Haematologica 2000; 85:1291-1307 Malignant trends in

The pathologist’s view point. Part I – indolent lymphomas STEFANO ALDO PILERI, STEFANO ASCANI,* ELENA SABATTINI, GIULIO FRATERNALI-ORCIONI, SIMONETTA POGGI, MILENA PICCIOLI, PIER PAOLO PICCALUGA, BARBARA GAMBERI, PIER LUIGI ZINZANI, LORENZO LEONCINI,° BRUNANGELO FALINI# Service of Pathologic Anatomy and Haematopathology, Institute of Haematology and Clinical “L. & A. Seràgnoli”, Bologna University, Policlinico S. Orsola, Bologna; *Institute of Pathologic Anatomy, Perugia Uni- versity, Ospedale S. Maria, Terni, Italy, °Institute of Pathologic Anatomy, Siena University, Siena; #Hematopathology Laboratory, Institute of Hematology, Perugia University, Perugia, Italy

ABSTRACT

Background and Objectives. The REAL/WHO classi- still insufficient for this approach to be applied fication constitutes a new tool for the better under- in all cases, a biologically correct clas- standing and treatment of malignant lymphomas. sification is not currently feasible. Despite this, The authors focus on the key features of B-cell lym- many hematopathologists agree that by pooling phomas with an indolent behavior, aiming to con- morphologic, immunophenotypic, molecular, tribute to the cross-talk between pathologists and and clinical findings it is possible to enumerate clinicians. a large series of distinct entities that can be rec- Data Sources and Methods. Each lymphoma entity is ognized and diagnosed in routine practice. In analyzed on the basis of the most representative 1994, on the basis of the work of the Interna- contributions in the literature and the authors’ expe- tional Lymphoma Study Group (ILSG), a list of rience gained in studying more than 20,000 lym- “real”, clearly characterized anatomo-clinical phoid tumors over a 20-year period. entities that can be readily recognized with cur- Results. Guidelines for diagnosis and areas of inter- rently available techniques was published as a est for future clinico-pathologic studies are identified proposal for an up-to-date practical classifica- and discussed. Within this context, selected data tion of malignant lymphomas: this list was obtained by the application of novel markers are termed the Revised European American Lymphoma presented. (REAL) Classification (Table 1).18 This classifi- Interpreation and Conclusions. The present know- cation has recently been adopted by the World ledge and organization of malignant lymphomas now Health Organization (WHO) as an operative make the development of tailored a feasi- guideline for studying and diagnosing malig- ble goal. nant lymphomas; furthermore, its methodolo- ©2000, Ferrata Storti Foundation gy has been extended to the categorization of hematopoietic non-lymphoid tumors.19 Key words: , morphology, phenotype, genoty- pe, behavior. Histologic grade and clinical aggressiveness Before the advent of immunophenotyping and molecular biology techniques that have ince the 1970s, several very different clas- allowed the identification of many lymphoid sifications of malignant lymphomas have 1-10 as separate entities, it was thought Sbeen used around the world. The result- that non-Hodgkin’s lymphoma constituted a ing lack of uniform diagnostic criteria for lym- single generic disease with various degrees of phoid tumors has given rise to considerable aggressiveness that could be revealed on the problems both for pathologists and clinicians, basis of morphology and clinical findings. This and has seriously hampered comparison of the 11-17 concept encouraged the convinction that it studies reported in the literature. In theory, should be possible to devise a single grading sys- as with all other tumor types, lymphomas tem capable of predicting the clinical course of should be classified on the basis of their sup- the disease. This was the principle behind the posed histogenesis so as to provide maximum ,8 in which lymphoid information on their biology, natural history tumors were divided into three prognostic and response to . In practice, however, groups (indicated by grades of clinical malig- since our knowledge of the immune system is nancy) on the basis of the survival of the Correspondence: Prof. Stefano Aldo Pileri, M.D., Servizio di Anatomia Pato- patients recruited in the original study. Never- logica ed Ematopatologia, Istituto di Ematologia e Oncologia Medica ‘L. & theless, each of these wide-ranging categories A. Seràgnoli’, Università di Bologna, Policlinico S. Orsola, via Massarenti 9, is actually known to contain a large number of 40138 Bologna, Italy. Phone: international +39-051-6364562 – Fax: inter- national +39-051-6363606 – E-mail: [email protected] conditions that differ greatly as regards their eti-

