(Carcinoid Tumors) of the Jejunum and Ileum

AAPA Macroscopic Examination Guidelines: Utilization of the CAP Cancer Protocols at the Surgical Gross Bench

Protocol for the Examination of Specimens from Patients with Neuroendocrine Tumors (Carcinoid Tumors) of the Jejunum and Ileum.

This protocol applies to segmental resections and ileocolectomies of well-differentiated neuroendocrine tumors of the jejunum and ileum. This protocol does not apply to well- differentiated tumors of the duodenum and ampulla, poorly differentiated neuroendocrine carcinoma (including small and large cell), other epithelial tumors including mixed neuroendocrine-non-neuroendocrine neoplasms, lymphoma, gastrointestinal stromal tumors, and non-gastrointestinal stromal tumor sarcomas.

Based on: AJCC Cancer Staging Manual, Eighth Edition pTNM requirements CAP Cancer Protocol: Jejunum/IleumNET 1.0.0.2 CAP Protocol Web Posting Date: February 2020 AAPA Macroscopic Examination Template Edition 3.0 AAPA Web Posting Date:

Revision History: 1st Edition – April 2017 2nd Edition – September 2018

Summary of Changes: This protocol is revised to the 8th edition of the AJCC Cancer Staging Manual and the current version of the CAP Cancer Protocol Jejunum / Ileum NET 1.0.0.2.

This is a major revision to the protocol. Extensive changes have been made throughout the document.

Procedures Covered in this Protocol: Segmental resections of the jejunum and ileum Ileocolectomy (may include ileum, cecum, appendix, ascending colon)

Authors: Molly A. Person, PA(ASCP)CM*, Patrice Brown, PA(ASCP)CM, Lainee Bulawa, PA(ASCP)CM, Heather Gaburo, PA(ASCP)CM, Courtney Hyland, PA(ASCP)CM, Monica Kendall, PA(ASCP)CM, Darryl Kinnear, PA(ASCP)CM, Alysha Martini, PA(ASCP)CM, Dennis Strenk, PA(ASCP)CM, Constance Thorpe, PA(ASCP)CM, Jon Wagner, PA(ASCP)CM

*Denotes primary author. All other contributing authors are listed alphabetically.

Previous Lead Contributors: Constance Thorpe, PA(ASCP)CM Katie Johnson, PA(ASCP)CM

Art Director Jesse McCoy, BFA, MHS, PA(ASCP)CM

Photograph Curator: Lainee Bulawa, MS, PA(ASCP)CM

Illustrator: Matthew Brownstein Endocrine – Jejunum and Ileum Jejunum/IleumNET 1.0.0.2

Structured Macroscopic Report Jejunum and Ileum Neuroendocrine Tumor

Procedure ____ Segmental resection, small intestine ____ Ileocolic resection ____ Other (specify): ______Not specified

Nature of Specimen Received ____ Fresh +____ Frozen section (intraoperative consultation) ____ Tissue collected for ancillary diagnostic studies, (specify test): ______In fixative Fixative type: ______+ Cold ischemic time: ______+ Fixation time (specify if for entire specimen or specific components): ______Other transport media (specify): ______

Specimen Orientation +____ Specimen oriented +____ Oriented by surgeon (specify): ______+____ Oriented based on anatomic landmarks +____ Not oriented

Specimen Integrity ____ Intact ____ Disrupted / fragmented: +____ Margin disrupted (specify):______+____ Tumor disrupted ____ Other (specify): ______

Ink Key +____ Ink applied (provide anatomic site and color applied): ______

Overall Dimensions (cm) ___ x ___ x ___ cm

Specimen Anatomical Components and Size in Three Dimensions (if applicable, list specimen components) ____ Small intestine: ___ (length) x ___ (diameter) cm ____ Mesentery: ___ x ___ x ___ cm ____ Cecum: ___ (length) x ___ (diameter) cm ____ Appendix: ___ (length) x ___ (diameter) cm ____ Right colon: ___ (length) x ___ (diameter) cm ____ Omentum: ___ x ___ x ___ cm ____ Other (specify): ______, ___x ___ x ___ cm

Macroscopic Tumor ____ Absent ____ Present

Tumor Focality ____ Unifocal ____ Multifocal (specify number of tumors): ___

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____ Indeterminate (explain): ______

Tumor Site ____ Jejunum ____ Ileum ____ Small intestine, not otherwise specified ____ Other (specify):______

Macroscopic Tumor Size Greatest dimension: ____ cm +Additional dimensions: ___ x ___ cm or ranging from ___ - ___ cm ____ Indeterminate (explain): ______

