂammation or ulceration

333 Crypt Dysplasia

• Atypical crypt nuclei with surface maturation – Lomo et al. Am J Surg Path. 2006 • Not new observation: previously categorized as indefinite or, rarely, dysplasia • Insufficient follow up data to determine clinical significance (is it different from indefinite?) – DNA abnormalities on flow similar to low grade – 1/18 patients had LG or HG at same procedure

Diagnostic Categories

• Negative for dysplasia • Indeﬁnite for dysplasia • Low grade dysplasia • High grade dysplasia • Carcinoma – Intramucosal carcinoma – Invasive carcinoma

Crypt Dysplasia

• Problematic as clinical term – Introducing new term to diagnostic lexicon – Mostly trading term “indeﬁnite” for crypt dysplasia • Impact on surveillance minimal if treated as indeﬁnite or low grade • Term not yet accepted in many US academic centers • If used, must ensure clinicians know meaning

334 Subsquamous Intestinal Metaplasia Alert to Pathologist

• Surveillance after radiofrequency • Subsquamous intestinal metaplasia (SSIM) ablation or endoscopic mucosal resection – Gray et al. Am J Gastro. 2011 • • New lining squamous Biopsies not always deep enough to detect • Grading dysplasia challenging as surface not • Sub-squamous Intestinal Metaplasia seen (SSIM) or“Buried” IM • High grade dysplasia and cancer reported • Buried neoplasia – Titi et al. Gastroenterology. 2012 • Not visible with endoscope • SSIM also frequent in EMR specimens where no ablation occurred

335 Implications of SSIM

• Patient’s cannot be assured their cancer risk has been eliminated by EMR or ablation of entire Barrett ﬁeld. – Konda et al. Endoscopy, 2012-metastatic CA • After ablation, should biopsy squamous mucosa as well as apparent Barrett • Pathologists must document in report • Surveillance must continue, despite ablation

Summary: When Is Surveillance Summary: Continued Justified in Barrett Esophagus? • In US: surveillance still performed despite • Crypt dysplasia not completely accepted growing evidence of ineffectiveness as diagnostic term – Most cancers do not develop in surveillance – program Lack of follow up data on significance • Crucial role of pathologist to not increase – Most have been called indefinite in past surveillance pool or shorten interval • Subsquamous Intestinal Metaplasia unnecessarily – Pathologists must document and grade – Recognize tall/short blue cells – Significant limitation of ablation therapies – IM at cardia/normal GEJ is not Barrett – Goblet cell required to diagnose Barrett

Summary, continued

• Future: • Endomicroscopy with targeted biopsies of both Barrett epithelium and dysplastic foci to replace random biopsies • Molecular diagnosis to identify those at risk – DNA methylation, risk stratiﬁcation biomarker panels • Change in surveillance practices as better methods become available

336 337 338 339 340 Common Morphologic Patterns Tumor Patterns in Soft Tissue Tumors • “MFH-like” pattern

• Highly cellular spindle cell pattern John R. Goldblum, M.D.

Chairman, Department of Pathology, Cleveland Clinic • Myxoid soft tissue lesions Professor of Pathology, Cleveland Clinic Lerner College of Medicine • Round cell pattern

Malignant Fibrous Histiocytoma

• Storiform-pleomorphic

• Myxoid

• Giant cell

• Inflammatory

• Angiomatoid

Malignant Fibrous Histiocytoma

• Storiform-pleomorphic

• Myxoid

• Giant cell

• Inflammatory

341 MFH: Fact or Fiction? “MFH-like” Pattern 159 Pleomorphic Tumors • Pleomorphic sarcoma with a specific

line of differentiation

Pleomorphic sarcoma Pseudosarcomas • Dedifferentiated sarcoma with specific (N=20; 12.6%) differentiation • Pseudosarcoma with “MFH-like” pattern (N=97; 61%) • Undifferentiated pleomorphic sarcoma Unclassifiable ?MFH (so-called MFH) (N=42; 26.4%)

Pleomorphic Leiomyosarcoma Pleomorphic Rhabdomyosarcoma

• Middle-aged-elderly • Elderly patients

• Extremities, retroperitoneum, • Deep soft tissue of large vessels extremities • Pleomorphic cells with • Large spindled cells with cytoplasmic eosinophilia deeply eosinophilic cytoplasm • HHF-35, desmin, fast • Strong SMA, desmin, h- myosin, myogenin caldesmon • 5-yr metastatic rate: >90% • 5-yr metastatic rate: 60-70%

Pleomorphic Liposarcoma Pleomorphic MPNST

• Elderly patients • Origin from a major nerve OR

• Deep soft tissue of • Origin in a pre-existent extremities (thigh), neurofibroma OR retroperitoneum • S100+ sarcoma in an NF1 • Multivacuolated patient pleomorphic lipoblasts • 5-yr survival: 30-40% • 5-yr metastatic rate: 30-50%

342 Extraskeletal Osteosarcoma Undifferentiated Pleomorphic Sarcoma (formerly MFH)

• Elderly patients • Elderly patients

• Deep soft tissue of • Deep soft tissue of extremities extremities

• Malignant bone/osteoid • “vimentin only,” scattered produced by cytologically SMA+ malignant cells • Diagnosis of exclusion • 5-yr survival: 20-30%

Kaplan-Meier plots of metastasis rates in 88 soft tissue sarcoma of AJCC staging system (ed 5) stage II or III originally diagnosed as MFH; 26 tumors showedFig myogenic 1. differentiation and 62 did not (P = .006). “MFH-like” Pattern Dedifferentiated Sarcoma

• Well-differentiated liposarcoma

• Chondrosarcoma

• Chordoma

• Dermatofibrosarcoma protuberans Fletcher C D et al. JCO 2001;19:3045-3050

“MFH-like” Pattern Pseudosarcomas • Sarcomatoid carcinoma

• Sarcomatoid mesothelioma

• Melanoma

• Ki-1 anaplastic lymphoma

343 CAM5.2

Pseudosarcomas

Tumor Useful markers

Sarcomatoid CA AE1/3, CAM 5.2, CK 5/6, p63

Sarcomatoid mesoth CAM 5.2

Melanoma S-100

(+/- melanocytic markers)

Anaplastic lymphoma CD30; ALK-1

Highly Cellular Spindle Cell Tumors

• Cellular schwannoma • MPNST • Fibrosarcoma • Leiomyosarcoma • Monophasic synovial sarcoma

344 Cellular Schwannoma Definition

• Highly cellular schwann cell proliferation composed predominantly/ exclusively of Antoni A areas

• Absence of well-formed Verocay bodies

345 S100

Cellular Schwannoma Cellular Schwannoma Worrisome Features Behavior Recurred Bony erosion Mets F/U (mean) • High cellularity Woodruff (14) 0% 7% 0% 2.2 yrs • Mitotically active (usual < 4/10HPF) • Nuclear atypia Fletcher (18) 5% 11% 0% 14 yrs • Focal necrosis White (58) 5% 19% 0% 7 yrs • Bony erosion

Lodding (29) 0% 3% 0% 7 yrs * THE PERFECT PSEUDOSARCOMA*

346 Desmin

S100

Cellular Schwannoma vs MPNST

Cellular MPNST schwannoma Encapsulated + -

Cellularity 3+ 3+

Necrosis rare common

Pleomorphism 1+ - 2+ 3+

Mitoses 1+ - 2+ 3+

Divergent diff. - + (10%)

S-100 diffuse focal (60%)

347 348 MPNST vs Monophasic SS Immunohistochemical Quandary

• Not all MPNST stain for S100 (60-70%, focal)

• Some MSS stain for S100 (30%, focal)

• Not all MSS stain for AE1/AE3 and/or EMA (70-90%, focal)

• Some MPNST stain for AE1/AE3 and/or EMA (30%, focal)

TLE1

MSS MPNST SS 91/94 (97%)

CK7 88%* 0% MPNST 4/88 (5%)

CK19 88%* 0%

* focal staining in some • often more cells than AE1/AE3 or schwannomas and SFT/HPC EMA; positive in cases that were negative for AE1/AE3 and EMA

TLE1 CK7 Smith AJCP 1999 Terry J et al. AJSP 2007

Synovial Sarcoma: Utility of TLE1 Synovial Sarcoma: Utility of TLE1

Sensitivity Specificity (X;18)+ SS MPNST

TLE1 35/35 (100%) 1/43 (2.3%) TLE1 100% 96%

AE1/AE3 24/35 (70.6%) 7/43 (16.6%) AE1/AE3 71% 85%

EMA 18/35 (64.3%) 3/43 (7.3%) EMA 64% 91%

BCL-2 30/35 (96.8%) 11/43 (26.2%) BCL-2 97% 71% Jagdis et al AJSP 2009 Jagdis et al AJSP 2009

349 Cellular Spindle Cell Pattern Synovial Sarcoma t(X;18)(p11;q11) S100 CK7/19 SMA TLE1

SSX1 Cellular schwannoma + (diffuse) - - -

MPNST 60% (focal) - - -

SYT SSX2 MSS 30% (focal) + - + (SS18)

Leiomyosarcoma - - + -

SSX4 Fibrosarcoma - - +/- -

SYT or SS18 (18q11) - Break Apart Probe

SYNOVIAL SARCOMA POSITIVE

Myxoid Soft Tissue Tumors Myxoid Soft Tissue Lesions • Morphology is most useful Benign Malignant • Nodular fasciitis • Myxoid liposarcoma • Cellularity and cellular arrangement • Myxoma • Myxofibrosarcoma (myxoid MFH) • intramuscular • Atypia • juxta-articular • Myxoid chondrosarcoma • Vascular pattern • cutaneous • Low-grade fibromyxoid sarcoma • Nerve sheath tumors • neurofibroma • All other sarcomas • Limited use of IHC (S100) • neurothekeoma • schwannoma • FISH extremely useful

350 351 Myxoid/Round Cell Liposarcoma

t(12;16)(q13;p11) t(12;22)(q13;q12)

DDIT3 (CHOP)/FUS DDIT3 (CHOP)/EWSR1

352 Extraskeletal Myxoid Chondrosarcoma LGFMS / HSCT

t(7;16)(q33;p11) t(11;16)(p11;p11) t(9;22)(q22;q12) t(9;17)(q22;q11)

NR4A3/EWSR1 NR4A3/TAF2N CREB3L2/FUS CREB3L1/FUS *EWSR1 involved in up to 45% of soft tissue myoepitheliomas

Myxoid Soft Tissue Tumors

Tumor Defect Genes Myxoma GNAS1 activating mutations

LGFMS/HSCT t(7;16)(q33;p11) CREB3L2/FUS t(11;16)(p11;p11) CREB3L1/FUS

MLS/RCLS t(12;16)(q13;p11) DDIT3/FUS t(12;22)(q13;q12) DDIT3/EWSR1

ESMCS t(9;22)(q22;q12) NR4A3/EWSR1 t(9;17)(q22;q11) NR4A3/TAF2N

MyxoFS None characteristic

Round Cell Tumors Benign

• Glomus Tumor

• Eccrine Spiradenoma

• Giant Cell Tumor

353 Round Cell Pattern

• ES/PNET • Mesenchymal CS

• Alv RMS • Round cell LS

• DSRCT • Poorly diff SS

• Neuroblastoma • “Ewing-like” • CIC-DUX • Small cell/Merkel cell • BCOR-CCNB3 • Lymphoma • CIC-FOXO4

PAS/D

354 ES/PNET: Histologic Spectrum ES/PNET: Ultrastructure

ES PNET

Cell shape regular irregular

Chromatin fine coarse

Nucleoli pinpoint prominent

Glycogen abundant scant

Rosettes absent present

ES/PNET: Ultrastructural Spectrum

Feature ES PNET

Organelles scarce abundant

Dense-core granules absent abundant

Neurotubules absent abundant

Neuritic processes absent abundant CD99

355 MIC - 2 Gene Product (CD99) ES/PNET: Cytogenetics Diagnosis Positive (%)

ES/PNET 93% t(11;22)(q24;q12) – FLI1;EWSR1 Translocation Small Cell Osteosarcoma 12% EWSR1 FLI1 NH COOH Desmoplastic Round Cell Tumor 20% 2 ETS DNA binding domain Neuroblastoma 0% Rhabdomyosarcoma 15% t(21;22)(q22;q12) ERG/EWSR1 Small Cell Carcinoma 9% t(7;22)(p22;q12) ETV1/EWSR1 t(17;22)(q12;q12) FEV/EWSR1 T-LL/ALL 92% t(2;22)(q33;q12) E1AF/EWSR1

Very rare involvement of FUS in lieu of EWSR1

EWSR1 (22q12) Break-apart probe

t(11;22), t(21;22) – EWSR1/PNET (FLI1/EWSR1, ERG/EWSR1) t(11;22) - DSRCT (WT-1/EWSR1) t(12;22) – Clear Cell Sarcoma (ATF1/EWSR1) t(9;22) – ES Myxoid Chondrosarcoma (NR4A3/EWSR1) t(12;22) – Myxoid/Round Cell Liposarcoma (DDIT3/EWSR1) t(2;22) – Angiomatoid fibrous histiocytoma (CREB1/EWSR1) t(6;22) – Soft tissue myoepithelioma (Pou5F1/EWSR1)

356 Rhabdomyosarcoma International Classification Favorable Prognosis • Botryoid • Spindle cell Intermediate Prognosis • Embryonal Poor Prognosis • Alveolar

Newton WA et al, Cancer 1995

MyoD1/Myogenin

• part of the Myo-D superfamily of genes

• encode for DNA binding proteins which activate transcription of skeletal muscle- specific genes

• results in commitment to skeletal muscle differentiation

• can detect skeletal muscle differentiation at an earlier stage than with markers currently available Myogenin

357 Myogenin in Pediatric Tumors Alveolar Rhabdomyosarcoma

Tumor Cases stained

Rhabdomyosarcoma (69) 69/69 (100%) • Alveolar (48) • Embryonal (20) t(2;13)(q35;q14) t(1;13)(q36;q14) • Spindle cell (1)

Other pediatric sarcomas (50) 0/50 (0%) • ES/PNET (16) • DSRCT (6) PAX3/FOXO1A PAX7/FOXO1A • Neuroblastoma (4) • Others (24) *up to 25% lack t(2;13) or t(1;13) Kumar S et al, Mod Pathol 2000

FOXO1A Break Apart Probe

358 Desmoplastic Round Cell Tumor Desmoplastic

• Gender: 90M / 19F Round Cell Tumor • Age: 6-49 years (mean: 22 years) CK 86% • Sites: • Abdominal cavity 103 Vim 97% • Thoracic region 4 WT-1 95% • Cranial fossa 1 • Extremity 1 Des 90% • Prognosis: poor, but may respond to aggressive therapy NSE 81%

Gerald W et al, J Clin Oncol 1998 CD99 20%

t(11;22)(p13;q12) “Ewing-like” Sarcoma

CIC-DUX BCOR-CCNB3

EWSR1+ CIC-FOXO4 Desmoplastic Round Cell Tumor

359 CIC-DUX Tumor Immunophenotype

CD99 focal + S100 - Desmin - Myogenin -

Cytokeratin (AE1/AE3) - TLE1 - WT1 + ERG + EWSR1 FISH -

CD99

CIC-DUX Sarcomas Round Cell Pattern CD99 TdT Myo Cam 5.2 Des WT1 Takeaway Points ES/PNET + - - 20% - - • Round cell sarcoma which is “Ewing-like” but EWSR1-negative A-RMS 15% - + 50% + -

• Has a fairly characteristic constellation of histologic Lymphoma >90% + - - - - features which, if recognized, should prompt the appropriate molecular work-up DSRCT 20% - - + + +

• Aggressive clinical behavior CIC-DUX focal + - - - - +

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Common Morphologic Patterns In Soft Tissue Tumors With An Emphasis On Useful Ancillary Diagnostic Techniques

John R. Goldblum, M.D.

Chairman, Department of Pathology, Cleveland Clinic Professor of Pathology, Cleveland Clinic Lerner College of Medicine 9500 Euclid Avenue L25 Cleveland OH 44195 Ph 216-444-8238 Fx 216-445-2142 [email protected]

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Recognizing patterns at low magnification is one of the keys to organizing differential diagnoses in soft tissue sarcomas. Each pattern elicits a differential diagnosis, and often a combination of light microscopic and immunohistochemical features will allow one to classify a neoplasm with a given morphologic pattern. Below is a description of some of the more common morphologic patterns seen in soft tissue sarcomas with some practical points on the approach to tumors with these morphologic patterns.

"MFH-Like" Pattern The concept of malignant fibrous histiocytoma (MFH) has undergone significant change over the past five decades. The term was first introduced in 1963 to refer to a group of soft tissue tumors characterized by a storiform or cartwheel-like growth pattern, which were believed to be derived from histiocytes on the basis of early tissue culture studies demonstrating ameboid movement and phagocytosis of explanted tumor cells.1,2 Ultrastructural studies both endorsed and refuted the histiocytic origin of these tumors, however. With the advent of immunohistochemistry and the accessibility of numerous monoclonal antibodies directed against various structural proteins of specific cell types, the phenotype of this tumor was shown to be more closely aligned with a fibroblast than a histiocyte.3–6 Furthermore, many, but not all, lesions labeled as “malignant fibrous histiocytoma” could, upon close scrutiny, be subclassified as lineage-specific sarcomas, an observation that led some to question the existence of MFH as a distinct entity.7 The extent to which such lesions can be subclassified as sarcomas of alternative type is, in large part, dependent on definitional criteria and the number of ancillary studies a pathologist is willing to bring to bear on the evaluation of a pleomorphic sarcoma. There is still no general agreement as to what percentage of pleomorphic sarcomas, when subjected to rigorous evaluation, remain unclassified. These discrepancies, nonetheless, underscore the fact that the criteria by which a pleomorphic tumor is provisionally labeled as a undifferentiated pleomorphic sarcoma (UPS/MFH) as well as the criteria by which some are reclassified differ from institution to institution. Whatever the true incidence of this lesion, there is agreement that the term MFH should be used synonymously with undifferentiated pleomorphic sarcoma (UPS) which, by a combination of sampling and immunohistochemistry, shows no definable line of differentiation and by electron microscopy manifests fibroblastic/myofibroblastic features.8 At this point, we use the term undifferentiated pleomorphic sarcoma in our diagnostic reports, but also state that this is

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synonymous with so-called MFH in parentheses so as to avert any misunderstanding with clinicians who continue to be familiar with that term. Whenever I encounter a soft tissue neoplasm with a "MFH-like" pattern, I consider several broad possibilities before concluding that the lesion is in fact an undifferentiated pleomorphic sarcoma (UPS). First, I consider whether the lesion in question is some type of pleomorphic sarcoma with a specific line of differentiation that can be identified through light microscopy and/or immunohistochemistry. The rationale behind attempting to more precisely classify a pleomorphic sarcoma is discussed below. Second, I consider the possibility that the lesion could be a component of a dedifferentiated sarcoma, particularly when dealing with a sarcoma in the retroperitoneum. Thorough sampling is often required in order to recognize the low-grade sarcoma from which the dedifferentiated "MFH-like" areas arose. Importantly, I also want to exclude the possibility that the lesion is a non-mesenchymal neoplasm. The most common consideration is that of a sarcomatoid carcinoma, particularly when the lesion arises on a mucosal surface, skin or within a parenchymal organ. Other considerations might include (depending upon site and other clinical factors) sarcomatoid mesothelioma, melanoma and even anaplastic lymphoma. Finally, if all of these can be excluded, then one can arrive at the conclusion that the lesion is a UPS.