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Table 1. The REAL classification in the form adopted by the as low and high grade forms, in exact contrast WHO. to the findings of a validation study on the REAL Classification promoted by the U.S. National Can- B-cell neoplasms cer Institute and S. Salvatore Foundation in March Precursors B-cell neoplasms 1994, which demonstrated that mantle cell lym- • Precursor B-lymphoblastic /lymphoma phomas are associated with a 5-year survival Mature (peripheral) B-cell neoplasms rate of less than 30%, while that of ALCL is • B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma about 80%.20 • B-cell prolymphocytic leukemia • Lymphoplasmacytic lymphoma Clinical categorization of non-Hodgkin’s • Splenic marginal zone B-cell lymphoma (± villous lymphocytes) lymphomas • • Plasma cell myeloma/ The large series of different entities that can be • Extranodal marginal zone B-cell lymphoma of MALT type distinguished on morphologic, immunopheno- • • typic and biological grounds and that are gen- • Nodal marginal zone B-cell lymphoma (± monocytoid B cells) erally included under the umbrella term non- • Diffuse large B-cell lymphoma • Burkitt’s lymphoma/Burkitt cell leukemia Hodgkin’s lymphoma can be ordered on the basis of various principles, including their sup- posed normal counterpart within the immune T- and NK-cell neoplasms system, their morphologic appearance and their Precursor T-cell clinical characteristics. For the practical oncol- • Precursor T-lymphoblastic leukemia/lymphoma ogist, the most rational criterion is their pre- Mature (peripheral) T-cell neoplasms dictable behavior. Thus, patients with lymphoid • T-cell prolymphocytic leukemia tumors can be divided into two different main • T-cell granular lymphocyte leukemia • Aggressive NK-cell leukemia groups on the basis of the characteristics of the • Adult T-cell lymphoma/leukemia (HTLV1+) process at the time of presentation and the • Extranodal NK/T-cell lymphoma, nasal type patients’ life expectancy, as proposed by Longo • Enteropathy-type T-cell lymphoma 21,22 • Hepatosplenic γδ T-cell lymphoma et al.: indolent and aggressive lymphomas. At • Subcutaneous panniculitis-like T-cell lymphoma times, a further distinction between aggressive • /Sézary syndrome • Anaplastic large cell lymphoma T/Null cell, primary cutaneous type and very aggressive lymphomas is made, • Peripheral T-cell lymphomas, not otherwise specified (NOS) depending on the expected survival - weeks or • Angioimmunoblastic T-cell lymphoma months - in untreated cases. This clinical group- • Anaplastic large cell lymphoma T/Null cell, primary systemic type ing is not mentioned in the REAL Classification. In fact, on the basis of what has been reported Hodgkin’s disease (Hodgkin’s lymphoma) in the literature, the ILSG members agreed that • Nodular lymphocyte predominance Hodgkin’s disease the aggressiveness of malignant lymphomas • Classical Hodgkin’s disease varies significantly among different histologic Hodgkin’s disease Lymphocyte-rich classical Hodgkin’s disease categories and within each category among dif- Mixed cellularity Hodgkin’s disease ferent patients. Such a variability is due to the Lymphocyte depletion Hodgkin’s disease influence exerted - singly or in combination - by Only major categories are included. biological factors, such as cytokinesis (i.e. cell proliferation and loss), oncogene activation, presence of hybrid fusion genes, development of ology, presentation, natural history, epidemiol- multidrug resistance, microambient, or correla- ogy and