+Macroscopic Appearance of Tumor +____ Necrotic +____ Color: ______+____ Consistency: ______+____ Well-circumscribed +____ Other:______

Macroscopic Extent of Tumor (involvement of adjacent structures) ____ Confined to mucosa/submucosa ____ Involves muscularis propria ____ Involves visceral peritoneum or adjacent adipose tissue ____ Involves adjacent structures or other organs (specify): ______

Macroscopic Margin Involvement Note: use this section only if all margins are uninvolved and can be assessed. ____ All margins are uninvolved by macroscopic tumor Margins examined: ______Note: Margins may include proximal, distal, radial, or mesenteric, and others. Distance of tumor to closest margin: ___ mm or ___ cm Specify margin: ______Margin(s) involved by macroscopic tumor Specify margin(s): ______

Note: individual margin reporting required if any margins are involved or margin involvement cannot be assessed.

Proximal Margin ____ Cannot be assessed ____ Uninvolved by macroscopic tumor ____ Involved by macroscopic tumor

Distal Margin ____ Cannot be assessed ____ Uninvolved by macroscopic tumor ____ Involved by macroscopic tumor

Radial or Mesenteric Margin ____ Cannot be assessed ____ Uninvolved by macroscopic tumor ____ Involved by macroscopic tumor

Other Margin(s) (required only if applicable)

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Specify margin(s): ______Cannot be assessed ____ Uninvolved by macroscopic tumor ____ Involved by macroscopic tumor

Mesenteric Masses ____ Not identified ____ Large mesenteric mass(es) (>2 cm) present + Specify number: ____ + Size: ___ x ___ x ___ cm or Range: ___ - ___ cm +___ Encases superior mesenteric vessels +____ Mesenteric tumor deposit(s) (<2 cm) + Specify number: ____ + Size: ___ x ___ x ___ cm or Range: ___ - ___ cm +___ Encases superior mesenteric vessels ____ Cannot be determined (explain):______

+Macroscopic Appearance of Uninvolved Tissue / Organ(s) +____ Ischemic changes (e.g., wall attenuation, dusky discoloration, etc.) +____ Small bowel stenosis, lumen narrows to ___ cm over ___ cm length +____ Mesenteric fibrosis +____ Mucosal ulceration +____ Perforation: ___ x ___ x ___ cm; Location:______+____ Other: ______

Lymph Node Examination ____ No lymph nodes submitted or identified ____ Number of lymph nodes identified: ______Number of lymph nodes macroscopically involved: ___ +Size of lymph nodes in three dimensions: ___ x ___ x ___ cm or range in size: ___ - ___ cm ____ Number of lymph nodes cannot be determined (explain): ______

+____ Tissue Submitted for Research (specify, if possible): ______

(+) Data elements preceded by this symbol are optional elements which may be clinically important but are not yet validated or regularly used in patient management.

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Recommended Block Allocation Key:

Small bowel resection: A1: Proximal mucosal margin, (perpendicular or en face) A2: Distal mucosal margin, (perpendicular or en face) A3-A4: Small bowel mass, with deepest invasion A5: Mass to radial margin (if present and applicable, radial margin may be submitted as a separate shave if distant from the mass) A6: Mass and adjacent uninvolved mucosa A7: Mesenteric mass (if present) to mesenteric margin, perpendicular A8: Additional Mesenteric mass (if present) A19-12: Lymph nodes (12 minimum recommended for adequate staging)

Note: Often neuroendocrine tumors of the ileum and jejunum will be small (~1 cm) and there may not be sufficient mass to submit 5 cassettes of tumor, as outlined above. This serves as an example representation of structures and relationships to be sampled as primary tumor allows, and if all mentioned structures are present.

Ileocolectomy: A1-2: Small bowel mass, with deepest invasion A3: Mass to radial margin (if present and applicable) A4: Mass and adjacent uninvolved mucosa A5: Mass to nearest mucosal margin, perpendicular OR nearest mucosal margin, shave (if far) A6: Mesenteric mass to mesenteric margin, perpendicular A7: Additional mesenteric mass (if present) A8-11: Lymph nodes (12 minimum recommended for adequate staging) A12: Representative uninvolved cecum / ascending colon A13: Appendix (if present)

References:

1. Lester SC. Manual of Surgical Pathology, 3rd ed. Philadelphia, PA: Saunders/Elsevier; 2010.

2. Shi C, Adsay V, Bergsland EK, et al. Protocol for Examination of Specimens From Patients With Neuroendocrine Tumors (Carcinoid Tumors) of the Jejunum and Ileum. CAP Cancer Protocols. Jejunum/IleumNET 1.0.0.2. February 2020.