Pleomorphic sarcoma with a specific line of differentiation

A variety of pleomorphic sarcomas may have areas which resemble UPS. In some cases, determining the specific line of differentiation may rely on random sampling of a small area within a large tumor. Although a specific type of pleomorphic sarcoma may be suggested by histologic features, immunohistochemical stains are often required to confirm the diagnosis. Although it could be argued that subtyping pleomorphic sarcomas is nothing more than an academic exercise, there is some evidence to suggest that pleomorphic sarcomas with myogenic differentiation are more clinically aggressive than those without myogenic differentiation9–11 (see below). The only criterion for rendering a diagnosis of pleomorphic liposarcoma is the recognition of multivacuolated pleomorphic lipoblasts. The major difficulty in such cases is separating pleomorphic sarcomas that infiltrate fat and isolate individual cells from those with true lipoblasts. Pleomorphic leiomyosarcoma is composed of cells with distinct cytoplasmic eosinophilia. At least focally, most cases have areas with a fascicular arrangement and cells with blunt-ended nuclei with a perinuclear vacuole and deeply eosinophilic cytoplasm. Pleomorphic rhabdomyosarcoma is recognized by the presence of large cells with eosinophilic

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cytoplasm and cross striations which can be confirmed by the immunohistochemical demonstration of skeletal muscle differentiation (desmin, MyoD1, myogenin). A definitive diagnosis of pleomorphic malignant peripheral nerve sheath tumor can be difficult unless the pleomorphic sarcoma clearly arises from a benign nerve sheath tumor or arises from a peripheral nerve in a patient with type 1 neurofibromatosis. The only criterion for recognizing extraskeletal osteosarcoma is the production of osteoid or bone by cytologically malignant cells. As mentioned, several studies have suggested that pleomorphic sarcomas with myogenic differentiated are clinically more aggressive than those without myogenic differentiation.9–11 Fletcher and colleagues reviewed 100 cases diagnosed as "MFH" and concluded (based upon morphology and immunohistochemistry) that 70 of these tumors were non-myogenic pleomorphic sarcomas, whereas 30 showed evidence of myogenic differentiation.10 Of these 30 cases, 20 were classified as leiomyosarcoma, 9 as myogenic sarcoma, not otherwise specified, and 1 as rhabdomyosarcoma. The pleomorphic sarcomas with myogenic differentiation were significantly more likely to be deep-seated when compared to those without myogenic differentiation (83% versus 63%; p=0.04), and they were also more likely to be grade 4 tumors (97% versus 67%; p=0.02). Similarly, in 2003, Deyrup et al compared 42 cases of pleomorphic sarcoma with myogenic differentiation to 50 pleomorphic sarcomas without myogenic differentiation.9 The only significant difference between these two groups was that the tumors with myogenic differentiation were significantly more likely to be grade 3 sarcomas (88% versus 74%; p=0.038). There was a significant difference in overall survival, as those sarcomas with myogenic differentiation had shorter overall survival. Overall survival was also directly correlated with the number of myogenic markers found to be positive, as those with 3 markers found in a given tumor were more aggressive than those with fewer markers. Massi et al evaluated 65 pleomorphic sarcomas of the extremities and compared 31 sarcomas with myogenic differentiation (22 leiomyosarcomas, 4 rhabdomyosarcomas and 5 myofibrosarcomas) with 34 non-myogenic pleomorphic sarcomas.11 The tumors were classified into diagnostic categories based upon morphology, immunohistochemistry and, in some cases, ultrastructural analysis. Tumor site and myogenic differentiation were both found to be independent predictors of disease relapse for localized tumors by multivariate analysis, but only myogenic differentiation was found to be an independent predictor of overall survival by multivariate analysis. In summary, there are at least three studies which strongly suggest the adverse prognostic effect of myogenic differentiation in a pleomorphic sarcoma. However, there are a number of practical issues which make the assessment of myogenic differentiation in a

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pleomorphic sarcoma rather difficult. First, these are relatively uncommon tumors, and there are inconsistencies with regard to diagnostic criteria. There is a relatively small number of cases subjected to multivariate analysis in order to control for tumor site, tumor size and tumor depth. Exactly how many myogenic markers should be positive to designate a tumor as having myogenic differentiation is unclear, and the extent of staining needed to designate a myogenic marker as positive is similarly unclear.

Pleomorphic sarcoma as a result of dedifferentiation The process of tumor progression or dedifferentiation involves the transformation of a low-grade sarcoma to a higher-grade sarcoma, which usually (but not always) resembles a UPS. The most common scenario is the progression of a low-grade well-differentiated liposarcoma to a pleomorphic sarcoma (dedifferentiated liposarcoma). Other low-grade neoplasms can also dedifferentiate, including chondrosarcomas, chordomas and parosteal osteosarcomas. Certainly in a limited biopsy specimen it can be impossible to prove that a pleomorphic sarcoma is part of a dedifferentiated sarcoma if the low-grade component is not represented. However, in a retroperitoneal sarcoma where dedifferentiated liposarcoma is always a strong consideration, it can often be suggested that the high-grade sarcoma could be part of a dedifferentiated liposarcoma. There is some evidence to suggest that dedifferentiated sarcomas (dedifferentiated liposarcoma in particular) are more indolent tumors than de novo pleomorphic sarcomas arising in the same location. For example, in the study of 32 cases of dedifferentiated liposarcoma by McCormick et al, 19 of 32 dedifferentiated liposarcomas (59%) arose in the retroperitoneum or paratesticular region, most of which arose de novo (94%).12 These patients were followed for a mean of 5.6 years, and metastasis was detected in only 4 of 27 cases with follow-up (15%), a rate much lower than would be expected for de novo pleomorphic sarcomas in that location. Similarly, Henricks and colleagues studies 155 cases of dedifferentiated liposarcoma, most of which arose in the retroperitoneum, spermatic cord or scrotum (77%) and most of which showed de novo dedifferentiation (86%).13 Similar to the results reported by McCormick et al, these authors found a metastatic rate of only 18% for dedifferentiated liposarcomas of the retroperitoneum and an overall metastatic rate of 17% for dedifferentiated liposarcomas at all sites. Thus, it does appear that dedifferentiated liposarcoma has a lower metastatic rate than other pleomorphic sarcomas arising at the same site. If this is true, then it is logical to presume that it is of clinical importance to distinguish a dedifferentiated liposarcoma from a de novo pleomorphic sarcoma of some other type.

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The utility of MDM2 and CDK4 analysis in distinguishing dedifferentiated liposarcoma from other types of pleomorphic sarcoma is controversial, but the preponderance of evidence suggests that this analysis is useful. Inasmuch as both MDM2 and CDK4 are amplified and often immuno-positive in well-differentiated liposarcomas, it is reasonable to presume that these markers would be similarly expressed in dedifferentiated liposarcomas since the latter are derived from the former. In fact, there are a number of studies that confirm the frequent amplification and immunoexpression of these markers in dedifferentiated liposarcoma. For example, Binh et al found amplification of MDM2 and/or CDK4 in 53 of 55 (96%) dedifferentiated liposarcomas, using either RT-PCR or a-CGH.14 Using these same techniques, however, some of the simulators of dedifferentiated liposarcoma were also found to have amplification of these genes, including myxofibrosarcoma (8/13 cases; 62%), leiomyosarcoma (5/13 cases; 16%), MPNST (2/6 cases; 33%) and "MFH" (3/39 cases; 8%). By immunohistochemistry, almost all cases of dedifferentiated liposarcoma expressed MDM2 (52 cases), CDK4 (51 cases) or both markers (49 cases). However, some of the simulators of dedifferentiated liposarcoma also showed immunoexpression of these antigens. For example, 8 of 13 cases of myxofibrosarcoma stained for MDM2, 3 stained for CDK4 and 2 stained for both markers. Thus, although assessment of gene amplification and immunoexpression in dedifferentiated liposarcoma is highly sensitive, it lacks complete specificity. I polled many of my soft tissue pathology colleagues in the United States and in other countries, and it is clear there is a lack of uniformity in the approach to utilizing these techniques in the distinction of dedifferentiated liposarcoma from other types of pleomorphic sarcoma.

Pleomorphic non-mesenchymal neoplasms resembling a pleomorphic sarcoma It can be exceedingly difficult to distinguish a UPS from a sarcomatoid carcinoma. A reasonable approach would be to assume a pleomorphic malignant neoplasm arising in the skin, mucosal surface or parenchymal organ is a sarcomatoid carcinoma, until proven otherwise. A battery of epithelial markers including broad-spectrum, low- and high- molecular weight cytokeratins is required, but equivocal results are not uncommon for several reasons. First, not all sarcomatoid carcinomas show the immunohistochemical expression of epithelial markers. Second, virtually any type of sarcoma, including UPS, can on occasion express cytokeratins. Strong and diffuse cytokeratin expression, especially with multiple antibodies, strongly supports a diagnosis of sarcomatoid carcinoma, as does the recognition of an intraepithelial/intramucosal dysplastic component. In the end, some cases are not resolvable and can only be diagnosed as a pleomorphic malignant neoplasm, sarcoma versus carcinoma.

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p63 can also play a role in this immunohistochemical work-up. While p63 is frequently expressed in a variety of sarcomatoid carcinomas, it is actually very rarely expressed in soft tissue tumors. In the study by Jo and Fletcher in 2011, p63 was expressed in only 9% of 650 soft tissue tumors tested.15 For example, p63 was found in only 1 of 20 cases of AFX, 0 of 20 cases of dedifferentiated liposarcoma and 0 of 20 cases of leiomyosarcoma. It should also be kept in mind that sarcomatoid mesothelioma, melanoma and anaplastic lymphoma can on occasion mimic UPS; a panel of markers including CAM5.2, S-100 protein, melanocytic markers such as HMB45 and Melan A, CD30 and ALK1 can help resolve these issues. Following the exclusion of the aforementioned scenarios, one is left with UPS as a diagnosis of exclusion, the details of which are described below.

Undifferentiated pleomorphic sarcoma: a diagnosis of exclusion Depending on definitional criteria, UPS still accounts for a significant proportion of sarcomas occurring in late adult life. It manifests a broad range of histologic appearances, although the most common form consists of a mixture of storiform and pleomorphic areas. UPS is characteristically a tumor of late adult life, with most cases occurring in persons between the ages of 50 and 70 years.16 Tumors in children are exceedingly rare, and this diagnosis should always be made with caution in patients under 20 years of age. Approximately two-thirds occur in men, and whites are affected more often than blacks or Asians. The tumor occurs most frequently on the lower extremity, especially the thigh, followed by the upper extremity, usually as a painless, slowly enlarging mass. As mentioned previously, careful sampling and microscopic observation is necessary in retroperitoneal lesions to exclude dedifferentiated liposarcoma, a lesion which in our experience is far more common than de novo UPS in this location. Microscopically, the classic form of UPS has a highly variable morphologic pattern and shows frequent transitions from storiform to pleomorphic areas, although the emphasis in most tumors is on haphazardly arranged pleomorphic zones. Storiform areas consist of plump spindle cells arranged in short fascicles in a cartwheel, or storiform, pattern around slit-like vessels. Although such tumors resemble dermatofibrosarcoma protuberans, they differ by a less distinctive storiform pattern and by the presence of occasional plump histiocytic cells, numerous typical and atypical mitotic figures, and secondary elements including xanthoma cells and chronic inflammatory cells. Although this pattern is easily recognized, it is seldom seen throughout the entire tumor. Instead, most tumors have a combination of storiform and pleomorphic areas, with a preponderance on the latter. Least often, tumors have a fascicular

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growth pattern and resemble fibrosarcomas, except for scattered giant cells, and it may be an arbitrary distinction in some cases as to whether a given tumor should be designated as a UPS or fibrosarcoma. In contrast to the storiform areas, pleomorphic areas contain plumper fibroblastic cells and more rounded histiocyte-like cells arranged haphazardly with no particular orientation to vessels. Pleomorphism and mitotic activity are usually more prominent. The stroma and secondary elements vary considerably in the storiform and pleomorphic areas. Usually, the stroma consists of delicate collagen fibrils encircling individual cells but occasionally collagen deposition is extensive and widely separates cells. Rarely, the stroma contains metaplastic osteoid or chondroid material. If, however, bone or cartilage is extensive and/or appears immature, the tumor should be classified as an osteo- or chondrosarcoma. The vasculature, although elaborate, is seldom appreciated unless it becomes dilated and resembles that of a hemangiopericytoma. Some examples of this tumor have numerous giant cells, a lesion formerly referred to as the giant cell type of MFH or malignant giant cell tumor of soft parts.17,18 These tumors tend to be distinctly multinodular and composed of a mixture of spindled, rounded and osteoclast-type giant cells. Dense fibrous bands containing vessels often encircle the nodules, which frequently show secondary hemorrhage and necrosis. The cells display pleomorphism and prominent mitotic activity, and may contain ingested material such as lipid or hemosiderin. The nuclei of the osteoclast-type giant cells tend also to be of high nuclear grade. Focal osteoid or mature bone is present in up to 50% of these cases and is usually located at the periphery of the tumor nodules.

Immunohistochemical findings

The role of immunohistochemistry in the diagnosis of UPS has traditionally been an ancillary one, primarily serving as a means to exclude other pleomorphic tumors. Thus, the diagnosis continues to presuppose thorough sampling and evaluation of hematoxylin-eosin-stained sections. Despite the limited diagnostic applications of immunohistochemistry aside from excluding other lesions, there is ample evidence that these tumors do not display features of monocytes or macrophages but, rather, fibroblasts/myofibroblasts.8,19,20 Many of these tumors show focal immunoreactivity for smooth muscle actin, but stains for desmin and h-caldesmon are typically negative.21 Some examples also show rare cytokeratin-positive cells, which can cause confusion with those tumors in which a sarcomatoid carcinoma is a real consideration.22– 24 Thus, focal immunoreactivity for any number of intermediate filaments is, in my opinion, insufficient evidence of a specific line of differentiation and should not dissuade one from

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rendering a diagnosis of UPS. On the other hand, diffuse immunoreactivity is far more likely to reflect a specific line of differentiation.

Cytogenetic and molecular genetic findings

Over the past several decades, studies have reported a variety of cytogenetic abnormalities in "MFH" but of course the utility of this information is limited by the varying criteria for making this diagnosis.25,26 In general, pleomorphic sarcomas of all types are characterized by complex but nonspecific cytogenetic aberrations and, as such, this technique is not useful in distinguishing among these pleomorphic sarcomas.27 More recently, a number of studies utilizing comparative genomic hybridization have evaluated UPS and compared the findings to those of other pleomorphic sarcomas.28–31 Interestingly, several studies have found striking similarities between UPS and pleomorphic leiomyosarcoma suggesting a shared lineage.29,31

Clinical behavior

The vast majority of UPS are high-grade lesions having a local recurrence rate ranging from 19– 31%, a metastatic rate of 31–35%, and a 5-year survival of 65–70%.32–37 Both local recurrence and distant metastases often develop within 12–24 months of diagnosis. Only a minority of patients develop metastases after 5 years, with the common metastatic sites being lung (90%), bone (8%), and liver (1%). Regional lymph node metastases are decidedly uncommon. The factors that correlate consistently with metastasis, survival, or both are depth, tumor size, grade, necrosis, and local recurrence, although they are not necessarily independent variables. For example, size and depth appear to co-vary because large tumors tend to be deep tumors. In the study by Engellau and colleagues, necrosis and local recurrence were significant predictors of metastasis within the first two years of diagnosis and throughout a longitudinal follow-up period, whereas only tumor depth and local recurrence were significant predictors beyond 2 years.32

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1. Ozzello L, Stout AP, Murray MR. Cultural characteristics of malignant histiocytomas and fibrous xanthomas. Cancer. 1963;16:331–344.

2. O’Brien JE, Stout AP, Malignant fibrous xanthomas. Cancer. 1964;17:1445–1455.

3. Iwasaki H, Isayama T, Johzaki H, Kikuchi M. Malignant fibrous histiocytoma. Evidence of perivascular mesenchymal cell origin immunocytochemical studies with monoclonal anti-MFH antibodies. Am. J. Pathol. 1987;128(3):528–537.

4. Iwasaki H, Isayama T, Ohjimi Y, et al. Malignant fibrous histiocytoma. A tumor of facultative histiocytes showing mesenchymal differentiation in cultured cell lines. Cancer. 1992;69(2):437– 447.

5. Roholl PJ, Kleyne J, Elbers H, et al. Characterization of tumour cells in malignant fibrous histiocytomas and other soft tissue tumours in comparison with malignant histiocytes. I. Immunohistochemical study on paraffin sections. J. Pathol. 1985;147(2):87–95.

6. Roholl PJ, Kleyne J, Van Unnik JA. Characterization of tumor cells in malignant fibrous histiocytomas and other soft-tissue tumors, in comparison with malignant histiocytes. II. Immunoperoxidase study on cryostat sections. Am. J. Pathol. 1985;121(2):269–274.

7. Fletcher CD. Pleomorphic malignant fibrous histiocytoma: fact or fiction? A critical reappraisal based on 159 tumors diagnosed as pleomorphic sarcoma. Am. J. Surg. Pathol. 1992;16(3):213– 228.

8. Montgomery E, Fisher C. Myofibroblastic differentiation in malignant fibrous histiocytoma (pleomorphic myofibrosarcoma): a clinicopathological study. Histopathology. 2001;38(6):499– 509.