response to treatment. Moreover, each tion with micro-organisms (e.g. single variety of lymphoma displays its own spec- and ).23-34 For all these reasons, trum of degrees of morphologic and clinical the ILSG members preferred to avoid the attri- aggressiveness. As a result, it no longer appears bution of grades of malignancy, thinking that possible to categorize lymphoid tumors on the they cannot be defined by pure morphology or basis of a generic grading system that would be statistical analysis: the latter only informs on the tantamount to considering as a single entity dif- natural history of the disease or on its median ferent types of lung cancer, such as the carci- response to treatment and thus is neither pre- noid, squamous cell carcinoma, adenocarcino- dictive of the single patient’s outcome nor sup- ma and small cell carcinoma. Nor can the degree ports ad hoc therapeutic decisions (so-called tai- of malignancy of a lymphoma be realistically lored therapy).35,36 These concepts found sub- determined on the basis of cell size, as was envis- stantial confirmation at the VII International Con- aged by the Updated Kiel Classification (UKC).9,10 ference on Malignant Lymphomas (Lugano, 1-5 June Indeed, this principle would lead to mantle cell 1999).37,38 In particular, 1,093 cases were gath- lymphomas and anaplastic large cell lym- ered into indolent, aggressive and very aggressive phomas (ALCL) being interpreted, respectively, lymphomas according to the above mentioned

Haematologica vol. 85(12):December 2000 Pathologist’s view point: indolent lymphomas 1293 clinical criteria.38 When the outcome within each Table 2. List of indolent B-cell lymphomas. group was evaluated on the basis of individual histologic subtypes, it emerged that clinical Disseminated lymphomas/ grouping is a rather rough tool: for instance, • B-cell chronic lymphocytic leukemia • Lymphoplasmacytic lymphoma among aggressive lymphomas, the 5-year sur- • Hairy cell leukemia vival rates ranged from 78% for anaplastic large • Splenic marginal zone B-cell lymphoma (± villous lymphocytes) cell lymphomas to 14% for mantle cell lym- • Plasma cell myeloma/plasmacytoma phomas, with intermediate rates of 38% for dif- Extranodal lymphomas fuse large B-cell lymphomas and 68% for follic- • Extranodal marginal zone B-cell lymphoma of MALT type 38 ular lymphomas grade 3. Nodal lymphomas • Small lymphocytic lymphoma Indolent lymphomas • Follicular lymphoma Bearing in mind the above mentioned limita- • Nodal marginal zone B-cell lymphoma (± monocytoid B cells) tions on the absolute value of the terms indolent and aggressive, in the clinical setting indolent lym- phomas are considered to be those associated growth pattern.7,10,18 B-cell small lymphocytic with a survival measurable in years, indepen- lymphoma (B-SLL) is the solid equivalent of B- dently of whether or not any therapy is applied. CLL:18,39 it usually presents in the , These lymphoproliferative disorders have very but can infiltrate the bone marrow, also in the variable clinical presentations. Some are con- absence of an overt peripheral blood spread. stantly systemic diseases, often with leukemic Morphologically,7,10,18,40,41 B-CLL/SLL is mainly manifestations. Others have an extranodal pri- constituted by small lymphoid elements, with mary presentation and can remain localized for the contemporary presence of variable amounts long periods, even in the absence of any thera- of prolymphocytes and paraimmunoblasts (Fig- py. Yet others correspond to tumors with nodal ure 1a), that represent the proliferating com- presentation, which can have widespread partment of the process and which in some immune system involvement at the time of diag- areas can be numerous enough to give rise to nosis. This leads to the basic distinction of three pseudofollicles (Figure 1b). These latter fundamental subtypes of indolent lymphoma: – detectable only in tissue sections – seem to rep- disseminated leukemias/lymphomas, extranodal resent a worse prognostic indicator. In addition, forms and nodal ones. On pathologic grounds, prolymphocytes and paraimmunoblasts are indolent lymphomas display