3. Woltering EA, et al. Neuroendocrine Tumors of the Jejunum and Ileum. Amin MB, et al. AJCC Cancer Staging Manual. 8th ed. New York, NY: Springer; 2017:375-387.

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Specimen Handling / Dissection Guidelines

 Resection • Before opening: o Determine which portions of bowel are present (small bowel only vs. small bowel, cecum, appendix, etc.). o Measure all components (length and diameter of small bowel, appendix, large bowel; three dimensions of attached mesentery, omentum). o Identify any areas of disruption, if present. If defects are present, they should be measured and described, including relationship to margins and the tumor. If disruption precludes accurate margin assessment, this must be stated. Consultation with the surgeon should be considered, as well as the possibility of differentially inking areas of disruption to allow them to be distinguished from intact true margin, microscopically. o Examine the specimen to determine where the mass or masses are located to avoid cutting through them. o Palpate the attached mesentery for a mass or masses. • Open the specimen, being careful to avoid masses / nodules, which may be numerous. • Identify mucosal mass(es) present, count, and measure sizes. Provide three dimensions for a single mass and a number with size range (at minimum) for multiple masses. Mucosal masses larger than 1 cm confer a worse prognosis and higher T stage, T2. • Measure distances to margins, including mucosal, mesenteric, and radial (if present). The radial margin may be present on right hemicolectomy specimens and is identifiable where the pericolic fat appears slightly dull, rather than having glistening peritoneum. • Ink the radial margin or areas of serosal puckering overlying the mass. • Submit mucosal margins: o If mass(es) are far from the margin, shaved or en face. . If more than one mass is identified, consider submitting each margin entirely (full circumference). o If mass(es) are close to the margin, perpendicularly. • Cut across the masses to evaluate depth of invasion (confined to mucosa, involves submucosa, muscularis propria, serosa, adjacent structures) (Figure 1). • Submit cross sections of mass to show greatest depth of invasion. • Describe the cut surface (well-circumscribed / necrotic / color / consistency). • Look for areas of luminal stricture or dilation; measure and describe. • Section through the attached mesentery. Mesenteric masses are considered regional lymph node metastases. • If a mesenteric mass is present, provide three dimensions and submit sections. Mesenteric mass >2 cm confers the highest N stage, N2. • Comment on increased mesenteric fibrosis, if present. • Search for and submit all lymph nodes (12 minimum). • Describe non-neoplastic tissues and submit representative sections.

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References:

1. Shi C, Adsay V, Bergsland EK, et al. Protocol for Examination of Specimens From Patients With Neuroendocrine Tumors (Carcinoid Tumors) of the Jejunum and Ileum. CAP Cancer Protocols. Jejunum/IleumNET 1.0.0.2. February 2020.

2. Lester SC. Manual of Surgical Pathology, 3rd ed. Philadelphia, PA: Saunders/Elsevier; 2010.

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3. Woltering EA, et al. Neuroendocrine Tumors of the Jejunum and Ileum. Amin MB, et al. AJCC Cancer Staging Manual. 8th ed. New York, NY: Springer; 2017:375-387.

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Well-differentiated Neuroendocrine Tumors of the Jejunum and Ileum

Incidence, Epidemiology, and Morbidity / Mortality Well-differentiated neuroendocrine tumors (NETs) of the small intestine occur in the United States at a rate of 1.2 per 100,000. Of all primary malignancies of the small intestine, neuroendocrine tumors represent approximately 40%.

Small intestinal NETs most commonly occur in the 7th decade of life but have been reported in patients from 20 to 80 years old. Overall, neuroendocrine tumors are slightly more common in women, including NETs of the small intestine, with 53% of patients being women.

Most survival data related to staging applies to low-grade (G1 and G2) neuroendocrine tumors, as defined by mitotic count. Rarely, tumors are classified as “well-differentiated G3 NETs,” which usually behave as well-differentiated neoplasms, rather than high grade carcinomas. Additionally, there is no statistically significant difference in survival data between clinical stage I and II, II and III, III and IV. For pathologic stage, only stage III and IV show a significant difference. Patient survival rates are good – with rates of 95% survival or higher for stages I-III, and about 87% for stage IV at 3 years.