9. Deyrup AT, Haydon RC, Huo D, et al. Myoid differentiation and prognosis in adult pleomorphic sarcomas of the extremity: an analysis of 92 cases. Cancer. 2003;98(4):805–813.

10. Fletcher CD, Gustafson P, Rydholm A, Willén H, Akerman M. Clinicopathologic re- evaluation of 100 malignant fibrous histiocytomas: prognostic relevance of subclassification. J. Clin. Oncol. 2001;19(12):3045–3050.

11. Massi D, Beltrami G, Capanna R, Franchi A. Histopathological re-classification of extremity pleomorphic soft tissue sarcoma has clinical relevance. Eur J Surg Oncol. 2004;30(10):1131– 1136.

12. McCormick D, Mentzel T, Beham A, Fletcher CD. Dedifferentiated liposarcoma. Clinicopathologic analysis of 32 cases suggesting a better prognostic subgroup among pleomorphic sarcomas. Am. J. Surg. Pathol. 1994;18(12):1213–1223.

13. Henricks WH, Chu YC, Goldblum JR, Weiss SW. Dedifferentiated liposarcoma: a clinicopathological analysis of 155 cases with a proposal for an expanded definition of dedifferentiation. Am. J. Surg. Pathol. 1997;21(3):271–281.

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14. Binh MBN, Sastre-Garau X, Guillou L, et al. MDM2 and CDK4 immunostainings are useful adjuncts in diagnosing well-differentiated and dedifferentiated liposarcoma subtypes: a comparative analysis of 559 soft tissue neoplasms with genetic data. Am. J. Surg. Pathol. 2005;29(10):1340–1347.

15. Jo VY, Fletcher CDM. p63 immunohistochemical staining is limited in soft tissue tumors. Am. J. Clin. Pathol. 2011;136(5):762–766.

16. Weiss SW, Enzinger FM. Malignant fibrous histiocytoma: an analysis of 200 cases. Cancer. 1978;41(6):2250–2266.

17. Guccion JG, Enzinger FM. Malignant giant cell tumor of soft parts. An analysis of 32 cases. Cancer. 1972;29(6):1518–1529.

18. van Haelst UJ, de Haas van Dorsser AH. Giant cell tumor of soft parts. An ultrastructural study. Virchows Arch A Pathol Anat Histol. 1976;371(3):199–217.

19. Brecher ME, Franklin WA. Absence of mononuclear phagocyte antigens in malignant fibrous histiocytoma. Am. J. Clin. Pathol. 1986;86(3):344–348.

20. Wood GS, Beckstead JH, Turner RR, et al. Malignant fibrous histiocytoma tumor cells resemble fibroblasts. Am. J. Surg. Pathol. 1986;10(5):323–335.

21. Agaimy A, Gaumann A, Schroeder J, et al. Primary and metastatic high-grade pleomorphic sarcoma/malignant fibrous histiocytoma of the gastrointestinal tract: an approach to the differential diagnosis in a series of five cases with emphasis on myofibroblastic differentiation. Virchows Arch. 2007;451(5):949–957.

22. Lawson CW, Fisher C, Gatter KC. An immunohistochemical study of differentiation in malignant fibrous histiocytoma. Histopathology. 1987;11(4):375–383.

23. Litzky LA, Brooks JJ. Cytokeratin immunoreactivity in malignant fibrous histiocytoma and spindle cell tumors: comparison between frozen and paraffin-embedded tissues. Mod. Pathol. 1992;5(1):30–34.

24. Weiss SW, Bratthauer GL, Morris PA. Postirradiation malignant fibrous histiocytoma expressing cytokeratin. Implications for the immunodiagnosis of sarcomas. Am. J. Surg. Pathol. 1988;12(7):554–558.

25. Gazziola C, Cordani N, Wasserman B, et al. Malignant fibrous histiocytoma: a proposed cellular origin and identification of its characterizing gene transcripts. Int. J. Oncol. 2003;23(2):343–351.

26. Al-Agha OM, Igbokwe AA. Malignant fibrous histiocytoma: between the past and the present. Arch. Pathol. Lab. Med. 2008;132(6):1030–1035.

27. Mertens F, Fletcher CD, Dal Cin P, et al. Cytogenetic analysis of 46 pleomorphic soft tissue sarcomas and correlation with morphologic and clinical features: a report of the CHAMP Study Group. Chromosomes and MorPhology. Genes Chromosomes Cancer. 1998;22(1):16–25.

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28. Kageyama K, Moriyama T, Hizuka N, et al. Hypoglycemia associated with big insulin-like growth factor II produced during development of malignant fibrous histiocytoma. Endocr. J. 2003;50(6):753–758.

29. Larramendy ML, Gentile M, Soloneski S, Knuutila S, Böhling T. Does comparative genomic hybridization reveal distinct differences in DNA copy number sequence patterns between leiomyosarcoma and malignant fibrous histiocytoma? Cancer Genet. Cytogenet. 2008;187(1):1– 11.

30. Nishio J, Iwasaki H, Nabeshima K, et al. Establishment of a new human pleomorphic malignant fibrous histiocytoma cell line, FU-MFH-2: molecular cytogenetic characterization by multicolor fluorescence in situ hybridization and comparative genomic hybridization. J. Exp. Clin. Cancer Res. 2010;29:153.

31. Carneiro A, Francis P, Bendahl P-O, et al. Indistinguishable genomic profiles and shared prognostic markers in undifferentiated pleomorphic sarcoma and leiomyosarcoma: different sides of a single coin? Lab. Invest. 2009;89(6):668–675.

32. Engellau J, Anderson H, Rydholm A, et al. Time dependence of prognostic factors for patients with soft tissue sarcoma: a Scandinavian Sarcoma Group Study of 338 malignant fibrous histiocytomas. Cancer. 2004;100(10):2233–2239.

33. Le Doussal V, Coindre JM, Leroux A, et al. Prognostic factors for patients with localized primary malignant fibrous histiocytoma: a multicenter study of 216 patients with multivariate analysis. Cancer. 1996;77(9):1823–1830.

34. Salo JC, Lewis JJ, Woodruff JM, Leung DH, Brennan MF. Malignant fibrous histiocytoma of the extremity. Cancer. 1999;85(8):1765–1772.

35. Gibbs JF, Huang PP, Lee RJ, et al. Malignant fibrous histiocytoma: an institutional review. Cancer Invest. 2001;19(1):23–27.

36. Zagars GK, Mullen JR, Pollack A. Malignant fibrous histiocytoma: outcome and prognostic factors following conservation surgery and radiotherapy. Int. J. Radiat. Oncol. Biol. Phys. 1996;34(5):983–994.

37. Belal A, Kandil A, Allam A, et al. Malignant fibrous histiocytoma: a retrospective study of 109 cases. Am. J. Clin. Oncol. 2002;25(1):16–22.

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Myxoid Soft Tissue Tumors and Tumor-Like Lesions The pathologist need not panic when one comes across one of these lesions because these entities can be separated from one another when one pays attention to certain key features when evaluating these lesions. For example, evaluation of the cellularity of the lesion, as well as the arrangement of the cells with respect to one another is absolutely critical in distinguishing these lesions from one another. While some lesions are characterized by extremely low cellularity (intramuscular myxoma), others are characteristically much more cellular (nodular fasciitis). Similarly, these cells may stand apart from one another, with little cell-cell contact (myxoid liposarcoma), or they may be arranged into nests or chains (myxoid chondrosarcoma). Nuclear pleomorphism, although somewhat subjective, is also useful in this evaluation, given that some lesions totally lack nuclear pleomorphism (intramuscular myxoma), whereas others are characterized by a high degree of cytologic atypia (myxofibrosarcoma). Another often underappreciated feature in this evaluation is the presence or absence of an underlying vasculature. While some lesions are characteristically of low vascularity (intramuscular myxoma), others are characterized by an intricate vascular network that allows one to recognize the lesion as malignant (myxoid liposarcoma and myxfibrosarcoma). Occasionally, the evaluation of the myxoid stroma using histochemical techniques may also be useful. Hyaluronic acid and chondroitin sulfate are the most common mucosubstances found in these lesions, and one or the other of these substances is often typical of a particular lesion. For example, intramuscular myxoma, myxoid liposarcoma and myxofibrosarcoma are characterized by hyaluronic acid-rich myxoid stroma, whereas myxoid chondrosarcoma and chordoma are characterized by a chondroitin sulfate-rich myxoid stroma. Although Alcian blue (pH2.5) stains both hyaluronic acid and chondroitin sulfate, pretreatment with hyaluronidase will result in the loss of Alcian blue positivity if the stroma is made up of hyaluronic acid. In contrast, chondroitin sulfate-rich stroma is hyaluronidase resistant. Benign myxoid soft tissue lesions that enter into this differential diagnosis include nodular fasciitis, intramuscular myxoma and angiomyxoma, among others. The malignant lesions may include myxoid liposarcoma, myxofibrosarcoma, extra-skeletal myxoid chondrosarcoma and low-grade fibromyxoid sarcoma (Evans' tumor).

Nodular fasciitis is a self-limited, reactive lesion often mistaken for a sarcoma due to its high cellularity, rapid growth and brisk mitotic activity.1 In Montgomery and Meis' series of 53 cases, only 43% of the cases were correctly diagnosed, and 21% of these cases were misdiagnosed as some type of sarcoma2. This lesion typically presents as a rapidly growing mass of short duration, and is most common in young patients (average age: 34 years), although essentially

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any age group may be affected. The upper extremity is the most common site; other fairly common sites include the trunk, lower extremities and head and neck region (although virtually any superficial site in the body may be involved). Most lesions are less than 3.0 cm, but I have seen examples as large as 6.0 cm. Most cases are subcutaneous, but some are in skeletal muscle, causing further concern that the lesion is a sarcoma.

Histologically, nodular fasciitis has many appearances, varying dramatically in cellularity and amount of myxoid stroma both between lesions, and within the same lesion. Most cells are plump, immature “tissue culture” fibroblasts or myofibroblasts that show little nuclear pleomorphism. Mitoses are characteristically numerous, but atypical mitoses are not present. The cells are arranged into short, irregular bundles or fascicles, but never form long, sweeping fascicles, and are deposited in a hyaluronic acid-rich myxoid matrix that occasionally forms microcysts. Other helpful histologic features include cleft-like spaces, red blood cell extravasation, rare giant cells and, occasionally, metaplastic bone or cartilage. In older lesions, there is an increased amount of stromal fibrosis with a less prominent myxoid matrix. Immunohistochemically, the majority of cells stain for smooth-muscle actin, which may be a source of confusion if one is considering the possibility of leiomyosarcoma.2

The intramuscular myxoma typically occurs in middle-aged to elderly patients, and is extremely rare in childhood. These lesions present as a painless, palpable fluctuant mass within the deep soft tissues of the thigh, shoulder, buttocks or upper arm, although virtually any site may be involved.3 In addition, lesions with similar histology can occur in a cutaneous and juxta-articular location. Although usually solitary, multiple intramuscular myxomas have been found to be associated with fibrous dysplasia, and generally occur in the same anatomic region as the bony abnormalities.4 Rare patients also display melanotic pigmentation of the skin and endocrine abnormalities (Albright's syndrome). Myxomas occurring in a cutaneous location may be sporadic or associated with Carney's complex, characterized by an association with endocrine abnormalities, spotty pigmentation, cardiac myxomas, and psammomatous melanotic schwannomas, inherited in an autosomal dominant manner.5 The juxta-articular myxoma is another variant of myxoma, most commonly found in the area of the knee (90%).6 Males are affected significantly more commonly than females, typically between the third and seventh decades of life. In the series by Meis and Enzinger, 34% of the cases recurred, sometimes with multiple recurrences. Despite the tendency for these lesions to recur, these are best treated conservatively by local excision.

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Grossly, the intramuscular myxoma appears to be well-circumscribed, although a true fibrous capsule is not present. Histologically, these lesions characteristically are of low cellularity, composed of bland spindled or stellate cells with cytoplasmic processes. The cells tend not to touch one another, but rather are separated by abundant myxoid stroma composed of hyaluronic acid. Although some cells with vacuolated cytoplasm may be present and may resemble lipoblasts, these are macrophages that have imbibed products of the myxoid stroma resulting in cytoplasmic vacuolization. In addition, although grossly well circumscribed, there is often some infiltration into the surrounding skeletal muscle, with entrapment of atrophic skeletal muscle fibers. Although scattered blood vessels may be present, there is relatively little vascularity, and the vascularity lacks the organization of many myxoid sarcomas. These lesions are essentially cured by local excision, and have little tendency to recur, even if incompletely excised.

Angiomyxoma (aggressive angiomyxoma) typically occurs as a large, ill-defined mass within the pelvis, perineum, or genital tract in women.7,8 Rare cases have also been reported in men.9 Histologically, the lesion is composed of spindled or stellate cells that generally do not touch one another, and are separated by an abundant myxoid stroma composed primarily of hyaluronic acid. The cells lack nuclear atypia, and mitotic figures are difficult to identify. Mast cells are frequently prominent. In addition, these lesions are characterized by a prominent vascularity with vessels of different caliber, including thin-walled vessels and thick hyalinized vessels. Although histologically bland, these lesions are characterized by a high rate of local recurrence; metastases have not been reported.

Myxoid liposarcoma is the most common subtype of liposarcoma.10 This is a tumor of adult life and typically occurs in the deep soft tissues of the extremity, especially the thigh and popliteal region. At low power, the most striking feature is the very characteristic delicate plexiform capillary pattern that is found throughout the neoplasm. The spindled cells between the capillaries are primitive mesenchymal cells, and vary little from one another, without significant nuclear pleomorphism. The cells are evenly distributed, and typically do not touch one another. Interspersed between the primitive mesenchymal cells are the diagnostic lipoblasts, which occur in varying numbers. By definition, a lipoblast is characterized by a sharply defined lipid droplet that usually pushes the nucleus to a peripheral location and indents or scallops the nucleus. Vacuolated cells indistinguishable from lipoblasts may be found in a variety of benign and

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malignant lesions. Therefore, an appropriate histologic background must be present in order to establish that cell as a true lipoblast. Unless there is a significant round cell component, myxoid liposarcomas are generally considered to be low-grade sarcomas.

Round cell liposarcoma is considered to be a poorly differentiated form of myxoid liposarcoma for several reasons. First, it is not uncommon to see mixtures of both myxoid and round cell liposarcoma within the same tumor. Furthermore, the characteristic translocation found in myxoid liposarcoma, t(12;16)(q13;p11), is also present in round cell liposarcoma,11,12 and can be detected by either fluorescence in-situ hybridization13,14 or polymerase chain reaction.15 At the molecular level, this translocation results in fusion of the DDIT3 (CHOP) gene on 12q13 with the FUS gene on 16p11.16-18 Rarely, the DDIT3 gene is fused to the N-terminal portion of the EWS gene.19,20

It has been suggested that tumors that have “hypercellular” or “round cell” areas within an otherwise typical myxoid liposarcoma pursue a more aggressive clinical course.21-24 However, it is difficult to know where on the spectrum of cellularity these cases actually lie. Furthermore, it is unknown whether there is a critical amount of these “round cell” areas that are predictive of a worse prognosis. Smith et al studied 29 cases of myxoid/round cell liposarcoma of the extremities and found that those patients whose tumors had greater than 5% round cell component were more likely to develop metastases or die from their disease.25 Round cell areas were defined as those areas with a marked increase in cellularity in which the cells were round and separated by little or no myxoid stroma. In these areas, the mitotic index was generally increased, and a plexiform vascular pattern was difficult to recognize secondary to the overgrowth of primitive round cells. In addition, transitional areas, defined as areas of increased cellularity compared to typical myxoid liposarcoma, but in which the cells remained spindled, did not have overlapping nuclear borders, and retained an easily discernible plexiform vascular pattern, were not found to worsen clinical outcome in the absence of a round cell component. Kilpatrick et al. found similar findings, although they found a cut-off point of 25% round cell component to be prognostically important by multivariate analysis.26

The myxoid variant of MFH was first characterized by Weiss and Enzinger in 1977, and was defined as an MFH that has at least 50% of the tumor cells deposited in a hyaluronic acid-rich myxoid stroma.27 However, it is apparent that there is a wide spectrum of lesions ranging from superficially located, hypocellular, low-grade myxoid lesions (low-grade myxofibrosarcoma) to

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those that are more deeply located, of higher stage and more biologically aggressive. Mentzel et al. recently evaluated the clinicopathologic features of 75 cases of so-called myxofibrosarcoma,28 now the preferred term for this entity. Almost 70% of their cases were superficially located, either in the dermis or subcutaneous tissues, usually on the upper or lower extremities, and characterized by a nodular growth pattern, a myxoid matrix containing elongated, curvilinear capillaries and spindled or stellate-shaped tumor cells with hyperchromatic atypical nuclei. These superficially located low-grade lesions are the lesions that are most likely to be confused with benign myxoid lesions. Some of the cases were more deeply located and showed areas of increased cellularity and cytologic atypia, more typical of the classic “myxoid MFH” described by Weiss and Enzinger. The latter group of lesions was characterized by moderate cellularity in which the cells showed significant nuclear pleomorphism and hyperchromatism, with easily identified mitotic figures. Similar to myxoid liposarcoma, a characteristic vasculature was present throughout the neoplasm, although these blood vessels tended to be more coarse than those seen in myxoid liposarcoma. Frequently, the atypical cells condensed along the periphery of the blood vessels. Although the depth of the primary lesion did not influence the incidence of local recurrence, only those neoplasms of intermediate or high-grade metastasized. In addition, some cases of low-grade myxofibrosarcoma progressed to higher-grade lesions in recurrences.

In 1987, Evans described a tumor that typically presents as a large, well-circumscribed mass in the deep soft tissues, most commonly in the shoulder, thigh and inguinal region, which he termed “low-grade fibromyxoid sarcoma.”29 Histologically, this lesion is composed of bland spindled cells of low to moderate cellularity deposited in a fibrous and myxoid stroma. The cells often have a swirling growth pattern and occasionally condense in a perivascular location. Cytologically, there is little nuclear pleomorphism, and mitotic figures are difficult to identify. Similar to other myxoid sarcomas, low-grade fibromyxoid sarcoma often has a rich, regular vascular network that is useful in its distinction from a benign lesion. Also, despite the gross circumscription, there is microscopic infiltration of the surrounding tissues. Of the twelve patients described in Evans’ 1993 paper, nine had local recurrences, seven had evidence of distant metastasis, and four died of disease.30 Some authors have also reported histologic progression to a higher-grade lesion in recurrences.31 Low-grade fibromyxoid sarcoma (and the related hyalinizing spindle cell tumor with giant rosettes) is characterized by a t(7;16) with fusion of the CREB3L2 gene on chromosome 12 with the FUS gene on chromosome 16.32 Mertens and colleagues reported the presence of CREB3L2-FUS fusion in 22 of 23 (96%) cases of low-grade

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fibromyxoid sarcoma,33 and we utilize paraffin-embedded tissue for FISH, probing with a FUS breakapart probe to confirm this difficult diagnosis.