low grade histologic extremely useful for the correct interpretation of characteristics with a strong prevalence of small inadequately fixed samples, when small lym- cells and a blastic ratio of less than 25% of the phocytes assume a cleaved nuclear profile, total population. Furthermore, the number of which can lead to the wrong diagnosis of man- mitotic figures is low, according to the criteria of 18 9,10 tle-cell lymphoma. In the REAL Classification, the UKC, and constantly less than 5 mitoses the B-CLL/SLL category includes forms with fea- per high-power field. A common feature of many tures of plasmacytoid differentiation (Figure 1c) different histologic types of indolent lymphoma and listed in the UKC as lymphoplasmacytoid is the tendency to undergo histologic transfor- immunocytomas. These forms, in fact, do not dis- mation into a high-grade form, with a corre- play significant clinical, prognostic, morpho- sponding acceleration of the clinical course. In logic or phenotypic differences that can justify a the following the morphologic characteristics of clear-cut distinction from typical B-CLL.42 B-CLL B-cell indolent lymphomas – excluding plasma- also includes the rarer forms of B-prolympho- cytoma/plasma cell myeloma – will be cytic leukemia (B-PLL): this decision is based on described; the subclassification is reported in the observation that over time B-CLL tends to Table 2. enrich itself in prolymphocytic forms (the so- B-cell chronic lymphocytic leukemia/small called prolymphocytoid crisis) (Figure 1d), in lymphocytic lymphoma such a way that – even when FAB criteria40 are B-cell chronic lymphocytic leukemia (B-CLL) applied – the distinction between the two is a well-known process, whose incidence is leukemic forms can become quite arbitrary.39,43 often underestimated by pathologists, since the B-CLL and B-PLL probably represent the two diagnosis is more often based on the examina- extremes of a single disease, provided with dif- tion of peripheral-blood and/or bone marrow ferent degrees of aggressiveness. In the WHO smears.7,10,18 However, histopathology largely scheme,19 however, B-CLL and B-PLL are listed concurs to tumor staging and aggressiveness separately. Finally, a diffuse large B-cell lym- definition by showing the exact amount of bone phoma (DLBCL) can occasionally develop with- marrow infiltrate, cellular composition and in the context of B-CLL/SLL. Such an event is

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Table 3. Phenotypic profile of 90 indolent B-cell lymphomas.

CD3 CD5 CD23 CD10 CD20 CD79a Bcl-2 Bcl-6 Bcl-1 IRF4 BSAP

B-CLL – (20/20) + (20/20) + (20/20) – (20/20) + (20/20@) + (20/20) + (20/20) – (20/20) – (20/20*) + (19/19@) + (15/15) LPL – (15/15) – (15/15) + (3/15) – (15/15) + (15/15) + (15/15) + (15/15) – (15/15) – (15/15) + (15/15) + (15/15) MCL – (10/10) + (10/10) – (10/10) – (10/10) + (10/10) + (10/10) + (10/10) – (10/10) + (10/10) – (8/10§) + (9/9) FCL – (20/20) – (20/20) + (4/20) + (20/20) + (20/20) + (20/20) + (20/20) + (19/19) – (20/20) – (6/20#) + (15/16) MZL/E – (10/10) – (10/10) – (10/10) – (10/10) + (10/10) + (10/10) + (8/8) – (10/10) – (10/10) –/+w (7/7) –/+w (7/7) MZL/N – (4/4) – (4/4) – (4/4) – (4/4) + (4/4) + (4/4) + (4/4) – (4/4) – (4/4) –/+w (3/3) –/+w (4/4) MZL/S – (6/6) – (6/6) – (6/6) – (6/6) + (6/6) + (6/6) + (6/6) – (6/6) – (4/4) – (4/4) + (6/6) HCL – (5/5) – (5/5) – (5/5) – (5/5) + (5/5) + (5/5) + (5/5) – (5/5) – (5/5) – (5/5) + (3/5)

Abbreviations: B–CLL: B–cell chronic lymphocytic leukemia. LPL: Lymphoplasmacytic lymphoma. FCL: Follicle center cell lymphoma. MZL/E: Marginal zone lymphoma, extranodal. MZL/N: Marginal zone lymphoma, nodal. MZL/S: Marginal zone lymphoma, splenic. HCL: Hairy cell leukemia. +More than 75% of neoplastic cells posi- tive. +/–: 50–75% of neoplastic cells positive. –/+: 25–50% of neoplastic cells positive. —Neoplastic cells virtually negative. @Small lymphoid elements are weakly stained, if stained; prolymphocytes and paraimmunoblasts of pseudofollicular structures are strongly positive. §Scattered positive cells. #Positive only in cases with plasmacellular differentiation. W: weak staining.