Clinical Presentation Most (90%) of small bowel neuroendocrine tumors are metastatic at the time of diagnosis. This is likely due to small and often asymptomatic primary tumors, causing a delay in diagnosis until symptoms manifest, often due to a metastasis to the liver. For masses that present prior to metastases, symptoms are often associated with obstruction. Extensive local and distant fibrosis may occur due to production of fibroblast growth factor (a common feature of small intestinal NETs), leading to adhesion, obstruction, and even distant damage to the tricuspid and pulmonic (right-sided) cardiac valves.

A significant number of these small bowel NETs exhibit a hormone-induced complex of symptoms, including flushing, sweating, diarrhea, and wheezing, known as “carcinoid syndrome.” Often, patients experiencing carcinoid syndrome have hepatic metastases, allowing the secretory products to enter directly into circulation and avoid metabolism by the liver. This has given rise to the terminology of “carcinoid tumor,” which refers specifically to serotonin (and other humoral factors such as bradykinins, prostaglandins, tachykinins, substance P, and / or histamine) producing tumors associated with the clinical manifestations of carcinoid syndrome. It does not apply to neuroendocrine tumors universally, which is why usage of the term “carcinoid” has lost favor.

Imaging Considerations Small bowel imaging often presents a diagnostic challenge due to the frequency of inflammatory conditions such as Crohn’s disease and relative greater frequency of adenocarcinoma compared to neuroendocrine tumors (in regard to the entire bowel). Metastases to the small bowel are also seen, particularly with malignant melanoma. Overall, malignancies of the small bowel are rare compared to other locations, and the value of positron emission tomography / computed tomography (PET / CT) in diagnosis has been less thoroughly investigated. Neuroendocrine tumors are usually small and difficult to detect radiographically. When visualized, it is usually due to irregular narrowing of the lumen, kinking, or fixation of loops of bowel. Spiculated mesenteric masses may also be seen. Additionally, NETs are typically not FDG-avid, and novel PET radiotracers may show greater value. The true value of PET / CT is as an adjunct modality for detection of distant disease.

Both magnetic resonance imaging (MRI) and CT are recommended for staging neuroendocrine tumors due to their differing sensitivity and specificity. MR imaging is highly sensitive for detection of liver metastases. CT scans are more sensitive for detection of extrahepatic metastases.

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Echocardiograms may also be useful for the detection of carcinoid heart disease when multiple exams are available for comparison. Cardiac changes may be due to secretion of vasoactive hormones, such as serotonin and fibroblast growth factor, impacting cardiac valves.

Pertinent Anatomy Small bowel neuroendocrine tumors most frequently associated with clinical symptoms, are usually found in the distal ileum within 60 cm of the ileocecal valve and tend to be multiple. They are thought to arise in the intraepithelial endocrine cells (enterochromaffin-like or ECL cells, most commonly). This is in contrast to the more common appendiceal neuroendocrine carcinomas, which are thought to arise from subepithelial endocrine cells.

Surgical resections of neuroendocrine tumors of the jejunum and ileum will likely consist of either a segment of small bowel with attached mesentery, or an ileocolectomy, with ileum, cecum, appendix, variable segment of right colon, and attached mesentery. For resections of small bowel, the majority of the specimen will be peritonealized, with true margin present at the proximal and distal mucosal margins, and the resection site of the mesentery (Figure 2). Mesenteric masses are not uncommon and are significant for patient staging and prognosis. Additionally, they may encase the mesenteric vessels and approach the mesenteric margin, both of which are important points to document in the gross description.

For right hemicolectomy specimens, the possibility of a true radial margin is introduced, where the cecum and right colon have undergone secondary retroperitonealization. While the mass will be arising in the fully peritonealized terminal ileum for application to this protocol, distance to radial margin should not be neglected, especially in cases of large masses or those with extensive adhesions or matting.

If the specimen is received disrupted, the defects should be thoroughly described, including their size, location, and relationship to the tumor or margins (see “specimen handling”).

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Figure 2: Examples of circumferential / mesenteric margin types include the terminal ileum, cecum / ascending colon, and transverse colon, respectively.

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Macroscopic Features of Tumor (Figures 3A-3D) Gastrointestinal neuroendocrine tumors tend to have a firm, tan-yellow cut surface due to relatively high lipid content. They may also be pink-red due to vascularity. Lesions are most often solid and well-circumscribed and may appear like a submucosal “button.” Often, multiple nodules will be present, and the entire length of the specimen should be carefully examined. When occurring in any part of the gastrointestinal tract, tumors larger than 2.0 cm confer a higher likelihood of lymph node metastases. For NETs arising in the jejunum and ileum, however, metastases occur in most patients with tumors larger than 1.0 cm, and in 12% of patients with tumors less than a centimeter.