Similar to myxoid liposarcoma, extraskeletal myxoid chondrosarcoma also occurs primarily in the deep soft tissues of the extremities.34 Macroscopically, the neoplasm occurs as a multinodular, well-circumscribed mass, which frequently shows large areas of hemorrhage. Histologically, this is a lesion of moderate cellularity in which the cells tend to touch one another and are arranged in small cords or strands. These cells show little nuclear pleomorphism, low mitotic activity, and have a moderate amount of eosinophilic cytoplasm. The vascularity is not prominent, in contrast to myxoid liposarcoma and myxofibrosarcoma. The myxoid matrix in this case is composed of chondroitin sulfate. Immunohistochemically, these cells may be S100 protein positive, although Dei Tos et al found that only 7 of 39 cases (18%) stained for this antigen,35 although it is usually unnecessary to perform immunostains. In addition, this tumor has been found to harbor a characteristic translocation, t(9;22)(q22;q12), which involves a rearrangement of the EWS gene on 22q12 with the NR4A3 gene (formerly known was CHN or TEC).36 The resultant NR3A3-EWS fusion transcript can be detected by RT-PCR or FISH.37 In the series by Meis-Kindblom et al, older patient age, larger tumor size and tumor location in the proximal extremity or limb girdle were adverse prognostic factors identified by multivariate analysis.38 Local recurrences and metastases were noted in 48% and 46% of patients, respectively. Some patients had prolonged survival even after the development of metastasis, although many of these patients eventually died as a result of tumor. Interestingly, this study did not identify a relationship between tumor cellularity and prognosis.

In conclusion, despite the fact that numerous benign and malignant soft tissue lesions may have a myxoid stroma, these lesions can be reliably separated from one another through the systematic evaluation of certain parameters, in conjunction with clinical features including age, site and rate of growth of the neoplasm, with little need for ancillary studies. However, molecular testing for translocations has become increasingly important.

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References

1. Bernstein KE, Lattes R. Nodular (pseudosarcomatous) fasciitis, a nonrecurrent lesion: Clinicopathologic study of 134 cases. Cancer 1982; 49:1668-1675. 2. Montgomery EA, Meis JM. Nodular fasciitis. Its morphologic spectrum and immunohistochemical profile. Am J Surg Pathol 1991; 15:942-948. 3. Kindblom LG, Stener B, Angervall L. Intramuscular myxoma. Cancer 1974; 34;1734- 1744. 4. Ireland DCR, Soule EH, Ivins JC. Myxoma of somatic soft tissues. A report of 58 patients, three with multiple tumors and fibrous dysplasia of bone. Mayo Clin Proc 1973; 48:401-410. 5. Carney JA, Headington JT, Su WPD. Cutaneous myxomas. A major component of the complex of myxomas, spotty pigmentation, and endocrine overactivity. Arch Dermatol 1986; 122:790-798. 6. Meis JM, Enzinger FM. Juxta-articular myxoma. A clinical and pathological study of 65 cases. Hum Pathol 1992; 23:639-646. 7. Steeper TA, Rosai J. Aggressive angiomyxoma of the female pelvis and perineum. Report of nine cases of a distinctive type of gynecologic soft tissue neoplasm. Am J Surg Pathol 1983; 7:463-475. 8. Begin LR, Clement PB, Kirk ME, Jothy S, McCaughey WTE, Ferenczy A. Aggressive angiomyxoma of pelvic soft parts: A clinicopathologic study of nine cases. Hum Pathol 1985; 16:621-628. 9. Tsang WYW, Chan JKC, Lee KC, Fisher C, Fletcher CDM. Aggressive angiomyxoma. A report of four cases occurring in men. Am J Surg Pathol 1992; 16:1059-1065. 10. Weiss SW, Goldblum JR. Myxoid variant of liposarcoma. In: Soft Tissue Tumors, 4th Edition, CV Mosby, St. Louis, MO, 2001. 11. Gibas Z, Miettinen M, Limon J, et al. Cytogenetic and immunohistochemical profile of myxoid liposarcoma. Am J Clin Pathol 1995;103:20-26. 12. Tallini G, Akerman M, Del Cin P, et al. Combined morphologic and karyotypic study of 28 myxoid liposarcomas. Implications for a revised morphologic typing (a report from the CHAMP group). Am J Surg Pathol 1996;20:1047-1055. 13. Mezzelani A, Sozzi G, Pierotti MA, Pilotti S. Rapid differential diagnosis of myxoid liposarcoma by fluorescence in-situ hybridization on cytological preparations. J Clin Pathol 1996;49:308-309. 14. Aoki T, Hisaoka M, Kouho H, Hashimoto H, Nakata H. Interphase cytogenetic analysis of myxoid soft tissue by fluorescence in-situ hybridization and DNA flow cytometry using paraffin-embedded tissue. Cancer 1997;79:284-293. 15. Kuroda M, Ishida T, Horiuchi H, et al. Chimeric TLS/FUS-CHOP gene expression and the heterogeneity of its junction in human myxoid and round cell liposarcoma. Am J Pathol 1995;147:1221-1227. 16. Aman P, Ron D, Mandahl N, et al. Rearrangement of the transcription factor gene CHOP in myxoid liposarcomas with t(12;16)(q13;p11). Genes Chromosomes Cancer 1992;5:278-285. 17. Crozat A, Aman P, Mandahl N, Ron D. Fusion of CHOP to a novel RNA-binding protein in human myxoid liposarcoma. Nature 1993;363:640-644. 18. Rabbitts T, Forster A, Larson R, Nathan P. Fusion of the dominant negative transcription regulator CHOP with a novel gene FUS by translocation t(12;16) in malignant liposarcoma. Nature Genet 1993;4:175-280. 19. Panagopoulos I, Hoglund M, Mertens F, et al. Fusion of the EWS and CHOP genes in myxoid liposarcoma. Oncogene 1996;12:489-494.

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20. Dal Cin P, Sciot R, Panagopoulos I, et al. Additional evidence of a variant translocation t(12;22) with EWS/CHOP fusion in myxoid liposarcoma: clinicopathological features. J Pathol 1997;182:437-441. 21. Azumi N, Curtis J, Kempson RL, Hendrickson MR. Atypical and malignant neoplasms showing lipomatous differentiation: A study of 111 cases. Am J Surg Pathol 1987;11(3):161-183. 22. Enzinger FM, Winslow DJ. Liposarcoma: A study of 103 cases. Virch Arch Pathol Anat 1962;335:367-388. 23. Evans HL. Liposarcoma: A study of 55 cases with a re-assessment of its classification. Am J Surg Pathol 1979;3(6):507-523. 24. Evans HL. Liposarcomas and atypical lipomatous tumors: A study of 66 cases followed for a minimum of 10 years. Surg Pathol 1988;1(1):41-54. 25. Smith TA, Easley KA, Goldblum JR. Myxoid/round cell liposarcoma of the extremities: A clinicopathologic study of 29 cases with particular attention to extent of round cell liposarcoma. Am J Surg Pathol 1996;20(2):171-180. 26. Kilpatrick SE, Doyon J, Choong PFM, Sim FH, Nascimento AG. The clinicopathologic spectrum of myxoid and round cell lipoma. A study of 95 cases. Cancer 1996;77:1450- 1458. 27. Weiss SW, Enzinger FM. Myxoid variant of malignant fibrous histiocytoma. Cancer 1977; 39;1672-1689. 28. Mentzel T, Calonje E, Wadden C, et al. Myxofibrosarcoma: Clinicopathologic analysis of 75 cases with emphasis on the low-grade variant. Am J Surg Pathol 1996;20(4):391- 405. 29. Evans HL. Low-grade fibromyxoid sarcoma: A report of two metastasizing neoplasms having a deceptively benign appearance. Am J Clin Pathol 1987; 88:615-619. 30. Evans HL. Low-grade fibromyxoid sarcoma: A report of twelve cases. Am J Surg Pathol 1993; 17(6):595-600. 31. Goodlad JR, Mentzel T, Fletcher CDM. Low-grade fibromyxoid sarcoma: Clinicopathological analysis of 11 new cases in support of a distinct entity. Histopathology 1995;26:229-237. 32. Panagopoulos I, Storlazzi CT, Fletcher CD, et al. The chimeric FUS/CREB3L2 gene is specific for low-grade fibromyxoid sarcoma. Genes Chromosomes Cancer 2004;40:218- 228. 33. Mertens F, Fletcher CD, Antonescu CR, et al. Clinicopathologic and molecular genetic characterization of low-grade fibromyxoid sarcoma and cloning of a novel FUS/CREB3L1 fusion gene. Lab Invest 2005;85:408-415. 34. Enzinger FM, Shiraki M. Extraskeletal myxoid chondrosarcoma: An analysis of 34 cases. Hum Pathol 1972;3:421-435. 35. Dei Tos AP, Wadden C, Fletcher CDM. Extraskeletal myxoid chondrosarcoma: An immunohistochemical reappraisal of 39 cases. Appl Immunohistochem 1997;5(2):73-77. 36. Hinrichs SH, Jaramillo MA, Gumerlock PH, et al. Myxoid chondrosarcoma with a translocation involving chromosomes 9 and 22. Cancer Genet Cytogenet 1985;14:219- 226. 37. Brody, RI, Ueda T, Hamelin A, et al. Molecular analysis of the fusion of EWS to an orphan nuclear receptor gene in extraskeletal myxoid chondrosarcoma. Am J Pathol 1997;150:1049-1058. 38. Meis-Kindblom JM, Bergh P, Gunterberg B, Kindblom L-G. Extraskeletal myxoid chondrosacroma. A reappraisal of its morphologic spectrum and prognostic factors based on 117 cases. Am J Surg Pathol 1999;23:636-650.

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Fibrosarcoma-Like Pattern Highly cellular spindled mesenchymal neoplasms arranged into a fascicular growth pattern are not uncommonly encountered in the deep soft tissues, mediastinum or retroperitoneum. When presented with such a lesion, the most common differential diagnosis usually includes cellular schwannoma, MPNST, leiomyosarcoma, monophasic synovial sarcoma and fibrosarcoma. Through a combination of light microscopy and immunohistochemistry, one can usually detect a specific line of cellular differentiation in such tumors. As a matter of fact, a diagnosis of fibrosarcoma, once a very common diagnosis to make in the realm of soft tissue pathology, has become a diagnostic rarity.

Cellular Schwannoma Cellular schwannoma is one of several variants of schwannoma that may cause diagnostic confusion, in this case because of its high cellularity, mitotic activity, and presence of bony destruction. These lesions typically occur in middle-aged patients (although the age range is broad), with a slight female predilection. They are most commonly found within the paravertebral region of the posterior mediastinum, retroperitoneum, and pelvis. A small number of patients have been found to have neurofibromatosis.

Grossly, these lesions are typically encapsulated, and may or may not be associated with an identifiable nerve, either grossly or microscopically. Degenerative changes, including cyst formation, hemorrhage and necrosis may be seen. Histologically, the neoplasm is composed of slender, elongated spindled cells with wavy contours that may be arranged in short intersecting fascicles, or in longer sweeping fascicles reminiscent of the herringbone pattern seen in fibrosarcoma. By definition, the lesion is composed almost entirely of Antoni A areas, and although abortive nuclear palisades may be seen, true Verocay bodies are not formed. Although at first glance this lesion may be difficult to differentiate from other spindle cell sarcomas, a variety of histologic features may serve as useful diagnostic clues. This lesion typically has cellularity that is disproportionate to the degree of mitotic activity and cytologic atypia that is present. It should be noted that the cellular schwannoma can have some mitotic activity, although it is typically less than that seen in MPNSTs (<4 MF/10 HPF). Similarly, focal cytologic atypia has been identified in a small percentage of cellular schwannomas, but is typically not to the degree both in quality and quantity as is seen in MPNSTs. Other useful features to recognize this lesion include (1) the presence of paracapsular and/or perivascular lymphoid

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aggregates; (2) the focal presence of Antoni B areas; (3) prominent perivascular hyalinization and, very importantly (4) diffuse and strong immunoreactivity for S100 protein.

It is critical to distinguish the cellular schwannoma from a MPNST, given the differences in both therapy and prognosis. Tumor cellularity is not useful in that both of these lesions are highly cellular. As mentioned previously, although nuclear pleomorphism and mitotic activity may be seen in cellular schwannoma, MPNSTs typically have more extensive nuclear pleomorphism and mitotic figures, including atypical mitoses. Divergent differentiation, often in the form of rhabdomyoblasts, is found in approximately 10% of MPNSTs1, but is not seen in cellular schwannomas. Finally, S100 protein immunoreactivity is a useful adjunct in this differential diagnosis, as cellular schwannomas show diffuse and strong S100 protein positivity, whereas only about 60% of MPNSTs show S100 protein positivity, typically in a focal distribution.2 Four large studies of cellular schwannoma with significant follow-up (total of 119 cases) have been published.3-6 Although up to 5% of tumors have locally recurred, none of the patients have developed metastatic disease or died due to their tumor. Importantly, erosion of adjacent bone has been noted in approximately 13% of the patients in these series, and may contribute to the erroneous diagnosis of a sarcoma.

Ultrastructurally, these cells have been found to have the characteristic features of schwann cells, with elongated bipolar cytoplasmic extensions, interdigitating cytoplasmic processes, and multilayering of basal lamina.7

Other Variants of Schwannoma Ancient Schwannoma Ancient schwannomas are recognized by their extensive degenerative changes, which include cyst formation, hemorrhage, hyalinization and calcification. Significant nuclear atypia may also be noted, but importantly, the degree of mitotic activity is not proportionate to the degree of nuclear atypia that is present, suggesting a degenerative phenomenon.8 These degenerative findings typically occur in lesions that have been present for a long duration, and thus are more commonly found in more deeply situated tumors.

Plexiform Schwannoma Rare schwannomas may also be arranged in a plexiform architecture, reminiscent of what one encounters in the plexiform neurofibroma. Frequently, the individual nodules closely resemble

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those seen in the cellular schwannoma. It is important to distinguish these lesions from plexiform neurofibroma, as they lack an association with neurofibromatosis type I, and also are not known to undergo malignant transformation.9,10

Glandular Schwannoma Rare peripheral nerve sheath tumors also contain clear-cut epithelial differentiation in the form of glands. Although most of these lesions have been described in patients with neurofibromatosis type I and presumably have arisen from neurofibromas, there have been several reports of glandular differentiation in schwannomas.11,12 However, the existence of this entity has been disputed, as some authors believe that these reports represent schwannomas containing entrapped skin adnexal structures.13 Woodruff and Christensen identified 11 cases of glandular peripheral nerve sheath tumors and found that 92% of the tumors were histologically malignant and 74% of the patients had neurofibromatosis type I. The authors did not identify any cases of glandular schwannoma.13

Multiple Schwannomas (schwannomatosis) Rarely, schwannomas may occur multifocally, either in the form of multiple cutaneous schwannomas coursing along a nerve14 or occurring in multiple different sites and often associated with intracranial tumors.15,16 While some authors believe that this entity (schwannomatosis) is distinct from neurofibromatosis type II (NF-2), others believe that this might represent an unusual variant of NF-2.17

Neuroblastoma-like Schwannoma Another rare variant of schwannoma is one in which the cells have a rounded morphology and are often centered around a collagen core forming rosette-like structures, giving a superficial resemblance to neuroblastoma.18 However, both ultrastructural and immunohistochemical examination reveal that these cells have the characteristic features of schwann cells, and this is supported by the benign clinical outcome in all cases.

Melanotic Schwannoma Another rare form of schwannoma has been variably referred to as the melanocytic schwannoma19 or the psammomatous melanotic schwannoma.20 Interestingly, over 50% of the patients with this unusual tumor have evidence of Carney's syndrome (myxomas in a variety of sites, spotty pigmentation and endocrine overactivity).20 The most striking histologic features are

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the heavy pigment deposits which stain positively for Fontana's stain and negatively for iron, as well as the presence of psammoma bodies, which may be extensive in some cases. Immunohistochemically, the cells strongly express S100 protein and HMB-45. Ultrastructurally, the cells resemble typical schwann cells, except for the presence of premelanosomes and melanosomes. Although most of these tumors act in a benign fashion, rare cases may metastasize.20

Finally, the issue of malignant transformation in a schwannoma is an interesting one, as Woodruff et al could only find 9 acceptable cases reported in the literature.21 None of the patients had evidence of neurofibromatosis type I, and the majority of the patients died of their disease. Histologically, the benign component in these tumors was classical schwannoma, whereas the malignant component consisted of an epithelioid malignant peripheral nerve sheath tumor in 7 cases, and showed evidence of neuroepithelial differentiation in 2 cases. Thus, although exceedingly rare, schwannomas do have the capacity to undergo malignant transformation.

Synovial Sarcoma Synovial sarcoma is the third most common type of sarcoma (after so-called MFH and liposarcoma), and typically affects adolescents and young adults (most common between 15-40 years).22 By far the most common location is the extremities, particularly in proximity to large joints (especially the knees), but distal extremity synovial sarcomas are not uncommon. These tumors can also occur on the upper extremities, head and neck region and the trunk. Although these tumors are often intimately related to tendons, tendon sheaths and bursal structures, they are exceedingly rare within joint cavities.

Histologically, synovial sarcomas are composed of variable mixtures of epithelial cells, spindled cells and cells that have features intermediate between these two (transitional cells). Thus, the classic biphasic synovial sarcoma is fairly easily recognized, as it is composed of a distinct population of these three cell types. However, when the epithelial elements are predominant (epithelial type of synovial sarcoma), these lesions may be extremely difficult to recognize and separate from carcinomas, melanomas or mesotheliomas. At the other extreme, when the epithelial elements are difficult to identify or completely absent (monophasic fibrous type of synovial sarcoma), then this lesion becomes difficult to differentiate from other highly cellular spindle cell sarcomas.