commonly termed Richter’s syndrome and its finding seems to have prognostic relevance, the mechanisms are still controversial: in fact, in cases with a high content of CD23+ elements some cases the supervening large B-cell tumor running a more aggressive clinical course. represents the blastic transformation of the Immunoglobulin (Ig) expression is exceedingly same B-CLL clone, while in others it corresponds weak, thus preventing its detection on tissue sec- to a de novo neoplasm, whose occurrence might tions in most instances. However, cases with be facilitated by the defective immune response plasmacytoid differentiation are characterized of B-CLL patients.18 On phenotypic grounds by a more abundant Ig production, with (Table 3), B-CLL is characterized by the expres- detectable amounts at the intracytoplasmic lev- sion of B-cell markers, such as CD19 (often el. More recently, some additional markers have weak), CD22, and CD79a.18 Conversely to that been proposed and found useful for the diag- which is observed in other B-cell tumors, the nosis of B-CLL/SLL, such as cyclin D1, bcl-2 pro- small cell component is very weakly stained for tein, bcl-6 product, multiple myeloma oncogene CD20 in tissue sections, prolymphocytes and 1/interferon regulatory factor-4 (MUM1/IRF4), paraimmunoblasts representing the only con- and PAX-5 gene product/B cell-specific activator sistently positive components (Figure 1e).44 This protein (PAX5/BSAP).18,49-51 The search for cyclin finding seems clinically relevant in the light of D1 is often employed in the differential diagno- the increasing therapeutic usage of anti-CD20 sis between B-CLL/SLL and MCL, since the lat- antibodies.45 The profile of the tumor is further ter shows regular overexpression of the molecule characterized by the presence of CD5 and CD23 due to the occurrence of t(11;14) or bcl-1 gene at the cell membrane level (Figures 1f and 1g). rearrangement.18,52 On rare occasions, however, As these molecules may be expressed at different cyclin D1 positivity can also occur in B-CLL, a densities and are rather sensitive to fixation, their fact that strengthens the relevance of CD23 detection is greatly facilitated by the application detection.53 The bcl-2 product is always strong- of effective antigen retrieval techniques.46 In par- ly expressed by B-CLL/SLL: this finding does not ticular, CD5 and CD23 along with other mark- correspond to the presence of t(14;18), but ers allow the distinction of B-CLL/SLL from oth- indicates a certain protection of neoplastic cells er lymphoid tumors with different origin and from apoptosis.18 The latter finding along with behavior (Table 1), such as mantle-cell lym- the low proliferative activity (as shown by the Ki- phoma (MCL) (CD5+, CD10-, CD23-, DBA.44-, 67 marking) is responsible for the typical slow CD68-), immunocytoma (IC) (CD5-, CD10-, progression of the tumor. Bcl-6 is never CD23+/-, DBA.44-, CD68-), marginal-zone lym- expressed by lymphomatous cells:49 its presence phoma (MZL) (CD5-, CD10-, CD23-/+, or absence is very useful for distinguishing DBA.44–/+, CD68+/-), hairy cell leukemia (HCL) between neoplastic pseudofollicles (bcl-6-, (CD5-, CD10-, CD23-/+, DBA.44+, CD68+/-), and CD10-, bcl-2+, CD5+, CD23+) and residual ger- follicle center lymphoma (FCL) (CD5-, CD10+, minal centers (bcl-6+, CD10+, bcl-2-, CD5-, CD23-, DBA.44-, CD68-). As far as concerns B- CD23-). The application of the newly developed CLL, CD23 expression is indeed much stronger antibodies raised against the transcription fac- in prolymphocytes and paraimmunoblasts than tors IRF4 and BSAP produce opposite pat- in the small cell component (Figure 1g):47,48 this terns.50,51 Small lymphoid elements are BSAP+