Figure 3, A-D demonstrating neuroendocrine tumors of the ileum and jejunum. A. A mucosal nodule and mesenteric mass are visible. B. Close up of a typical primary neuroendocrine tumor of the small bowel. C. Neuroendocrine tumor of the terminal ileum with a polypoid and ulcerated appearance. Green discoloration present only proximal to the lesion is suggestive of obstruction. D. Cut surface of the neuroendocrine tumor of the ileum shown in C, causing puckering of the muscularis. Photos courtesy of Eleni Constantopoulos, PA(ASCP), Mayo Clinic, Rochester, MN.

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Treatment / Therapy Guidelines Due to the propensity of metastases to occur in the setting of very small primary tumors, treatment of jejunoileal neuroendocrine tumors includes complete resection and regional lymphadenectomy. Often complete resection is adequate and systemic therapy is not required. However, if there is clinically significant tumor burden, treatment with Octreotide or Ianreotide is recommended, but will likely only benefit patients with somatostatin positive disease. If there is significant tumor burden in the liver, the patient may be treated with directed therapies such as arterial embolization, chemoembolization, or radioembolization.

Prognosis and Prognostic Features Large mesenteric masses (greater than 2 cm) or encasement of the mesenteric vessels most likely indicates a poor prognosis, but more information is needed to establish an additional N category as a prognostic variable. As currently defined by the AJCC, a large mesenteric mass indicates an N2 stage, giving a minimum overall stage group of III.

Metastases are more common for patients with primary tumors larger than a centimeter, conferring a tumor stage of T2 or higher and minimal overall stage group of II.

Biomarkers are useful in the diagnosis, monitoring, and prognosis of neuroendocrine tumors. Chromogranin A is a commonly used biomarker which may reflect tumor load, be used to monitor response to treatment, and correlates to a poor prognosis if elevated. Unfortunately, elevated levels can also occur in patients with a variety of other conditions. Neurokinin A (NKA) plasma levels of greater than 50 pg/mL may confer a worse prognosis than lower NKA values, and patients whose NKA levels fall below 50 pg/mL with treatment have an excellent prognosis.

Metastatic Sites The most common site of metastasis at presentation is the liver. Neuroendocrine tumors also show a strong affinity for spread via the lymphatic system, with regional lymph nodes including the superior mesenteric, retropancreatic, inferior pancreaticoduodenal, and hepatic artery lymph nodes. Overall, the metastases are predominantly seen in distant and regional lymph nodes (72.4%), liver (19.5%), lung (0.5%), and bone (0.3%). Uncommonly, metastases to the ovary and peritoneum may occur.

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References

1. Amin MB, Edge S, Greene F, et al, (Eds.) AJCC Cancer Staging Manual. 8th ed. New York, NY: Springer International Publishing; 2017.

2. Butros SR, et al. Positron Emission Tomography and Computed Tomography of the Hollow Viscera. Gore RM, Levine MS. Textbook of Gastrointestinal Radiology. 4th ed. Philadelphia, PA: Elsevier, Inc; 2015:96-111.

3. Neuroendocrine and Adrenal Tumors. NCCN Clinical Practice Guidelines in Oncology. National Comprehensive Cancer Network. March 5, 2019. Version 1.2019. NCCN.org. Accessed December 12, 2019.

4. Overman MJ, Kunitake H. Epidemiology, clinical features, and types of small bowel neoplasms. January 18th, 2019. UpToDate.com. Accessed January 21, 2020.

5. Shi C, Adsay V, Bergsland EK, et al. Protocol for Examination of Specimens From Patients With Neuroendocrine Tumors (Carcinoid Tumors) of the Jejunum and Ileum. CAP Cancer Protocols. Jejunum/IleumNET 1.0.0.1. June 2017.

6. Strosberg JR. Clinical characteristics of well-differentiated neuroendocrine (carcinoid) tumors arising in the gastrointestinal and genitourinary tracts. August 15, 2019. UpToDate.com. Accessed January 21, 2020.

7. Strosberg JR. Clinical features of carcinoid syndrome. August 15, 2019. UpToDate.com. Accessed January 21, 2020.

8. Thomas S, et al. Malignant Neoplasms and Wall Thickening of the Small Bowel. Sahani DV, Samir AE. Abdominal Imaging. 2nd ed. Philadelphia, PA: Elsevier, Inc; 2017: 236-248.