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The monophasic fibrous type of synovial sarcoma is probably the most common subtype and can usually be recognized through a combination of histologic and immunohistochemical features. At low magnification, one is often impressed with a “marbled” appearance that is due to alternating areas of low and high cellularity. The spindled cells are often arranged into irregular fascicles, but typically lack the regular herringbone pattern seen in fibrosarcoma. Although the cells are generally spindled, some may have more of an ovoid appearance. Nuclear pleomorphism is typically minimal, and mitotic figures can be identified fairly easily. Other features that should suggest a diagnosis of monophasic fibrous type of synovial sarcoma include the presence of calcification or ossification, a conspicuous mast cell infiltrate, and a hemangiopericytomatous vasculature.

Immunohistochemistry is extremely useful in arriving at this diagnosis. Virtually all monophasic synovial sarcomas stain for cytokeratins, epithelial membrane antigen, or both.23-26 Guillou et al.26 found that all but one of 100 cases of synovial sarcoma stained for at least one of these epithelial markers. In their hands, a significantly greater percentage of cases stained for EMA, although in our laboratory, we have found AE1/AE3 or CAM 5.2 to be more consistently positive. In addition, Guillou et al. noted the relative frequent S100 protein immunoreactivity in all subtypes of synovial sarcoma. Thus, not all spindle cell sarcomas that stain for S100 protein are necessarily malignant peripheral nerve sheath tumors. Given the fact that rare cases of MPNST stain for cytokeratins (and in fact may be S100 protein negative), we have found cytokeratin subsets useful in this regard. Smith et al.27 found that virtually all monophasic synovial sarcomas stained for cytokeratins 7, 19, or both, whereas staining for either of these antigens is extremely rare in cases of MPNST. More recently, gene expression profiling of synovial sarcomas revealed consistent expression of the TLE1 gene.28 Subsequently, an antibody to TLE1 was developed which, according to Terry et al, shows a high degree of sensitivity and specificity for synovial sarcoma.29 In our experience, this antibody is exceedingly useful in confirming a diagnosis of synovial sarcoma, and it is very easy to interpret, since, in my experience, virtually all cells in every synovial sarcoma I have tested shows strong nuclear immunoreactivity. Finally, it must be kept in mind that a significant percentage of synovial sarcomas (including poorly differentiated synovial sarcomas) show membranous immunoreactivity for CD99, a feature which can cause confusion with the Ewing's family of tumors.30

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A consistent, specific translocation, most commonly a balanced reciprocal translocation, t(X;18)(p11;q11), is found in virtually all synovial sarcomas, regardless of subtype.31 This translocation involves the fusion of the SYT gene (also known as SS18) on chromosome 18 with either the SSX1 or SSX2 gene on the X chromosome (both at Xp11) or very rarely with SSX4 (also Xp11).32-34 This fusion can be detected by RT-PCR or FISH. In our practice, we utilize an SYT breakapart probe utilizing paraffin-embedded tissue, an ancillary test used in virtually any case in which we suspect a diagnosis of synovial sarcoma.

Synovial sarcoma is generally viewed and treated as a high-grade sarcoma. However, in a large series of cases reported by Bergh et al, the authors identified features that could place patients in either low or high-risk groups.35 Adverse prognostic factors with regard to metastasis and survival included older age, larger tumor size, the presence of poorly differentiated areas, high Ki-67 values, the presence of necrosis, vascular invasion and prior local recurrence.

Malignant Peripheral Nerve Sheath Tumor Another lesion that frequently enters into the differential diagnosis is malignant peripheral nerve sheath tumor (MPNST). Unless this lesion clearly arises from a nerve trunk, a neurofibroma, or occurs in a patient with von Recklinghausen's disease, it may be extremely difficult to recognize and differentiate from these other lesions. At low magnification, similar to the appearance of monophasic synovial sarcoma and cellular schwannoma, there are alternating hypo- and hypercellular regions resulting in a marbled appearance. The spindled cells are arranged into an irregular fascicular pattern, similar to that seen in fibrosarcoma, but other architectural patterns may be present, including areas of nuclear palisading, myxoid zones, and a perivascular targetoid growth pattern. The tumor cells are wavy or angulated, but show more nuclear pleomorphism than that seen in cellular schwannoma. Mitotic figures are usually numerous (>4 MF/10 HPF). Immunohistochemically, about 60% of MPNSTs stain for S100 protein, typically with only focal positivity.

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References

1. Ducatman BS, Scheithauer BW. Malignant peripheral nerve sheath tumors with divergent differentiation. Cancer 1984;54:1049-1057. 2. Weiss SW, Langloss JM, Enzinger FM. Value of S100 protein in the diagnosis of soft tissue tumors with particular reference to benign and malignant schwann cell tumors. Lab Invest 1983;49:299-308. 3. Lodding P, Kindblom LG, Angervall L, Stenman G. Cellular schwannoma: A clinicopathologic study of 29 cases. Virch Arch Pathol Anat 1990; 416:237-248. 4. White W, Shiu MH, Rosenblum MK, Erlandson RA, Woodruff JM. Cellular schwannoma: A clinicopathologic study of 57 patients and 58 tumors. Cancer 1990; 66:1266-1275. 5. Fletcher CDM, Davies SE, McKee PH. Cellular schwannoma: A distinct pseudosarcomatous entity. Histopathology 1987; 11:21-35. 6. Woodruff JM, Godwin TA, Erlandson RA, Susin M, Martini N. Cellular schwannoma: A variety of schwannoma sometimes mistaken for a malignant tumor. Am J Surg Pathol 1981; 5:733-744. 7. Woodruff JM. Cellular schwannoma. Bone and Soft Tissue Specialty Conference, Case 1. USCAP Meeting, Toronto, Canada, 1995. 8. Dahl I. Ancient neurilemmoma (schwannoma). Acta Pathol Microbiol Scand 1977;85(6):812-818. 9. Fletcher CDM, Davies SE. Benign plexiform (multinodular) schwannoma: A rare tumor unassociated with neurofibromatosis. Histopathology 1986;19:971-980. 10. Hirose T, Scheithauer BW, Sano T. Giant plexiform schwannoma: A report of two cases with soft tissue and visceral involvement. Mod Pathol 1997;10:1075-1081. 11. Brooks JJ, Draffen RM. Benign glandular schwannoma. Arch Pathol Lab Med 1992;116:192-195. 12. Fletcher CDM, Madziwa D, Heyderman E, et al. Benign dermal schwannoma with glandular elements - true heterology or a local “organizer” effect? Clin Exp Dermatol 1986;11:475-485. 13. Woodruff JM, Christensen WN. Glandular peripheral nerve sheath tumors. Cancer 1993;72:3618-3628. 14. Buenger KM, Porter NC, Dozier SE, Wagner RF. Localized multiple neurilemmomas of the lower extremity. Cutis 1993;51:36-38. 15. Purcell SM, Dixon SL. Schwannomatosis: An unusual variant of neurofibromatosis or a distinct clinical entity? Arch Dermatol 1989;125:390-393. 16. Shishibo T, Niimura M, Ohtsuka F, Tsuru N. Multiple cutaneous neurilemmomas as a skin manifestation of neurilemmomatosis. J Am Acad Dermatol 1984;10:744-754. 17. Reith JR, Goldblum JR. Multiple cutaneous plexiform schwannomas: Report of a case and review of the literature with particular reference to the association with types 1 and 2 neurofibromatosis and schwannomatosis. Arch Pathol Lab Med 1996;120:399-401. 18. Goldblum JR, Beals TF, Weiss SW. Neuroblastoma-like neurilemmoma. Am J Surg Pathol 1994;18:266-273. 19. Fu YS, Kaye GI, Lattes R. Primary malignant melanocytic tumors of the sympathetic ganglia with an ultrastructural study of one. Cancer 1975;36:2029-2041. 20. Carney JA. Psammomatous melanotic schwannoma: A distinctive heritable tumor with special associations including cardiac myxoma and the Cushing syndrome. Am J Surg Pathol 1990;14:206-222. 21. Woodruff RM, Selig AM, Crowley K, Allen PW. Schwannoma with malignant transformation. A rare, distinctive peripheral nerve tumor. Am J Surg Pathol 1994;18(9):882-895.

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22. Weiss SW, Goldblum JR. Synovial sarcoma. In: Enzinger and Weiss's Soft Tissue Tumors, 5th Ed. Elsevier, New York 2008. 23. Ordonez NG, Mahfouz SM, Mackay B. Synovial sarcoma. An immunohistochemical and ultrastructural study. Hum Pathol 1990;21:733-749. 24. Schmidt D, Thum P, Med C, Harms D, Treuner J. Synovial sarcoma in children and adolescents. A report from the Kiel Pediatric Tumor Registry. Cancer 1991;67:1667- 1672. 25. Fetsch JF, Meis JM. Synovial sarcoma of the abdominal wall. Cancer 1993;72:469-477. 26. Guillou L, Wadden C, Kraus MD, Dei Tos AP, Fletcher CDM. S100 protein reactivity in synovial sarcomas - A potentially frequent diagnostic pitfall. Immunohistochemical analysis of 100 cases. Appl Immunohistochem 1996;4(3):167-175. 27. Smith TA, Machen SK, Fisher C, Goldblum JR. Utility of cytokeratin subsets in distinguishing monophasic synovial sarcoma from malignant peripheral nerve sheath tumor. Am J Clin Pathol 1999;112:641-648. 28. Nielsen TO, West RB, Linn SC, et al. Molecular characterization of soft tissue tumours: a gene expression study. Lancet 2002;359:1301-1307. 29. Terry J, Saito T, Subramanian S, et al. TLE1 as a diagnostic immunohistochemical marker for synovial sarcoma emerging from gene expression profiling studies. Am J Surg Pathol 2007;31:240-246. 30. Folpe AL, Schmidt RA, Chapman D, Gown AM. Poorly differentiated synovial sarcoma: immunohistochemical distinction from primitive neuroectodermal tumors and high-grade malignant peripheral nerve sheath tumors. Am J Surg Pathol 1998;22:673-682. 31. Dal Cin P, Rao U. Jani-Sait S, Karasousis C, Sandberg AA. Chromosomes in the diagnosis of soft tissue tumors. I. Synovial sarcoma. Mod Pathol 1992;5:357-362. 32. de Leeuw B, Balemans M, Olde Weghuis D, Geurts van Kessel A. Identification of two alternative fusion genes, SYT-SSX1 and SYT-SSX2, in t(x;18)(p11.2;q11.2)-positive synovial sarcomas. Hum Mol Genet 1995;4:1097-1099. 33. Fligman I, Lonardo F, Jhanwar SC, Gerald WL, Woodruff J, Ladayni M. Molecular diagnosis of synovial sarcoma and characterization of a variant SYT-SSX2 fusion transcript. Am J Pathol 1995;147:1592-1599. 34. Argani P, Zakowski MF, Klimstra DS, Rosai J, Ladanyi M. Detection of the SYT-SSX chimeric RNA of synovial sarcoma in paraffin-embedded tissue and its application in problematic cases. Mod Pathol 1998;11;65-71. 35. Bergh P, Meis-Kindblom JM, Gherlinzoni F, et al. Synovial sarcoma: identification of low and high-risk groups. Cancer 1999;85:2596-2602.

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Round Cell Tumor Pattern The differential diagnosis of small round cell tumors is broad. On occasion, one can encounter a benign round cell tumor (e.g. glomus tumor, giant cell tumor of tendon sheath, cutaneous adnexal tumors of various types), which can be mistaken for a high-grade round cell sarcoma. However, most of the time when one encounters a round cell pattern, the differential diagnosis includes a broad group of malignant round cell tumors which includes the Ewing's family of tumors (EFT), alveolar rhabdomyosarcoma, neuroblastoma, lymphoblastic lymphoma, Merkel cell carcinoma, small cell carcinoma, poorly differentiated synovial sarcoma, mesenchymal chondrosarcoma, round cell liposarcoma, desmoplastic small round cell tumor, small cell osteosarcoma and others. Although the light microscopic features are useful in narrowing this differential diagnosis, in virtually every case, ancillary studies including immunohistochemistry and molecular genetic studies are required in order to more precisely classify the round cell tumor. Given that this has important therapeutic and prognostic implications, simply designating a given tumor as a round cell tumor, not otherwise specified, is not acceptable in most cases. However, there are rare cases that cannot be precisely classified.

Ewing’s/Peripheral Neuroectodermal Tumor (Ewing's Family of Tumors or EFT) A review of the literature over the past twenty years reveals a remarkable evolution in the concepts regarding classic osseous and extra-osseous Ewing's sarcoma. Through an accumulation of data, it has become apparent there is a spectrum of tumors that ranges from classic Ewing's sarcoma to classic peripheral neuroectodermal tumor (PNET). Since its initial description by James Ewing in 1921, Ewing's sarcoma was felt to arise only in bone, and it was not until 1975 when Angervall and Enzinger described the first cases of extra-osseous Ewing's sarcoma.1 PNET, since its initial description by Arthur Purdy Stout in 1918 as a round cell tumor of the ulnar nerve,2 has been documented in soft tissue unassociated with nerve,3 as well as within bone.4 As discussed below, these entities have histologic, immunohistochemical, ultrastructural, cytogenetic and molecular genetic features that are overlapping, supporting the histogenetic relationship among these neoplasms.

EFT: Clinical Features Most patients with EFT are adolescents or young adults, the majority of whom are less than 30 years of age.5 Although the mean age for PNET is similar to that of ES, there tends to be a broad age range for the former, with a significant number of patients over the age of 40 years. In

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contrast, patients with classic extraskeletal ES are rarely over 40 years of age. Both tumors are slightly more common in males than in females.

PNET most commonly arises in the extremities. In my experience, the most common anatomic sites are the upper thigh and buttock, followed by the upper arm and shoulder. Tumors intimately attached to a major nerve may give rise to signs and symptoms related to diminished neurologic function. The principal sites of classic extraskeletal ES are the paravertebral region and chest wall, generally in close association with the vertebrae or the ribs. These tumors may also arise in the soft tissues of the lower extremities and rare in the pelvic and hip regions, the retroperitoneum and the upper extremities. However, it is important to note that virtually every anatomic site has been documented to be involved by this family of tumors. In general, the tumor presents as a rapidly growing, deeply situated mass measuring between 5 and 10 cm. Superficially located cases do occur but are quite rare.

EFT: Pathologic Findings Classic ES is composed of solidly packed uniform small cells arranged in a lobular pattern separated by dense fibrous septa. At low magnification, there is nuclear uniformity, and nucleoli are inconspicuous, as are mitotic figures. The nuclear chromatin is fine and powdery, and there is a thin rim of pale cytoplasm often filled with glycogen. Hemorrhage and necrosis are prominent features. On the other end of the spectrum, classic PNET is characterized by more irregularity in nuclear size and shape, as well as a coarsening of the chromatin, more prominent nucleoli and increased mitotic figures. In addition, rosettes of varying types, including Homer Wright, Flexner-Wintersteiner and perivascular rosettes are typically seen. However, in between these ends of the spectrum is a variety of architectural and cytologic features in which it is unclear whether the lesion is best classified as a classic ES or PNET. The entity of "atypical Ewing's sarcoma" has been proposed to include some of these cases between the two ends of the histologic spectrum.6 However, the cut-off between ES and atypical ES, and between ES and PNET, is unclear and arbitrary. Fortunately, however, this distinction is not necessary since a number of more recent studies have shown that there is no significant difference in prognosis based upon where a given tumor lies on this morphologic spectrum.7

Ultrastructurally, typical ES is a primitive neoplasm composed of uniform round cells devoid of specific features and characterized by abundant deposits of cytoplasmic glycogen. On the other hand, PNET shows ultrastructural evidence of some degree of neural differentiation, including

390

rare dense core granules, neuritic cell processes, neurofilaments and neural tubules. Similar to that seen histologically, there is a spectrum of ultrastructural features between the two extremes that can be seen.

EFT: Immunohistochemical Features For many years, a diagnosis of either ES or PNET was essentially an immunohistochemical diagnosis of exclusion. However, it is clear that the product of the MIC2 gene (CD99) is the most sensitive marker on this family of tumors.8 The MIC2 gene is a pseudoautosomal gene located on the short arms of the sex chromosomes, and its product is a membranous glycoprotein that can be detected immunohistochemically using a variety of differenti antibodies (including 12E7 and O13). Although initially believed to be highly specific for the EFT, it is apparent that virtually all other round cell tumors in the differential diagnosis, on rare occasion, show membranous immunoreactivity for the MIC2 gene product, including lymphomas, particular T-lymphoblastic lymphoma and precursor B-lymphoblastic lymphoma, Merkel cell carcinoma, small cell carcinoma, alveolar rhabdomyosarcoma, small cell osteosarcoma, desmoplastic small round cell tumor and mesenchymal chondrosarcoma.9-12 Notably, however, childhood neuroblastomas have not been reported to stain for this antigen. Thus, although immunostains for CD99 are highly sensitive for recognizing the EFT, this marker should be used as part of a panel of immunostains, given the lack of complete specificity.

There are a few other notable immunohistochemical findings that one should be aware of for EFT. For example, up to 20% of tumors have focal immunoreactivity for low-molecular-weight cytokeratins,13 although these tumors do not express cytokeratins 7 or 19, a useful finding for distinguishing EFT from poorly differentiated synovial sarcoma.14 Desmin may also rarely be found in EFT, but there is no ultrastructural evidence of skeletal muscle differentiation in these tumors.15

Cytogenetic and Molecular Genetic Findings Approximately 90-95% of EFT are characterized by rearrangements of the EWS gene on 22q12 and ETS-related oncogenes, most commonly FLI-1 on 11q24.16 Less commonly, the EWS gene is fused with other ETS-related genes, including ERG on 21q22,17 ETV-1 on 7p22,18 E1AF on 17q1219 or FEV on 2q33.20 The translocation breakpoints are restricted to introns 7-10 of the EWS gene and introns 3-9 on the ETS-related gene, with the most common fusion being between exon 7 of EWS and exons 5 or 6 of FLI-1.21 These translocations result in a novel

391

chimeric gene that encodes for a chimeric transcript in protein, the function of which is largely unknown. Given the limitations of traditional cytogenetic techniques for detecting these translocations, the ability to detect fusion transcripts by molecular genetic techniques (including RT-PCR and FISH) using fixed, paraffin-embedded tissues has greatly facilitated the diagnosis of these tumors. In our practice, we utilize an EWSR1 breakapart probe as a routine part of the work-up for a suspected EFT.