Haematologica vol. 85(12):December 2000 Pathologist’s view point: indolent lymphomas 1295 and IRF4-, while prolymphocytes and paraim- of hemolytic anemia. On occasions, LPL gives munoblasts (as easily seen in pseudofollicles) rise to amyloid deposits in several organs and appear BSAP–/+ and IRF4+ (Figures 1h and 1i). apparatuses (Figure 2c):66-68 in the heart, these The latter finding – detected by our group in can cause severe arrhythmia with possible sud- large series of cases – fits with the recent obser- den death.69 At immunohistochemistry (Table vation that more than 50% of B-CLL cases might 3), neoplastic cells strongly express B-cell mark- be derived from memory B-cells as suggested by ers, including CD20 (Figure 2d), while they reg- the occurrence of bcl-6 and IgV gene muta- ularly lack CD5, CD10 and CD68.18 CD23 is tions.54-57 In fact, IRF4 – that is the product of found in a proportion of cases.18 Monotypic the MUM-1 gene – is physiologically expressed immunoglobulins are present at high density by B-lymphocytes following cell both at the surface and intracytoplasmic level.18 selection (i.e. by some centrocytes in the light The bcl-2 gene product is expressed indepen- zone of the germinal center, plasmacytoid ele- dently of the occurrence of t(14;18) causing pro- ments and plasma cells). Cases without bcl-6 tection against apoptosis. The Ki-67 marking is and IgV gene mutations might stem from naive usually low, with the exception of the cases that B-cells, do express CD38, carry trisomy 12, undergo blastic transformation. The search for occur more often in males and run a rather the bcl-6 protein gives negative results in the aggressive clinical course.54-57 Cytogenetic aber- neoplastic component, while it allows easy rations other than trisomy 12 are encountered detection of residual germinal centers, which in and consist in chromosome 17 abnormalities,58 turn are bcl-2-.49,70 Cyclin D1 is never overex- 14q+, and a deletion in the 13q14 chromoso- pressed.52 Neoplastic cells carry BSAP and IRF4, mal region, involving a not yet identified tumor the latter matching with plasmacytoid/plasma- suppressor gene and occurring in approximate- cellular differentiation (Figures 2e and 2f).50,51 ly 60% of B-CLL cases.59 At the molecular level, The above mentioned phenotypic profile differs mutation or loss of heterozygosity of the p53 significantly from that of other small B-cell lym- gene usually occurs in association with Richter’s phoid tumors, thus representing a basic tool for syndrome.59 C-myc, bcl-1, and bcl-2 genes have LPL recognition. Cytogenetic and molecular not been found to be clearly associated with the biology studies have shown the occurrence of disease, although t(11;14) has occasionally t(9;14)(p13;q32) in about 50% of cases.71 The been observed in “atypical” B-CLL.60,61 chromosomal breakpoints of the translocation Lymphoplasmacytic lymphoma involve the IgH locus on chromosome 14q32 and – on chromosome 9p13 – the genomic This has practically become a category by way 18 region containing the PAX-5 (paired homeobox- of exclusion. In fact, it comprises neoplasms 5) gene. Since this gene encodes BSAP, it is not that do not display characteristics that would surprising to find expression of this molecule in allow their inclusion among B-CLL/SLL, MCL, 18 LPL, which might contribute to tumor develop- MZL or FCL. The immunocytoma of the REAL ment.51 Recently, a 7q deletion has been classification18 – termed only lymphoplasmacyt- 19 described in small cell lymphomas with immu- ic lymphoma (LPL) in the WHO scheme – actu- nocytic morphology.72 ally corresponds to the lymphoplasmacytic immuno- cytoma of the UKC,9,10 since it is made up of ele- Hairy cell leukemia ments that range from small lymphocytes to Hairy cell leukemia (HCL) consists morpho- mature plasma cells by way of lymphoplasma- logically of small-medium sized B-cells, with vari- cytoid forms (Figure 2a). The production and ably shaped, round, oval or cleaved nuclei with a accumulation of intracytoplasmic Ig leads to the fairly wide cytoplasmic rim provided with the formation of frequent hyaline inclusions in the characteristic villous projections that give the form of Russell’s and/or Dutcher’s bodies (Fig- form its name7,10,18 (Figure 3a). Occasionally, neo- ure 2b)7,10,18 and, on occasions, to phenomena plastic cells are a bit larger and show a small, but of crystallization and phagocytosis by reactive distinct central nucleolus: this condition is com- histiocytes.62 On clinical grounds, the tumor is monly called HCL variant and seems to have a more aggressive than that of B-CLL/SLL,63 may more aggressive clinical course.73,74 The neoplas- transform into a diffuse large B-cell lymphoma tic population is generally confined to the periph- (immunoblastic)64 and frequently shows fea- eral blood, bone marrow and the red pulp of the tures corresponding to the original description , while the lymph nodes are only very rarely of Waldenström’s disease,65 such as diffuse involved.7,10,18 In the bone marrow, hairy cells bone marrow involvement and a monoclonal produce interstitial infiltration with progressive IgM/κ component in the serum. The latter may replacement of normal hematopoietic series and produce a hyperviscosity syndrome or autoim- low cellular density, due to the wideness of their mune phenomena, more frequently in the form cytoplasm (Figure 3b). Phenomena of edema and