9. Touroutoglou N, Arcenas A, Ajani JA. Neuroendocrine Tumors of the Gastrointestinal Tract. Cancer Network. https://www.cancernetwork.com/gastrointestinal-cancer/neuroendocrine-tumors- of-gastrointestinal-tract. Published April 2, 2005. Accessed March 5, 2020.

10. Woltering EA, et al. Neuroendocrine Tumors of the Jejunum and Ileum. Amin MB, et al. AJCC Cancer Staging Manual. 8th ed. New York, NY: Springer; 2017:375-387.

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APPENDIX I TNM Criteria

Definition of Primary Tumor (pT) pT Category pT Criteria pTX Primary tumor cannot be assessed pT0 No evidence of primary tumor pT1* Invades the lamina propria or submucosa and less than or equal to 1 cm in size pT2* Invades the muscularis propria or greater than 1 cm in size pT3* Invades through the muscularis propria into subserosal tissue without penetration of overlying serosa pT4* Invades visceral peritoneum (serosa) or other organs or adjacent structures

Used with the permission of the American Joint Committee on Cancer (AJCC), Chicago, Illinois. The original source for this material is the AJCC Cancer Staging Manual, Eighth Edition (2017) published by Springer Science and Business Media LLC, www.springer.com.

*Note: For any T add (m) for multiple tumors [TX(#) or TX(m), where X=1-4, and # = number of primary tumors identified**]; for multiple tumors with different T, use the highest.

** Example: if there are two primary tumors, only one of which invades through the muscularis propria into subserosal tissue without penetration of overlying serosa (jejunal or ileal), we define the primary tumor as either T3(2) or T3(m).

Definition of Regional Lymph Node (pN) pN Category pN Criteria pNX Regional lymph nodes cannot be assessed pN0 No regional lymph node metastasis has occurred pN1 Regional lymph node metastasis less than 12 nodes pN2 Large mesenteric masses (>2 cm) and/or extensive nodal deposits (12 or greater), especially those that encase the superior mesenteric vessels

Definition of Distant Metastasis (pM) (required only if confirmed pathologically) pM Category pM Criteria M0 No distant metastasis pM1 Distant metastasis pM1a Metastasis confined to liver pM1b Metastases in at least one extrahepatic site (e.g., lung, ovary, nonregional lymph node, peritoneum, bone) pM1c Both hepatic and extrahepatic metastases

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AJCC Prognostic Stage Groups When T is… And N is… And M is… Then the stage group is…. T1 N0 M0 I T1 N1, N2 M0 III T1 N0, N1, N2 M1 IV T2 N0 M0 II T2 N1, N2 M0 III T2 N0, N1, N2 M1 IV T3 N0 M0 II T3 N1, N2 M0 III T3 N0, N1, N2 M1 IV T4 N0 M0 III T4 N1, N2 M0 III T4 N0, N1, N2 M1 IV

TNM Descriptors: pT(m)NM: For multiple primary tumors, the T score is modified, with the suffix "(m)" recorded in parentheses following the pT indicator (pT indicating primary tumor). Each tumor should be assessed independently of the others. Variability in histologic features should be considered with adequate sampling performed to either assure or exclude histologic variability. With regard to the T score, for staging purposes, the pT score is established based on the primary tumor showing the greatest size and / or degree of invasion. ypTMN: The “y” prefix indicates those cases in which classification is performed during or following initial multimodality therapy (i.e., neoadjuvant chemotherapy, radiation therapy, or both chemotherapy and radiation therapy). The cTNM or pTNM category is identified by a “y” prefix. The ycTNM or ypTNM categorizes the extent of tumor actually present at the time of that examination. The "y" categorization is not an estimate of tumor prior to multimodality therapy (i.e., before initiation of neoadjuvant therapy). If neoadjuvant therapy renders macroscopic examination of tumor involvement inconclusive, dissection methods should be considered, which allow for mapping of the treated area, emphasizing the areas of potential deepest invasion. aTNM: The "a" prefix designates the stage determined at autopsy. rTNM: The "r" prefix indicates a recurrent tumor when staged after a documented disease-free interval.

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APPENDIX II Ancillary Testing

Clinical Testing

There are multiple markers available that are useful for monitoring the clinical care of patients with well-differentiated neuroendocrine tumors of the small bowel.