Alveolar Rhabdomyosarcoma Alveolar rhabdomyosarcoma (ARMS) is another important lesion to distinguish from EFET, given the different therapeutic modalities used to treat these tumors. Although there may be some overlap in the age distribution, ARMS often occurs in patients younger than seen in EFT. This tumor has a predilection for the deep soft tissues of the extremities, although it may arise in many other sites, including the head and neck, trunk, perineum, pelvis and retroperitoneum.

Histologically, ARMS is composed largely of ill-defined aggregates of poorly differentiated round or oval tumors cells that frequently show central loss of cellular cohesion and formation if irregular "alveolar" spaces. The individual cellular aggregates are separated and surrounded by a framework of dense, frequently hyalinized fibrous septa that surround dilated vascular channels. The cells at the periphery of the alveolar spaces are well preserved and adhere in a single layer to the fibrous septa, whereas those in the center of the alveolar spaces tend to be more loosely arranged or freely floating. These centrally located cells are often poorly preserved and show evidence of degeneration and necrosis. There are also "solid" forms of ARMS that lack an alveolar growth pattern entirely and are composed of densely packed groups of tumor cells resembling the round cell areas of EFT. These solidly cellular areas are more commonly encountered at the periphery of the tumor and probably represent the most active and most cellular stage of growth. However, even in these solid areas, there is a regular arrangement of fibrous septa that surround the primitive round cells. Rhabdomyoblasts may be found, but in some cases, they may be extremely difficult to identify.

Immunohistochemistry is extremely useful in making the diagnosis of ARMS. Although it is true that most of these tumors do express desmin and muscle-specific actin (HHF-35), there are some ARMS that do not express either of these antigens. In my experience, myogenin is the best marker in recognizing ARMS. Both MyoD1 and myogenin are members of the MyoD family of genes, which encode a series of DNA binding proteins that are involved in the initiation of

392

myogenic differentiation.22 These genes are expressed at the earliest stages of commitment of a mesenchymal cell to striated muscle, and antibodies to these genes appear to be the most sensitive markers of skeletal muscle differentiation.23 Although myogenin is expressed by virtually all subtypes of rhabdomyosarcoma, it is clear that ARMS tends to express this antigen more diffusely and strongly than the other subtypes of rhabdomyosarcoma. It is also important to recognize that some examples of ARMS may show membranous immunoreactivity for CD99.

Again, cytogenetic and molecular genetic features of ARMS may be extremely useful in confirming this diagnosis. The most common translocation is a t(2;13)(q35;q14), resulting in the fusion of the PAX3 gene on chromosome 2 with FOXO1a (formerly known as FKHR) gene on chromosome 13.24 Less commonly, ARMS may show a t(1;13), resulting in a PAX7-FOXO1a fusion. In our practice, we utilize a FOXO1a breakapart probe on fixed, paraffin-embedded tissues to detect this translocation. However, it must be kept in mind that only about 75% of ARMS have either a t(2;13) or t(1;13), and 25% of ARMS lack either of these translocations.

Once a diagnosis of rhabdomyosarcoma is established, it is important to properly subtype the tumor, given the significant prognostic implications. However, pathologists are not particularly good at subclassifying rhabdomyosarcomas, as there is a high degree of inter- and intraobserver variation in classifying these tumors.25 The Intergroup Rhabdomyosarcoma Study (IRS) has proposed the International Classification of Rhabdomyosarcoma (ICR), which seems to be the most reproducible classification scheme as well as the scheme which predicts prognosis best.26 Tumors having a superior prognosis include botryoid and spindle cell variants of embryonal rhabdomyosarcoma. The usual type of embryonal rhabdomyosarcoma has an intermediate prognosis, whereas ARMS has a poor prognosis.

Desmoplastic Small Round Cell Tumor (DSRCT) DSRCT is a relatively uncommon entity that typically involves the abdominal or pelvic peritoneum of young males and pursues an aggressive clinica course. Most patients with this tumor are 15 to35 years of age, although patients younger and older than this classic age range have also been reported. In a study of 109 patients with this tumor by Gerald et al,27 the patients ranged in age from 6 to 49 years, with a mean age of 22 years. Males far outnumber females at a ratio of approximately 4:1.

393

Most patients present with a large abdominal and/or pelvic mass with extensive peritoneal involvement, usually without an identifiable visceral site of origin. However, this tumor has been noted to arise in virtually every other anatomic location and does not necessarily arise in association with a mesothelial-lined surface. Although there was some speculation that this lesion could be related to a mesothelial neoplasm (mesothelial blastoma), given the fact that these tumors can arise in non-mesothelial locations and given the absence of convincing immunohistochemical or ultrastructural evidence of mesothelial differentiation, the histogenesis of this unusual tumors remains unknown.

Histologically, the neoplasm is composed of sharply outlined islands of tumor cells that are separated by a desmoplastic stroma containing myofibroblasts and prominent vascularity. There is often a suggestion of peripheral palisading, occasionally with central necrosis. The individual cells are relatively uniform, small and round to oval with hyperchromatic nuclei, inconspicuous nucleoli and scanty cytoplasm. Mitotic figures are easily identified. Rarely, cells with peripherally located nuclei and increased eosinophilic cytoplasm with a perinuclear clear zone (rhabdoid morphology) are seen. Despite this classic histology, the morphologic profile of this tumor continues to expand, as Ordonez noted that up to one-third of these tumors have atypical histologic features.28

Immunohistochemically, these lesions have a characteristic polyphenotypic profile with coexpression of cytokeratins, vimentin, desmin and NSE. The pattern of desmin immunoreactivity is quite unique with a characteristic perinuclear globular pattern of staining. An antibody that recognizes the carboxyl terminus portion of the WT1 gene product has also been developed and is highly sensitive and reasonably specific in recognizing this tumor.29 It is also important to note that up to 20% of DSRCT do stain for CD99. Interestingly, a characteristic cytogenetic aberration has been associated with this neoplasm, t(11;22)(p13;q12), involving the Wilms' tumor gene on chromosome 11 and the EWS gene on chromosome 22. In virtually all suspected cases, we utilize an EWSR1 breakapart probe in an attempt to detect this translocation.

Neuroblastoma Neuroblastoma may also be difficult to differentiate from some of these other round cell tumors. Patients with neuroblastoma are typically younger than those with the other tumors, as 90% of patients are diagnosed before the age of 5 years. Neuroblastoma is exceedingly rare in

394

adolescents and adults. These patients often have elevated catecholamine metabolite levels in their urine. Neuroblastomas arise from either the adrenal gland or extra-adrenal sympathetic ganglia, although metastases may be seen in virtually any location.

Histologically, neuroblastomas are composed virtually entirely of small round undifferentiated cells, typically with dark nuclei and clumped chromatin, inconspicuous nucleoli and scanty cytoplasm. The tumor cells are divided into small lobules by fibrovascular septa. Typically, some cells with peripherally located larger nuclei with vesicular chromatin and increased eosinophilic cytoplasm (representing immature ganglion cells) are seen. Neuroblastomas are characterized by rosettes of various types, including Homer Wright rosettes, with the cells deposited in a fibrillary background. Calcifications are often seen, and mature ganglion cells may be present.

Immunohistochemically, neuroblastomas do not stain for CD99, actin, desmin or myogenin. Although the vast majority of neuroblastomas do stain for neural markers, particularly NSE, the expression of these neural markers is not specific. Miettinen et al reported a high degree of sensitivity of the monoclonal antibody NB84 in recognizing neuroblastoma,30 but we have had no experience utilizing this antibody in our clinical practice. Neuroblastomas are characterized by a consistent cytogenetic abnormality with deletion of the short arm of chromosome 1 (1p-).31 Amplification of the N-myc gene is frequently seen in neuroblastoma and is of important prognostic value.32

Mesenchymal Chondrosarcoma Mesenchymal chondrosarcoma is a rare neoplasm that typically occurs in young adults with a peak age in the second to third decade of life. Approximately 20% of these neoplasms arise in an extraosseous location and are most common in the cranial or spinal meninges, orbit and soft tissues of the thigh.33 Histologically, Histologically, the neoplasm is characterized by a biphasic appearance of small nests or nodules of well-differentiated cartilage intimately admixed with undifferentiated round or slightly spindled cells with hyperchromatic nuclei and scanty cytoplasm. Frequently, a hemangiopericytoma-like vascular pattern is present. Although the biphasic appearance is characteristic, this may not be appreciated on a small biopsy, making distinction from other round cell tumors difficult. Immunohistochemically, mesenchymal chondrosarcomas do not express cytokeratins or myogenic markers, but variably express neural markers, including S100 protein. Membranous CD99 immunoreactivity is found in the majority of these tumors.34 In addition, there have been rare reports of mesenchymal chondrosarcoma with

395

the identical t(11;22) identified in the EFT, raising the possibility that these tumors are histogenetically related.35

Round Cell Liposarcoma Round cell liposarcoma is a poorly differentiated form of myxoid liposarcoma and typically behaves as a high-grade sarcoma. Histologically, the cells are relatively uniform, small and round with vesicular nuclei. The fine plexiform vascular pattern that is so characteristic of myxoid liposarcoma is inconspicuous in the round cell areas. This neoplasm may be very difficult to recognize, particularly on a needle biopsy, without a component of myxoid liposarcoma. In such cases, the application of FISH can be extremely useful, since (like myxoid liposarcoma) round cell liposarcoma is characterized most commonly by a t(12;16) involving the DDIT3 gene on chromosome 12 and the FUS gene of chromosome 16, for which breakapart probes are commercially available. Like myxoid liposarcoma, some cases of round cell liposarcoma may also harbor a t(12;22), and in such cases, using an EWSR1 probe can be useful.

Poorly Differentiated Synovial Sarcoma Poorly differentiated synovial sarcoma is composed of small round cells with little cytoplasm, often separated by a hemangiopericytoma-like vascular pattern. Unless one identifies other areas of classic biphasic or monophasic synovial sarcoma of lower grade, this lesion may be extremely difficult to separate from some of the other round cell sarcomas. In addition, the poorly differentiated form of synovial sarcoma is less likely to express cytokeratins.36 Further adding to the difficulty, some cases of poorly differentiated synovial sarcoma express membranous CD99 immunoreactivity, making distinction from EFT difficult.37 We have found the use to cytokeratin subsets useful in this regard, as a substantial portion of poorly differentiated synovial sarcomas stain for CK7 and 19, while EFT rarely, if ever, stains for the antigens.14 Detection of the t(X;18) using either RT-PCR or FISH (for the SYT gene) may be exceedingly useful in recognizing this tumor.38

396

References

1. Angervall L, Enzinger FM. Extraskeletal neoplasm resembling Ewing's sarcoma. Cancer 1975;36:240-251. 2. Stout AP. A tumor of the ulnar nerve. Proc NY Pathol Soc 1918;1:2-12. 3. Seemayer TA, Thelmo WL, Bolande RP, Wigglesworth FW. Peripheral neuroectodermal tumors. Perspect Pediatr Pathol 1975;2:151-172. 4. Jaffe R, Santamaria M, Yunis EJ, et al. The neuroectodermal tumor of bone. Am J Surg Pathol 1984;8:885-898. 5. Dehner LP. Primitive neuroectodermal tumor and Ewing's sarcoma. Am J Surg Pathol 1993;17(1):1-13. 6. Navarro S, Cavazzana AO, Llombart-Bosch A, Triche TJ. Comparison of Ewing's sarcoma of bone and peripheral neuroepithelioma: An immunocytochemical and ultrastructural analysis of 2 primitive neuroectodermal neoplasms. Arch Pathol Lab Med 1994;118:608-615. 7. Terrier P, Henry-Amar M, Trich TJ, et al. Is neuroectodermal differentiation of Ewing’s sarcoma of bone associated with an unfavorable prognosis? Eur J Cancer 1995;31:307- 314. 8. Stevenson AJ, Chatten J, Bertoni F, Miettinen M. CD99 (P30/32MIC2) neuroectodermal/Ewing's sarcoma antigen as an immunohistochemical marker: Review of more than 600 tumors and the literature experience. Appl Immunohistochem 1994;2(4):231-240. 9. Riopel M, Dickman PS, Link MP, Pearlman EJ. MIC-2 analysis in pediatric lymphomas and leukemias. Hum Pathol 1994;25:396-399. 10. Lumadue JA, Askin FB, Perlman EJ. MIC-2 analysis of small cell carcinoma. Am J Clin Pathol 1994;102:692-694. 11. Weidner N, Tjoe J. Immunohistochemical profile of monoclonal antibody O13: Antibody that recognizes glycoprotein p30/32MIC-2 and is useful in diagnosing Ewing's sarcoma and peripheral neuroepithelioma. Am J Surg Pathol 1994;18(5):486-494. 12. Perlman EJ, Dickman PS, Askin FB, et al. Ewing's sarcoma: Routine diagnostic utilization of MIC-2 analysis. Hum Pathol 1994;25:303-307. 13. Gu M, Antonescu CR, Guiter G, et al. Cytokeratin immunoreactivity in Ewing’s sarcoma. Prevalence in 50 cases confirmed by molecular diagnostic studies. Am J Surg Pathol 2000;24:410-416. 14. Machen SK, Fisher C, Gautam RS, Tubbs RR, Goldblum JR. Utility of cytokeratin subsets for distinguishing poorly differentiated synovial sarcoma from peripheral primitive neuroectodermal tumour. Histopathology 1998;33:501-507. 15. Parham DM, Dias P, Kelly DR, Rutledge JC, Houghton P. Desmin positivity in primitive neuroectodermal tumors of childhood. Am J Surg Pathol 1992;16(5):483-492. 16. Delattre O, Zucman J, Melot T, et al. The Ewing family of tumors - a subgroup of small round cell tumors defined by specific chimeric transcripts. N Engl J Med 1994;331:294- 299. 17. Sorensen PHB, Lessnick SL, Lopez-Terrada D, et al. A second Ewing’s sarcoma translocation, t(21;22), fuses the EWS gene to another ETS-family transcription factor, ERG. Nature Genet 1994;6:146-151. 18. Jeon I-S, Davis JN, Braun BS, et al. A variant Ewing’s sarcoma translocation (7;22) fuses the EWS gene to the ETS gene ETV-1. Oncogene 1995;10:1229-1234. 19. Kaneko Y, Yoshida K, Handa M, et al. Fusion of the ETS-family gene E1AF to EWS by t(17;22)(q12;q12) chromosome translocation in an undifferentiated sarcoma of infancy. Genes Chromosomes Cancer 1996;15:115-121.

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20. Peter M, Couturier J, Pacquement H, et al. A new member of ETS family fused to EWS in Ewing tumors. Oncogene 1997;14:1159-1164. 21. de Alava E, Kawai A, Healey JH, et al. EWS-FLI-1 fusion transcript structure is an independent determinant of prognosis in Ewing’s sarcoma. J Clin Oncol 1998;16:1248- 1255. 22. Weintraub H, Davis R, Tapscott S, et al. The MyoD gene family: Nodal point during specification of the muscle cell lineage. Science 1991;251:761-766. 23. Wang NP, Marx J, McNutt MA, et al. Expression of myogenic regulatory proteins (myogenin and MyoD1) in small blue round cell tumors of childhood. Am J Pathol 1995;147:1799-1810. 24. Shapiro DN, Sublett JE, Li B, Downing JR, Maeve CW. Fusion of PAX 3 to a member of the forkhead family of transcription factors in human alveolar rhabdomyosarcoma. Cancer Res 1993;53:5108-5112. 25. Asmar L, Gehan E, Newton WA, et al. Agreement among and within groups of pathologists in the classification of rhabdomyosarcoma and related childhood sarcomas: Report of an international study of four pathology classifications. Cancer 1994;74:2579- 2588. 26. Newton WA, Gehan EA, Webber BL, et al. Classification of rhabdomyosarcomas and related sarcomas: Pathologic aspects and proposal for a new classification - an Intergroup Rhabdomyosarcoma Study. Cancer 1995;76:1073-1085. 27. Gerald WL, Ladanyi M, de Alava E, et al. Clinical, pathologic and molecular spectrum of tumors associated with t(11;22)(p13;q12): desmoplastic small round-cell tumor and its variants. J Clin Oncol 1998;16:3028-3036. 28. Ordonez NG. Desmoplastic small round cell tumor. I: a histopathologic study of 39 cases with emphasis on unusual histological patterns. Am J Surg Pathol 1998;22:1303-1313. 29. Barnoud R, Sabourin J-C, Pasquier D, et al. Immunohistochemical expression of WT1 by desmoplastic small round cell tumor: a comparative study with other small round cell tumors. Am J Surg Pathol 2000;24:830-836. 30. Miettinen M, Chatten J, Paetau A, Stevenson A. Monoclonal antibody NB84 in the differential diagnosis of neuroblastoma and other small round cell tumors. Am J Surg Pathol 1998;22:327-332. 31. Brodeur GM, Green AA, Hayes FA, Williams KJ, Williams DL, Tsiatis AA. Cytogenetic features of human neuroblastomas and cell lines. Cancer Res 1981;41:4678-4686. 32. Brodeur GM, Seeger RC, Schwab M, et al. Amplification of N-myc in untreated human neuroblastomas correlates with advanced disease stage. Science 1984;224:1121-1124. 33. Nakashima Y, Unni KK, Shives TS, Swee RG, Dahlin DC. Mesenchymal chondrosarcoma of bone and soft tissue: a review of 111 cases. Cancer 1986;57:2444- 2453. 34. Granter SR, Renshaw AA, Fletcher CDM, Bhan AK, Rosenberg AE. CD99 reactivity in mesenchymal chondrosarcoma. Hum Pathol 1996;27:1273-1276. 35. Sainati L, Scapinello A, Montaldi A, et al. A mesenchymal chondrosarcoma of a child with the reciprocal translocation (11;22)(q24;q12). Cancer Genet Cytogenet 1993;71:144-147. 36. Folpe AL, Schmidt RA, Chapman D, Gown AM. Poorly differentiated synovial sarcoma: immunohistochemical distinction from primitive neuroectodermal tumors and high-grade malignant peripheral nerve sheath tumors. Am J Surg Pathol 1998;22:673-682. 37. Dei Tos AP, Wadden C, Calonje E, et al. Immunohistochemical demonstration of glycoprotein P30/32MIC2 (CD99) in synovial sarcoma. A potential cause of diagnostic confusion. Appl Immunohistochem 1995;3(3):168-173.

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38. Shipley J, Crew J, Birdsall S, et al. Interphase fluorescence in-situ hybridization and reverse transcription polymerase chain reaction as a diagnostic aide for synovial sarcoma. Am J Pathol 1996;148:559-567.