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Figure 1 (page 1296). B-cell chronic lymphocytic leukemia/small same as negatively shown by the application of the anti-cytoker- lymphocytic lymphoma: a) cellular composition; the red and yellow atin antibody MNF.116 (immunoalkaline phosphatase technique in arrows indicate a prolymphocyte and a paraimmunoblast, respec- paraffin sections; ×300); e) features of follicular colonization tively (Giemsa; ×600); b) pseudofollicular growth pattern (Giemsa; (Giemsa; ×400); f) the staining for bcl-6 reveals residual follicle ×50); c) features of lymphoplasmacytoid differentiation (Giemsa; center cells (immunoalkaline phosphatase technique in paraffin ×300); d) prolymphocytoid crisis (Giemsa; ×600); e) the expres- sections; ×100); g) content of CD4+ cells in a gastric marginal sion of CD20 is dim in small lymphocytes and strong in prolympho- zone lymphoma of the small cell type (immunoalkaline phos- cytes and paraimmunoblasts (immunoalkaline phosphatase tech- phatase technique in paraffin sections; ×100). nique in paraffin sections; ×250); f) neoplastic cells are positive at the determination of CD5 (immunoalkaline phosphatase tech- Figure 5. (page 1297). Splenic marginal zone B-cell lymphoma: a) nique in paraffin sections; ×250); g) CD23 staining is much the exact amount of neoplastic cells and their intrasinusoidal dis- stronger in prolymphocytes and paraimmunoblasts (immunoalka- tribution are not easily assessed in conventionally stained prepa- line phosphatase technique in paraffin sections; ×300); h) BSAP rations (Giemsa; ×500). b) CD20 immunostaining allows the clear- expression is limited to pseudofollicles (immunoalkaline phos- cut assessment of the tumor burden and distribution (immunoalka- phatase technique in paraffin sections; ×150); i) IRF4 positivity is line phosphatase technique in paraffin sections; ×300); c) circu- mostly expressed by small lymphocytes (immunoalkaline phos- lating neoplastic cells show the villous profile (May-Grünwald- phatase technique in paraffin sections; ×150). Giemsa; ×800); d) neoplastic cells grow around residual Malpighi- an corpuscles and infiltrate the red pulp (Giemsa; ×100); e) the Figure 2 (page 1296). Lymphoplasmacytic lymphoma: a) the tumors consists of small elements with monocytoid appearance, tumor consists of small lymphocytes, plasmacytoid elements and intermingled with some plasma cells and blasts (Giemsa; ×400); plasma cells (Giemsa; ×600); b) a Dutcher’s body (arrowed) f) IgD staining of a bone marrow biopsy (immunoalkaline phos- appears in the form of an “intranuclear inclusion” (P.A.S.; ×800); phatase technique in paraffin sections; ×400). c) amyloid deposits in a lymph node (hematoxylin and eosin; ×50); d) CD20 is strongly expressed (immunoalkaline phosphatase tech- Figure 6. (page 1297 and this one). Follicle center lymphoma: a) nique in paraffin sections; ×200); e) all neoplastic cells display grade I form: note the follicular aggregation and very low content strong positivity at BSAP determination (immunoalkaline phos- of centroblasts (one of which is arrowed) (Giemsa, ×300); b) phatase technique in paraffin sections from a bone marrow biopsy; grade II form: the content of centroblasts exceeds the value of ×300); f) the search for IRF4 produces a similar staining pattern 6/HPF (Giemsa, ×400); (page 1297). c) follicle center lymphoma (immunoalkaline phosphatase technique in paraffin sections from with a high content of signet-ring-like elements (hematoxylin and a bone marrow biopsy; ×400). eosin, ×400); d) CD10 expression by neoplastic cells; note the presence of stained elements in an interfollicular position Figure 3. (pages 1296 and 1297). Hairy cell leukemia: a) neoplas- (immunoalkaline phosphatase technique in paraffin sections; tic cells show variability of the nuclear profile and a rather wide ×250); e) the same with the anti-bcl-6 PG-B6 monoclonal antibody