5-hydroxyindoleacetic acid (5-HIAA) is a valuable marker for hormone secreting tumors. It can be measured in urine or plasma as a marker for increased serotonin production. The vast majority of symptomatic neuroendocrine tumors present with elevated serotonin. Similarly, repeat measurements of plasma serotonin levels serve as excellent markers for tumor burden and the severity of carcinoid syndrome. However, it has limited value when repeat collections are not available, and a single 5- HIAA value has a tenuous connection to severity. Its value for detection of tumors with sub-clinical hormone production is also limited, as shown by its high specificity (nearly 100%) but low sensitivity (approximately 35%).

Chromogranin A (CgA) is present in the secretory granules of all neuroendocrine cells and serves as a general neuroendocrine marker. Higher levels of CgA confer a worse prognosis, and changes over time may help signal recurrence or response to therapy. Unfortunately, CgA levels may vary significantly for reasons unrelated to the status of the patient’s NET. For instance, CgA may be elevated in the setting of proton-pump inhibitor use, chronic atrophic gastritis, and renal failure. It may also be elevated due to other neoplasia, such as pancreatic, small cell lung carcinoma, or prostate carcinoma. Levels may fluctuate based on time of collection and if the patient had fasted. Additionally, the upper limit of “normal” varies widely based on plasma vs. blood assessment, which may be a pitfall of surveying results over time. Therefore, while it is an important prognostic marker, its use over time may be limited.

Pancreastatin is a derivative of CgA, and when compared to CgA is a more reliable biomarker for NETs. Its levels are not impacted by proton pump inhibitor use, pernicious anemia, or type I gastric neuroendocrine tumors. It is, however, affected by food ingestion or medications and has been observed to be elevated in diabetics and hyperparathyroidism. Elevated pre-operative pancreastatin levels are associated with worse progression-free survival.

Neurokinin A (NKA) serves as a useful prognostic marker and measure of treatment response (see Prognosis and Prognostic Factors).

Immunohistochemistry

Ki-67 or mitotic rate is required to determine the histologic grade. While cytologic atypia in well- differentiated neuroendocrine carcinoma has no bearing on clinical behavior, mitotic rate and / or Ki- 67 index is a potential indicator for aggressive behavior and a CAP reporting requirement. Often, the grade determined based on Ki-67 labeling is higher than that assigned from the mitotic count. When both methods are performed, the higher of the two methods is assigned. Rarely, the Ki-67 index may be over 20% in well-differentiated NETs. These tumors are considered grade 3 well-differentiated NETs. While their morphology is likely typical of well-differentiated NETs, their behavior is more aggressive than grade 1 and grade 2, but not as aggressive as morphologically poorly-differentiated carcinomas. In cases where diagnosis is difficult, Chromogranin A and Synaptophysin may be used, with positive staining supporting the diagnosis of neuroendocrine tumor.

There is one FDA approved biomarker-directed therapy for metastatic small intestinal NETs, Lutetium 177 Dotatate. This treatment applies only to somatostatin receptor (SSTR) positive tumors, which is determined by immunohistochemistry.

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These tests can be performed on formalin fixed paraffin embedded tissue sections. The macroscopic description should provide the fixative used. 10% neutral buffered formalin is the preferred fixative. It is recommended that the duration of the fixation be provided as well.

References:

1. Modlin, IM, et all. Neuroendocrine Tumor Biomarkers: Current Status and Perspectives. Neuroendocrinology. 2014;100:265-277. DOI 10.1159/000368363.

2. Woltering EA, et al. Neuroendocrine Tumors of the Jejunum and Ileum. Amin MB, et al. AJCC Cancer Staging Manual. 8th ed. New York, NY: Springer; 2017:375-387.

3. Vanderbilt-Ingram Cancer Center. My Cancer Genome - Small Intestinal Neuroendocrine Tumor. https://www.mycancergenome.org/content/disease/small-intestinal-neuroendocrine- tumor/ Accessed March 2020.

4. Maqsood MH, Tameez Ud Din A, Khan AH. Neuroendocrine Tumor Therapy with Lutetium-177: A Literature Review. Cureus. 2019;11(1):e3986. Published 2019 Jan 30. doi:10.7759/cureus.3986

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APPENDIX III Frozen Section Considerations As is true with many specimens, the primary value of frozen assessment lies in confirming diagnostic material is received, and in margin assessment. Masses are often clearly identifiable and far from mucosal margins. However, it is not uncommon for a mesenteric mass to be near the mesenteric margin, and this should not be neglected for intra-operative consultation frozen assessment.