399 400 401 402 ! Chronic Hepatitis Symptoms: non-specific constitutional symptoms; jaundice Liver chemistry tests: mild inc. AST & ALT >>> Alk phos and TB low Histologic pattern: portal changes predominant; +/- fibrosis minimal lobular changes

• Chronic HCV hepatitis Diagnosis by serology • Chronic HBV hepatitis Biopsy for grade and stage • Autoimmune hepatitis • • • • Drug

John Hart, M.D. University of Chicago Medical Center [email protected]

S11-3050

• 35 y.o. male with fatigue; teacher of 7th & 8th grade math • TB = 1.8, AST = 100, ALT = 96, alk phos = 194 • HCV genotype 1a; HCV RNA = 347,724 IU • Platelet count = 66,000; albumin = 3.6 • Spider angiomas on chest; palpable spleen

• Liver biopsy performed

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• EBV serology negative • After 3 mo LCTs remained abnormal

• Liver biopsy performed

405

• DX: Mild portal & lobular inflammation • Comment: – There is no evidence of AIH – Not typical of EBV hepatitis – There is no fibrosis – Quantitative copper could be performed to rule out Wilson’s disease – Could this be celiac disease???

Chronic Hepatitis Symptoms: non-specific constitutional symptoms; jaundice Liver chemistry tests: mild ↑ AST & ALT >>> Alk phos and TB low Histologic pattern: portal changes predominant; +/- fibrosis; minimal lobular changes

• Chronic HCV hepatitis Diagnosis by serology • Chronic HBV hepatitis Biopsy for grade and stage • Autoimmune hepatitis • Mimics: – Wilson disease – Primary biliary cirrhosis – Non-specific portal inflammation – Biliary obstruction – Celiac disease – Lymphoma • • • Drug !

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• Hepatotoxicity in 1:2000 to 1:10,000 • Cause of Drug Rash with Eosinophilia and Systemic Symptoms [DRESS] syndrome • Latency is typically one to several weeks • Presenting symptom is diffuse maculopapular rash, followed by fever and N&V • Mild-to-moderate ALT elevations • Prolonged exposure after symptoms may result in irreversible, rapidly progressive liver failure • Steroids have been used but with uncertain effectiveness

409 Acute Hepatitis Symptoms: non-specific constitutional symptoms; jaundice Liver chemistry tests: AST & ALT >>> Alk phos Histologic pattern: lobular disarray with minimal portal changes; no fibrosis • Skin biopsy – drug hypersensitivity reaction • Lamotrigine d/c • Drugs and toxins • Started on steroids for DRESS syndrome • • • Rash improved and WBC down to 15,000 • • 6/17: TB = 2.3, AST = 293, ALT = 650, alk phos = 389; • INR = 1.4 • HAV infection • 6/30: TB = 1.3, AST = 28, ALT = 63, alk phos = 155, • Acute HCV infection DIAGNOSIS • INR = 1.0 • Acute HBV infection BY • Autoimmune hepatitis SEROLOGIC • Exotic infections TESTING

S10-6869 • 55 y.o. F with abdominal pain and headache • Noted to have mildly elevated LCTs at the time of thyroidectomy for Hashimoto’s thyroiditis 1 year ago • Status post cholecystectomy 20 years ago

• TB = 0.7, AST = 986, ALT = 630; alk phos = 107 • HAV, HBV, HCV, HEV, EBV, HHV-6, CMV negative • ANA = 1:160; anti-SMA & anti-dsDNA negative • No travel; no high risk behaviors; no ethanol • Synthroid, Citalopram, Gabapentin, MiraLAX, Citrucel

• Referred to the Univ. of Chicago for liver biopsy

410 411 DATE TB AST ALT AP 10/29/09 0.4 36 24 35 2/11/10 0.3 23 28 44 3/16/10 0.7 986 630 107 3/18/10 0.5 122 413 122 3/31/10 0.3 156 415 147

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413 Acute Hepatitis C07-28460 Symptoms: non-specific constitutional symptoms; jaundice Liver chemistry tests: AST & ALT >>> Alk phos Clinical History Histologic pattern: lobular disarray with minimal portal changes; no fibrosis • 78 year old female with osteoarthritis presented to her PCP with acute painless • Drugs and toxins jaundice. • • She is s/p cholecystectomy many years ago. • • She was on no prescription medications but • took a multivitamin and “Move Free”, a • HAV infection glucosamine / chondroitin supplement. • HEV infection • TB = 7.2 , AST = 1053, ALT = 1626, Alk phos = • Acute HCV infection DIAGNOSIS 354, GGT = 599. • Acute HBV infection BY • Serologic tests for HAV, HBV, HCV neg. • Autoimmune hepatitis SEROLOGIC • Stopped “Move Free” – jaundice resolved • Exotic infections TESTING

C07-28460 Clinical History

• 2 wks later - TB = 2.3, AST = 415, ALT = 678, Alk phos = 279 • Osteoarthritis worsened – resumed “Move Free” • 2 wks later - TB = 4.7, AST = 1177, ALT = 1206, Alk phos = 286. • Abdominal/pelvic CT scan no abnormalities • ANA, p-ANCA, anti-SMA negative • Liver biopsy performed

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415 ! " Mechanical Biliary Obstruction Signs & Symptoms: jaundice, abdominal pain Liver chemistry tests: Alk phos >>> AST & ALT Histologic pattern: bile duct proliferation, cholestasis, portal fibrosis

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• Lefkowitch JH.Bile ductular cholestasis: an ominous histopathologic sign related to sepsis and "cholangitis lenta". Hum Pathol. 1982 Jan;13(1):19-24.

Cholangitis lenta (bile ductular cholestasis

416 *$"!%"+ #$"'%

SN10-2952 #455/ #$ "+ # '"$#+ "0'#!- $ +## $#

• 86+" !"# $#)$' ! • :703- :435-:456-! #:789 • - %(. :4/830 • 1 /##$ # $"-'$ "+ $"$% #

417 " " ! !!'!( • MRI and MRCP reveal no evidence of • More than 20 cases reported in the literature biliary obstruction • Captopril, Enalipril, Lisonopril, Ramipril • Medications include Lisinopril • Modulation of eicosanoid metabolism results in toxic hepatic metabolite • Progressive cholestatic injury leading to bile duct epithelial damage, biliary necrosis and development of biliary cirrhosis

Ann Pharmacotherapy 1993; 27:228-31. Arch Pathol Lab Med 2003; 127:1493-7.

C04-17608 ! ( 46(,, ! &!$ +! *( *( ! * ( * ( ! ( * "% !$ *! " 7/.,3* 7/55* 7/16* 72/0 %% ( ! $" % (& ! ! ( ! $" % % ( ! $"

418 11-5702 &

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419 &$' % !

# • Poor prognostic feature: – Higher 3 month mortality – Spahr L et al. BMC Gastroenterology 2011;11:115. • Pathophysiology: – Extensive ballooning compromises bile canaliculi – Ethanol blocks intracellular/canalicular transport of bile acids – Concomitant pancreatic disease compresses extrahepatic bile ducts? • Dont perform ERCP to r/o biliary obstruction

!$ NOTE: Doesnt occur in NASH

420 06-26501 #586/ #$ "+ % "% • 87+0 0 )$* "''#!"# $#)$' ! • HCV RNA viral load = 44 million IU/mL • :508- :648-:599-! #:577 • ! #%(- $+!4 • HIV positive with a low CD4 count • %(. %( • # (+ '# • % # • 1 /##$ #

C10-24175 #$ "+ "" # '"$#+ "0 * ("#$+ " $ • 73+" !"# $#)$$## • !!" # !+"(## !("# • :403- :64-:74-! #:483 • -- %(. %( • # (+ '# • "'""% " • 1 / '" '###$ #

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422

Wilson PD. Polycystic kidney disease. N Eng J Med 2004 ; 350:151 - 64.

423 Mechanical Biliary Obstruction C16-15242 Signs & Symptoms: jaundice, abdominal pain % Liver chemistry tests: Alk phos >>> AST & ALT Histologic pattern: bile duct proliferation, cholestasis, portal fibrosis • 0/%))#"% % " $ " • Primary sclerosing cholangitis " • % + % " • Biliary atresia • 2./),' 2-,-' 2-.1' 2-0.. • Hepatic Langerhans cell histiocytosis • ' ' # • TPN toxicity • * # #* ( ( % • [Primary biliary cirrhosis] • Pancreatobiliary mass lesion/choledocholithiasis • Ischemic cholangiopathy • Cystic fibrosis • Mimics: – Cholangitis lenta (bile ductular cholestasis) – ACE inhibitor toxicity – Congenital hepatic fibrosis – Massive hepatic necrosis (fulminant hepatic failure)

424 & $% #,

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• )#+674$! % & • 30 y.o. F with a 1 month history of fever (101-103 F) 2 % $% #5$% + # 1 • She also developed a diffuse macular rash and significant ! %) !# $%% #3 arthralgias • ! $#(,#$ • Her only medication is an inhaler for seasonal asthma. • %/ %#(%(#$! $&)(# $$ • There is no family history of liver disease or recent travel • Laboratory evaluation: – TB = 0.3, AST = 111, ALT = 141, Alk phos = 71 – ANA negative; anti-SMA = 1:40; no hypergammaglobulinemia – Serologic tests for HAV, HBV, HCV negative – Serum ceruloplasmin and alpha-1-antitrypsin levels are normal

425 C08-17294 • 30 y.o. F with a 1 month history of fever (101-103 F) • She also developed a diffuse macular rash and significant arthralgias • Her only medication is an inhaler for seasonal asthma. • There is no family history of liver disease or recent travel • Laboratory evaluation: – TB = 0.3, AST = 111, ALT = 141, Alk phos = 71 (hepatitis) – ANA negative; anti-SMA = 1:40; no hypergammaglobulinemia – Serologic tests for HAV, HBV, HCV negative – Serum ceruloplasmin and alpha-1-antitrypsin levels are normal

• Histologic pattern: – Not clear • Diagnosis: – Hepatitis (weak) • Take home points: – As a last resort, Pubmed it

Elevated Parvo b19 IgM titer (8.78) Elevated Parvo b19 IgG titer (6.06)

426 Medical Liver Disease Interpretation by Histologic Pattern

John Hart, M.D. [email protected]

ACUTE HEPATITIS

Histologic Features: Lobular disarray predominates, with inconspicuous portal inflammation. In the resolving phase only scattered pigment laden Kupffer cells (highlighted by PAS/D may be evident

Differential Diagnosis: 1. Drug/toxin injury 2. Acute HAV hepatitis 3. Acute HBV hepatitis 4. Acute HCV hepatitis 5. Acute HEV hepatitis 6. Autoimmune hepatitis

CHRONIC HEPATITIS

Histologic Features: Portal inflammation predominates, with inconspicuous lobular necroinflammatory changes. Portal inflammation is more than sparse and involves most or all portal tracts

Differential Diagnosis: 1. Chronic HBV hepatitis 2. Chronic HCV hepatitis 3. Autoimmune hepatitis 4. Drug/toxin injury (rare) 5. Mimics of chronic hepatitis A. Wilson disease (periportal Mallory-Denk bodies) B. Primary biliary cholangitis (lymphocytic cholangitis, bile duct loss/ductular reaction) C. Celiac disease (positive serum TTG) D. Non-specific portal inflammation E. Lymphoma involving portal tracts

LOBULAR HEPATITIS

Histologic Features: Sinusoidal lymphocytic infiltrate without lobular disarray predominates. Mild portal inflammation is also present.

427

Differential Diagnosis: 1. EBV hepatitis 2. Drug toxicity (especially Dilantin)

CENTRILOBULAR HEPATOCYTE NECROSIS/DROPOUT

Histologic Features: Centrilobular hepatocyte necrosis and/or dropout. Portal inflammation is inconspicuous

Differential Diagnosis: 1. Drug/toxin exposure (in many cases a specific drug/toxin is not identified) 2. Ischemia (clinical history of an episode of profound hypotension) 3. Budd-Chiari syndrome (with centrilobular hemorrhage +/- hepatocyte atrophy) 4. Sinusoidal obstructive syndrome (Hx of stem cell transplantation, induction chemotherapy) 5. Autoimmune hepatitis (centrilobular plasma cell infiltrates, positive serum ANA)

RANDOM HEPATOCYTE NECROSIS

Histologic Features: Non-zonal patchy hepatocyte necrosis without prominent inflammation. Can be difficult to recognize in advanced cases with extensive necrosis.

Differential Diagnosis: 1. Adenovirus infection 2. Herpes simplex virus infection 3. Histoplasma infection

GRANULOMATOUS HEPATITIS

Histologic Features: granulomas with portal inflammation and/or lobular necroinflammatory activity

Differential Diagnosis: 1. Drug toxicity (many drugs, especially allopurinol, diltiazem, hydralazine, alpha-methyldopa) 2. Infections (particularly with fever or necrotizing granulomas, immunocompromised host) 3. Hepatic sarcoidosis (resembling chronic hepatitis, but with granulomas)

GRANULOMAS

Histologic Features: Portal or lobular granulomas (necrotizing or non-necrotizing) without significant portal or lobular inflammation

428

Differential Diagnosis: 1. Idiopathic (majority of cases) 2. Hepatic sarcoidosis (non-necrotizing) 3. Foreign material 4. Infection (particularly with fever or necrotizing granulomas, immunocompromised host) 5. Tumor

MECHANICAL BILIARY OBSTRUCTION

Histologic Features: Portal fibrosis, bile ductular proliferation, cholestasis

Differential Diagnosis: 1. Primary sclerosing cholangitis 2. Biliary atresia 3. Hepatic Langerhans cell histiocytosis 4. TPN toxicity [Primary biliary cholangitis (PBC) – looks more like chronic hepatitis] 5. Pancreatobiliary mass lesion/choledocholithiasis 6. Ischemic cholangiopathy 7. Cystic fibrosis 8. Mimics: Cholangitis lenta (bile ductular cholestasis) ACE inhibitor toxicity Congenital hepatic fibrosis Massive hepatic necrosis (fulminant hepatic failure)

BLAND CHOLESTASIS

Histologic features: Hepatocellular and/or canalicular cholestasis

Differential diagnosis: 1. Drug toxicity (estrogens, anabolic steroids, antibiotics, etc.) 2. Early mechanical biliary obstruction 3. TPN toxicity 4. Benign recurrent intrahepatic cholestasis

BILE DUCT PAUCITY

Histologic features: Loss of intralobular bile ducts, +/- cholestasis

Differential Diagnosis: 1. Drugs (numerous)

429 2. Primary biliary cholangitis (PBC) 3. Biliary atresia (late) 4. Alagille syndrome 5. Ischemic cholangiopathy 6. Liver allograft chronic rejection 7. Chronic graft versus host disease

430 431 432 433 434 THE CRITICAL ROLE OF THE PATHOLOGIST IN THE BLADDER CANCER EPIDEMIOLOGY (2016) MANAGEMENT OF BLADDER CANCER

Urinary bladder 18,010 2%

Urinary bladder 4,570 1.6%

Siegel et al. CaA Cancer J Clin 66:7-30, 2016 Thera, Cycladic Islands

BLADDER CANCER GENETICS

CHARACTERISTIC FEATURES BASAL p53-LIKE LUMINAL Squamous/sarcomatoid Usual UC Usual UC morphology Express high MWCK – Usually CK 5/6 negative Usually CK 5/6 negative CK5/6 CK20 negative CK20 positive CK20 positive CD44 positive CD44 negative CD44 negative TP53 mutated Activating FGFR3 Activating FGFR3 mutations mutations FOXA1 loss Activated wild-type p53

Zieger et al. Int J Cancer 125;2095, 2009 Choi W, et al. Cancer Cell 25:152-165, 2014

435 EORTC RISK TABLES Patients presenting with Ta/Tis/T1 tumors

RECURRENCE PROGRESSION*

• 6 - Number of tumors • 6 - CIS • 4 - Prior recurrence • 5 - Grade • 3 - Tumor size • 4 - T-category • 2 - Grade • 3 - Number of tumors • 1 - T-category • 3 - Tumor size • 1 - CIS • 2 - Prior recurrence

*Progression = development of muscle invasive disease Sylvester et al. Eur Urol 49:466, 2006

NORMAL UROTHELIUM WHO/ISUP 2004/2016 CLASSIFICATION

• NORMAL • FLAT LESIONS WITH ATYPIA – Reactive (inflammatory) atypia – Atypia of unknown significance – Dysplasia (low grade intraurothelial neoplasia) – Carcinoma in situ (high grade intraurothelial neoplasia) • PAPILLARY NEOPLASMS – Papilloma – Inverted papilloma – Papillary neoplasm of low malignant potential – Papillary carcinoma, low grade – Papillary carcinoma, high grade • INVASIVE NEOPLASMS

Cytokeratin 20

REACTIVE ATYPIA REACTIVE ATYPIA

436 DYSPLASIA DYSPLASIA

DYSPLASIA CIS “flat urothelial lesion…containing cytologically malignant cells”

Or Incipient Papillary Neoplasia? Cytokeratin 20

CIS - LARGE CELL CIS - LARGE CELL

437 CARCINOMA IN SITU CIS – “SMALL CELL” Abundant eosinophilic cytoplasm

CIS - DENUDING CIS – DENUDING – VON BRUNN’S NESTS

CARCINOMA IN SITU CARCINOMA IN SITU

No surface epithelium Pagetoid growth

438 SV: CIS - PAGETOID CARCINOMA IN SITU

Early invasion

CARCINOMA IN SITU – p53 IHC CARCINOMA IN SITU ~ 80% Positive

CK20

CARCINOMA IN SITU CARCINOMA IN SITU CK20

AMACR

P53

CK 20

439 REACTIVE ATYPIA UROTHELIAL CARCINOMA IN SITU - LONG TERM OUTCOME

SURVIVAL-TYPE 10-Year 15-Year

Progression-free 63% 59%

CK20 Cancer-specific 79% 74%

All-cause 55% 40%

p53 Cheng et al, Cancer 85:2469, 2000

CLASSIFICATION AND GRADING OF UROTHELIAL PAPILLOMA PAPILLARY UROTHELIAL NEOPLASMS

• Most are small BASED ON TWO KEY FEATURES • Essentially normal • Architectural disruption urothelium • Degree of cytologic atypia • Often vacuolated umbrella cells