Regarding the diagnosis of neuroendocrine tumors on frozen section, often only preliminary diagnoses are rendered. Final grading requires Ki-67 and mitotic count, which is impossible or significantly impaired (respectively) on frozen section. Subsequent Ki-67 may be performed on sections which were frozen but is suboptimal. Additionally, it is possible for lymphoma and small round blue cell tumors to mimic neuroendocrine carcinomas, a pitfall that is magnified on frozen section due to the decreased histologic quality when compared to paraffin embedded tissue.

References:

1. Graham RP. Frozen Section Handling of Neuroendocrine Tumors of the Small Bowel. Personal email communication, Jan 2020.

2. Woltering EA, et al. Neuroendocrine Tumors of the Jejunum and Ileum. Amin MB, et al. AJCC Cancer Staging Manual. 8th ed. New York, NY: Springer; 2017:375-387.

Endocrine – Jejunum and Ileum Jejunum/IleumNET 1.0.0.2

APPENDIX IV Narrative Macroscopic Description

Single mass:

Submitted [fresh / in formalin] in one container for gross and microscopic examination, labeled with the patient’s name, [ ], MRN, [ ], and “[Small bowel and cecum]” is a [small bowel resection / right hemicolectomy specimen], consisting of a ___ cm long, ___ cm diameter portion of small bowel, [___ x ___ cm portion of cecum, ___ x ___ cm appendix], ___ x ___ x ___ cm portion of mesentery. The specimen is oriented [based on anatomic landmarks / with a suture indicating ______]. Opening reveals a ___ x ___ x ___ cm [well-circumscribed / submucosal / ulcerated / polypoid] mass present within the small bowel, which [is confined to / extends to] the [mucosa / wall / serosa / mesenteric adipose]. The mass is ___ cm from the proximal mucosal margin, ___ cm from the distal mucosal margin and ___ cm from the mesenteric margin. The cut surface is [color / consistency / homogeneity]. A mesenteric mass [is / is not] identified, [___ x ___ x ___cm] [___ cm from the mesenteric margin]. The uninvolved small bowel [is dusky / has a site of stricture with the lumen narrowing to ___ cm / has a normal folding pattern]. Multiple lymph nodes are identified in the attached soft tissue.

Representative sections are submitted as follows:

A1: Proximal mucosal margin, (perpendicular or en face) A2: Distal mucosal margin, (perpendicular or en face) A3: Small bowel mass, with deepest invasion A4: Mass to radial margin (if present & applicable) A5: Mass and adjacent uninvolved mucosa A7: Mesenteric mass to mesenteric margin, perpendicular A8: Additional Mesenteric mass (if present) A9-11: Lymph nodes (12 minimum recommended for adequate staging) A12: Representative cecum / ascending colon A13: Appendix (if present)

Multiple masses:

Submitted [fresh / in formalin] in one container for gross and microscopic examination, labeled with the patient’s name, [ ], MRN, [ ], and “[Small bowel and cecum]” is a [small bowel resection / right hemicolectomy specimen], consisting of a ___ cm long, ___ cm diameter portion of small bowel, [___ x ___ cm portion of cecum, ___ x ___ cm appendix], ___ x ___ x ___ cm portion of mesentery. The specimen is oriented [based on anatomic landmarks / with a suture indicating ______]. Opening reveals multiple (___ [number]) [well-circumscribed / submucosal / ulcerated / polypoid] masses present within the small bowel, which [are entirely confined to / extend to] the [mucosa / wall / serosa / mesenteric adipose]. The nearest masses to margins are: ___ cm from the proximal mucosal margin, ___ cm from the distal mucosal margin and ___ cm from the mesenteric margin. The cut surface is [color / consistency / homogeneity]. A mesenteric mass [is / is not] identified, [___ x ___ x ___ cm] [___ cm from the mesenteric margin]. The uninvolved small bowel [is dusky / has a site of stricture with the lumen narrowing to ___ cm / has a normal folding pattern]. Multiple lymph nodes are identified in the attached soft tissue.

Representative sections are submitted as follows:

A1-2: Small bowel mass [#1], with deepest invasion A3: Mass [#1] to radial margin (if present & applicable) A4: Mass [#2] and adjacent uninvolved mucosa A5: Mass [#3] to nearest mucosal margin, perpendicular OR shave (if far)

Endocrine – Jejunum and Ileum Jejunum/IleumNET 1.0.0.2

A6: Mesenteric mass to mesenteric margin, perpendicular A7: Additional Mesenteric mass (if present) A8-11: Lymph nodes (12 minimum recommended for adequate staging) A12: Representative cecum / ascending colon A13: Appendix (if present)

Endocrine – Jejunum and Ileum Jejunum/IleumNET 1.0.0.2

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