PAPILLARY UROTHELIAL NEOPLASM OF PAPILLOMA LOW MALIGNANT POTENTIAL

440 PUNLMP LOW GRADE PUNLMP

LOW GRADE HIGH GRADE LOW GRADE

HIGH GRADE HIGH GRADE

441 HIGH GRADE PAPILLARY BLADDER – PAPILLARY UC

52/164 (32%) papillary UC were grade heterogeneous Cheng et al. Cancer 88:1663-1670, 2000

EARLY PAPILLARY CARCINOMA WHO 1973 vs WHO 2016

Papilloma Papilloma

Papillary urothelial Papillary ca, I neoplasm of low malignant potential

Papillary ca, II Papillary ca, low grade

Papillary ca, III Papillary ca, high grade

pTa BLADDER CA pTa BLADDER CA LONG TERM OUTCOME LONG TERM OUTCOME

Progression in stage Cancer-specific mortality

N=175 N=483 N=129 N=175 N=483 N=129

Pan et al, AJCP 133:788, 2010 Pan et al, AJCP 133:788, 2010

442 STAGING OF BLADDER CANCER (2010 TNM) BLADDER CANCER: • pTa Non-invasive, papillary OUTCOME AFTER CYSTECTOMY

• pTis Non-invasive, flat N=1,100 • pT1 Invasion of subepithelial connective tissue (lamina propria) • pT2 Invasion of muscularis propria – pT2a inner one-half – pT2b outer one-half • pT3 Invasion of perivesical tissue – pT3a microscopically – pT3b macroscopically • pT4 Invasion of adjacent structures Hautmann et al. Eur Urol 61:1039, 2012

TREATMENT OF T1 DISEASE TREATMENT OF T1 DISEASE

“ On the basis of clinical and administrative data, we estimate that between 31.2% and 46.8% of deaths potentially were avoidable.” 2008;102:270-275

Cancer 115:1011, 2009

RADICAL CYSTECTOMY FOR NON- MUSCLE INVASIVE BLADDER CANCER: EAU GUIDELINES 2013 UPDATE “It is reasonable to propose radical cystectomy to those patients with NMIBC who are at highest risk of progression” (“radical cystectomy should be considered”): • Multiple and/or large (> 3cm) T1, HG/G3 tumours • T1, HG/G3 tumours with concurrent CIS

15% 83% • Recurrent T1, HG/G3 tumours • T1, HG/G3 tumours with CIS in prostatic urethra Eur Urol 57:60-70, 2010 • Micropapillary variant of urothelial carcinoma

Babjuk et al. Eur Urol 64:639-653, 2013

443 DIAGNOSIS OF INVASION DIAGNOSIS OF INVASION Increased cytoplasm Irregular nests Retraction artifact Stromal response

DIAGNOSIS OF INVASION DIAGNOSIS OF INVASION Increased cytoplasm Retraction artifact

T1 SUBSTAGING T1 SUBSTAGING

N=1,515

HGPUC T1 ≤ 1mm vs > 1mm

HGPUC Ta vs T1 ≤ 1mm

pT1m: a single microscopic focus ≤ one HPF pT1e: a single microscopic focus > one HPF or more than one focus

Chang et al. Am J Surg Pathol 36:454, 2012 Cheng et al. J Clin Oncol 17:3182, 1999 van Rhijn et al. Eur Urol 61:378, 2012

444 T1 SUBSTAGING MUSCULARIS MUCOSAE (≤ 1 HPF vs > 1 HPF) DEVELOPMENT IN THE NORMAL URINARY Progression-free survivalN=301 Cancer-specific survivalN=301 BLADDER

PATTERN Ro (1987) Keep (1989) Paner (2007) P=0.012 P<0.001 Continuous 3% 13% 7% Discontinuous 21% 45% 41% Interrupted 70% 40% 52%* Absent 6% 0 *continuous + absent

Bertz et al. Histopathology 59:722, 2011.

MUSCULARIS MUCOSAE MUSCULARIS MUCOSAE

TRIGONE REGION MUSCULARIS MUCOSAE INVASION

445 MUSCULARIS MUCOSAE INVASION MUSCULARIS PROPRIA INVASION

MM vs MP INVASION pT1 – SUBSTAGING: MUSCULARIS MUCOSAE

“pT1a” “pT1b”

SURVIVAL ACCORDING TO MUSCULARIS MUCOSAE INVASION • 343 patients - initial treatment • 170 pT1 “Based on the available data, it is • Cases centrally recommended to provide an assessment reviewed of the depth and/or extent of subepithelial invasion inT1 cases.” • Substaging possible in 99 (58%) P<0.02 Grignon et al. Infiltrating urothelial carcinoma (p97) • Treated by: • TURBT with intravesical tx Angulo et al, J Cancer Res Clin Oncol 119:578, 1993

446 INTERNATIONAL COLLABORATION ON INTERNATIONAL COLLABORATION ON CANCER REPORTING (ICCR) CANCER REPORTING (ICCR) URINARY TRACT DATA SETS URINARY TRACT (BIOPSY/TRANSURETHRAL • Urinary tract (biopsy/transurethral resection) RESECTION) • Renal pelvis and ureter (resection) Substaging T1 Disease • Urinary bladder (resection) Required: No • Urethra (resection) Recommended: Yes MEMBERS Response type: Value list David Grignon (USA – Chair) Fadi Brimo (Canada) • Depth of invasion (mm) Brett Delahunt (New Zealand) Michael Koch (USA) and/or Antonio Lopez-Belatran (Spain) Victor Reuter (USA) Hema Samaratunga (Australia) Jonathan Shanks (England) • Total maximum dimension of invasive tumor (mm) John Srigley (Canada – ICCR) Toyonori Tsuzuki (Japan) and/or Theo van der Kwast (Canada) Murali Varma (Wales) • Invasion superficial to muscularis mucosae vs invasion www.iccr-cancer.org involving and/or deep to muscularis mucosae

T1 UC WITH LYMPHVASCULAR INVASION PROBLEMS WITH IDENTIFICATION OF LYMPHVASCULAR INVASION • 118 newly diagnosed T1; all with TURBT +/- intra-vesical tx (85%) • LVI diagnosis based on H&E alone • LVI diagnosed in 33 cases (28%)

“The general use of immunohistochemistry in the routine setting, however, cannot be recommended” Amin et al. Pathology Consensus Guidelines, International Consultation on Urologic Diseases, 2012 Cho et al. J Urol 182:2625-2631, 2009

GRADE AS A PREDICTOR OF OUTCOME UROTHELIAL CARCINOMA IN pT1 CA TREATED BY TURBT HISTOLOGIC VARIANTS (2016) • Divergent differentiation – Squamous differentiation – Glandular differentiation – Trophoblastic differentiation – Müllerian differentiation • Nested variant • Microcystic variant • Micropapillary variant • Plasmacytoid variant • Clear cell type • Lipid-rich • Lymphoepithelioma-like variant • Giant cell • Sarcomatoid carcinoma Kaubisch et al, J Urol 146:28-31, 1991

447 PROGNOSITIC SIGNIFICANCE OF RECOGNITION OF VARIANT HISTOLOGY VARIANT HISTOLOGY VARIANT NUMBER* PERCENT PERCENT NOT RECOGNIZED Squamous differentiation 32 32% <25% Small cell differentiation 16 16% 44% Glandular differentiation 13 13% <25% Micropapillary 12 12% 83% Nested 8 8% 87% Sarcomatoid 6 6% NA Lymphoepithelioma-like 3 3% 100% • Multi-institutional (5) Plasmacytoid 1 1% 100% • Radical cystectomy 2000 – 2008 Multiple types 10 10% NA • No neoadjuvant treatment * Variant histology present in 115/589 (20%) of TURBT cases reviewed (2004 – 2008)

Xylinas et al. Eur J Cancer 49:1889-1897, 2013 Shah et al. Urol Oncol 31:1650, 2013

UROTHELIAL CARCINOMA MICROPAPILLARY VARIANT MICROPAPILLARY TYPE • CLINICAL – Similar epidemiology to usual UC – High stage, 50% with + LN at diagnosis – Worse prognosis with high % MP • PATHOLOGY – Small, tight clusters of cells – Open spaces simulating lymphatic invasion – Deeply invasive – Suggested to be a form of glandular differentiation – Inversion of MUC1 staining to stromal aspect

MICROPAPILLARY VARIANT MICROPAPILLARY VARIANT

448 MICROPAPILLARY VARIANT UC - MICROPAPILLARY VARIANT

CD 34

UC - MICROPAPILLARY VARIANT: UC - MICROPAPILLARY VARIANT: DIAGNOSTIC CRITERIA DIAGNOSTIC CRITERIA

• High degree of agreement with “classical cases” (Kappa value 0.79) • Less agreement for equivocal cases • Key features for diagnosis included: • Extensive retraction artifact • Multiple nests within the same lacunar space • Epithelial ring forms • Peripheral nuclear orientation

Sangoi et al. Am J Surg Pathol 34:1367, 2010

UC - MICROPAPILLARY VARIANT UC - MICROPAPILLARY VARIANT URETER FROZEN SECTION

449 UC – MICROPAPILLARY VARIANT: OUTCOME UROTHELIAL CARCINOMA N=24 N=72 PLASMACYTOID VARIANT • CLINICAL – Described by Saphir in 1955 (“monocytoid SRC”) – Highly aggressive tumor – Linitis plastica-like; often no discrete mass but edematous mucosa in many • PATHOLOGY – Sheets of poorly cohesive cells – Distinct monocytoid/plasmacytoid morphology with variable numbers of true signet-ring cells

Lopez-Beltran et al. Hum Pathol Comperat et al. Pathology 42:650-654, – +/- typical UC component 42:1159-1164, 2010 2010 – CK ++ (variable CK7/CK20), p63+, LCA -

PLASMACYTOID CARCINOMA PLASMACYTOID CARCINOMA

450 PLASMACYTOID CARCINOMA PLASMACYTOID CARCINOMA

PLASMACYTOID VARIANT PLASMACYTOID CARCINOMA

E-CADHERIN CK 7 CK 20 CD 138

PATTERN OF SPREAD URETER MARGIN Rectum Fallopian Tube

Pelvic wall Lymph node

451 Plasmacytoid UC – Ureter Margin Frozen Section Plasmacytoid UC – Ureter Margin Frozen Section

PLASMACYTOID CARCINOMA IMPORTANCE OF PATHOLOGY

David P Wood Jr. 2009.

Keck et al. BMC Cancer 13:71, 2013

American Bald eagles “ice fishing” on Geist Reservoir, Indianapolis, IN

Thank you for your attention

452 THE CRITICAL ROLE OF THE PATHOLOGIST IN MANAGING BLADDER CANCER –SELECTED REFERENCES (2016)

HANDLING AND REPORTING

Edge S, Byrd D, Compton C, Fritz A, Greene F, Trotti AI, eds. AJCC Cancer Staging Manual. Seventh ed. New York: Springer; 2010.

Grignon DJ, Al-Ahmadie H, Algaba F, et al. Urinary Tract: Infiltrating Urothelial Carcinoma. In: WHO Classification of Tumours of the Urinary System and Male Genital Organs; Reuter V, Moch H, Humphrey P, Ulbright T, eds. IARC Press, Lyon, 2016

Amin MB, Smith SC, Reuter VE, Epstein JI, Grignon DJ, et al. Update for the practicing pathologist: The International Consultation On Urologic Disease-European Association of Urology consultation on bladder cancer. Mod Pathol 28:612-630, 2015

Amin M, Reuter V, Epstein J, et al. Consensus of Guidelines by the Pathology of Bladder Cancer Workgroup. In: Soloway M, Khoury S, editors. Bladder Cancer: 2nd International Consultation on Bladder Cancer - Vienna. Second ed. Paris: ICUD-EAU; 2012.(available free on line at: : http://www.icud.info/bladdercancer2nd.html

Grignon DJ. Tumors of the Urinary Bladder. In: Urological Pathology, Amin MB, Grignon DJ, Srigley JR, Eble JN, editors. Wolters Kluwer Lipincott Williams & Wilkins, Philadelphia, PA pp 340-460, 2014.

Amin M, Delahunt B, Bochner B et al. Protocol for the Examination of Specimens from Patients with Carcinoma of the Urinary Bladder. Chicago: College of American Pathologists. CAP Web site (updated 2013).

Babjuk M, Burger M, Zigeuner R, et al. EAU guidelines on non-muscle invasive urothelial carcinoma of the bladder: update 2013. Eur Urol 2013;64:639-653.

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Hautmann RE, de Petriconi RC, Pfeiffer C, Volkmer BG. Radical cystectomy for urothelial carcinoma without neoadjuvant or adjuvant therapy: long term results of 1,100 cases. Eur Urol 2012; 61:1039-1047.

Paner GP, Ro JY, Wojcik EM, Venkataraman G, Datta MW, Amin MB. Further characterization of the muscle layers and lamina propria of the urinary bladder by systematic histologic mapping: implications for pathologic staging of invasive urothelial carcinoma. Am J Surg Pathol 2007;31:1420-9.

453 Bertz S, Denzinger S, Otto W, et al. Substaging by estimating the size of invasive tumour can improve risk stratification in pT1 urothelial bladder cancer-evaluation of a large hospital-based single-center series. Histopathology 2011;59:722-732.

Van Rhijn BW, van der Kwast TH, Alkhateeb SS, et al. A new and highly prognostic system to discern T1 bladder cancer substage. Eur Urol 2012;61:378-384.

Orsola A, Trias I, Raventos CX et al. Initial high-grade T1 urothelial cell carcinoma: feasibility and prognostic significance of lamina propria invasion microstaging (T1a/b/c) in BCG-treated and BCG-non-treated patients. Eur Urol 2005;48:231-8

Bertz S, Denzinger S, Otto W, et al. Substaging by estimating the size of invasive tumor can improve risk stratification in pT1 urothelial bladder cancer – evaluation of a large hospital based single center series. Histopathology 2011;59:722-32.

Denzinger S, Burger M, Fritsche HM, et al. Prognostic value of histopathologic tumour growth patterns ath the invasion front of T1G3 urothelial carcinoma of the bladder. Scand J Urol Nephrol 2009;43:282-7.

Morris DS, Weozer AZ, Ye Z, et al. Understanding bladder cancer death: tumor biology versus physician proactice. Cancer 2009;115:1011.

Kulkarni GS, Hakenberg OW, Gschwend JE, et al. An updated critical analysis of the treatment strategy for newly diagnosed high-gtade T1 (previously T1G3) bladder cancer. Eur Urol 2010;57:60-70.

Fang AC, Ahmad AE, Whitson JM, et al. Effect of a minimum lymph node policy in radical cystectomy and pelvic lymphadenectomy on lymph node yields, lymph node positivity rates, lymph node density, and survivorship in patients with bladder cancer. Cancer. 2010;116:1901-8.

Karl A, Carroll PR, Gschwend JE et al. The impact of lymphadenectomy and lymph node metastasis on the outcomes of radical cystectomy for bladder cancer. Eur Urol 2009;55:826-35.

UROTHELIAL CARCINOMA AND HISTOLOGIC VARIANTS

Moch H, Reuter V, Humphrey P, Ulbright T. World Health Organization Histologic and Genetic Typing of Tumours of the Kidney, Urinary Bladder, Prostate Gland and Testis. Lyon, France: IARC Press; 2016.

Grignon DJ. Tumors of the Urinary Bladder. In: Urological Pathology, Amin MB, Grignon DJ, Srigley JR, Eble JN, editors. Wolters Kluwer Lipincott Williams & Wilkins, Philadelphia, PA pp 340-460, 2014.

454 Grignon DJ, Al-Ahmadie H, Algaba F, et al. Urinary Tract: Infiltrating Urothelial Carcinoma. In: WHO Classification of Tumours of the Urinary System and Male Genital Organs; Reuter V, Moch H, Humphrey P, Ulbright T, eds. IARC Press, Lyon, 2016.

Amin MB, Trpkov K, Lopez-Beltran A, Grignon D, and Members of the ISUP Immunohistochemistry in Diagnostic Urologic Pathology Group. Best practices recommendations in the application of immunohistochemistry in the bladder lesions: report from the International Society of Urologic Pathology consensus conference. Am J Surg Pathol 2014;38:e20-e34.

Paner GP, Annaiah C, Gulmann C, et al. Immunohistochemical evaluation of novel and traditional markers associated with urothelial differentiation in a spectrum of variants of urothelial carcinoma of the urinary bladder. Hum Pathol 2014;45:1473-1482.

Jung S, Wu C, Eslami Z, et al. The role of immunohistochemistry in the diagnosis of flat urothelial lesions: a study using CK20, CK 5/6, p53, CD138, and Her2/Neu. Ann Diagn Pathol 2014;18:27-32.

Pan CC, Chang YH, Chen KK, et al. Prognostic significance of the 2004 WHO/ISUP classification for prediction of recurrence, progression and cancer-specific mortality of non-muscle invasive urothelial tumors of the urinary bladder: a clinicopathologic study of 1,515 cases. Am J Clin Pathol 2010;133:788-795.

Gofrit ON, Pizov G, Shapiro A, et al. Mixed high and low grade bladder tumors – are they clinically high or low grade? J Urol 2014;191:1893-1696.

Pokuri VK, et al. Response (pT0) to Neoadjuvant Chemotherapy in Muscle-invasive Bladder Carcinoma. Clin Genitourin Cancer 2016;14:e59-65

Monn MF Contemporary bladder cancer: variant histology may be a significant driver of disease. Urol Oncol 2015;33:18.e15-20.

Seisen T1, Compérat E, Léon P, Roupret M. Impact of histological variants on the outcomes of nonmuscle invasive bladder cancer after transurethral resection. Curr Opin Urol 2014;24:524-31.

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38th Annual Seminar Pathology Review: Pulmonary, Neuro, and Hepatic Pathology for the General Pathologist February 12 – 17, 2017 • The Westin Snowmass Resort • Snowmass Village, Colorado

3rd Annual A Practical Approach to Surgical and Cytopathology April 6 - 8, 2017 • Hotel Del Coronado • Coronado, California

11 DAYS Gastrointestinal Pathology in Northern Europe July 16 – 27, 2017 • Book By March 8, 2017 Scandinavian & Russia aboard the Regal Princess Sail round-trip from Copenhagen, Denmark

7 DAYS A Practical Review of Soft Tissue Tumor and Gastrointestinal Pathology August 5 - 12, 2017 • Book By March 28, 2017 Inside Passage with Glacier Bay aboard the Ruby Princess Sail round-trip from Seattle, Washington

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Las Vegas • Fall 2017

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