rim of clear cytoplasm (hematoxylin and eosin; ×800); b) bone- (immunoalkaline phosphatase technique in paraffin sections; marrow interstitial infiltration by hairy cells; note the low cellular ×250); f) overexpression of the bcl-2 product (immunoalkaline density (Giemsa; ×300); c) neoplastic elements infiltrate the red phosphatase technique in paraffin sections; ×250); g) high Ki-67 pulp, sparing Malpighian corpuscles (Giemsa; ×50); d) DBA.44 marking in a diffuse form (immunoalkaline phosphatase technique staining highlights the typical “hairy” cytoplasmic profile, thus in paraffin sections; ×250); h) IRF4 antigen expression by neo- allowing the easy identification and counting of neoplastic cells plastic cells; note the presence of stained elements in an interfol- (immunoalkaline phosphatase technique in paraffin sections; licular position (immunoalkaline phosphatase technique in paraffin ×600). sections; ×250); i) follicle center lymphoma with features of plas- ma cell differentiation, as shown by the determination of kappa Ig Figure 4. (page 1297). Extranodal marginal zone B-cell lymphoma: light chain (immunoalkaline phosphatase technique in paraffin a) cytological details: centrocyte-like variant (Giemsa; ×400); b) sections; ×250); j) the same field at the search for BSAP cytological details: monocytoid-like variant (Giemsa; ×400); c) (immunoalkaline phosphatase technique in paraffin sections; lympho-epithelial lesions (hematoxylin and eosin; ×600); d) the ×250).

Haematologica vol. 85(12):December 2000 Pathologist’s view point: indolent lymphomas 1299 hemorrhage are frequently seen. In the spleen, lymph nodes and clearly evident in Malpighian the hairy cells substitute the red pulp with pseu- bodies in the spleen. B-cell lymphomas of the dosinus formation (Figure 3c) and spare marginal zone can be divided into three cate- Malpighi’s corpuscles for a long time. In addition gories: extranodal, nodal and splenic. The extra- to typical B-cell antigens, hairy cells express the nodal forms correspond to the mucosa-associ- receptor for interleukin 2 (CD25) and the CD103 ated lymphoid tissue (MALT) lymphomas integrin (a cell-adhesion molecule).18,75 Unfortu- described in the early eighties by Isaacson.85,86 nately, these antigens are negatively affected by These are primary B-cell lymphomas most fre- routine histopathology technical procedures. In quently found in the stomach, intestine, salivary paraffin sections, as well as being positive for gland, lung, respiratory airway, thyroid, ocular pan-B markers such as CD20 and CD79a, the adnexa and skin.87-90 It is interesting to note that, neoplastic cells often express the CD68 molecule with the exception of the intestine where MALT and are labeled by antibody DBA.44 (Figure is normally present as Peyer’s patches,91 in the 3d).76-78 The latter highlights the typical hairy pro- other sites the lymphoid tissue appears in an file of neoplastic cells, which otherwise is hardly acquired form following infective or more often visible. DBA.44, however, does not react or only autoimmune inflammatory processes.87-90 Neo- minimally reacts with the rare cases of HCL vari- plastic elements are usually small, have abun- ant. Immunoglobulins are rarely detected in rou- dant cytoplasm and a variable nuclear profile, at tine samples and, when detected, do not contain times resembling centrocytes (cc-like cells), at δ-heavy chain. The bcl-2 product is regularly times immunocytes (Ic-like cells) or B-monocy- expressed, BSAP is found in the majority of cas- toid elements (Figures 4a and 4b).18,85-90 The es, while the search for bcl-1, bcl-6 and IRF4 tumor shows regular plasma cell differentiation, always gives negative results (Table 3).49-53 The Ki- which at times is associated with amyloid 67 marking is extremely low, a finding in keeping deposit92 or is so striking as to lead to a misdi- with the very indolent behavior of the process. agnosis of extramedullary plasmacytoma.88 The More recently, monoclonal antibodies specific to number of mitotic figures is low. Lymphoma- human tartrate-resistant acid phosphatase have tous cells usually attack the epithelial compo- been produced and proposed as specific HCL nent – giving rise to lympho-epithelial lesions markers.79 HCL represents one of the tumors for (LELs) – and surround and colonize pre-existing which it is of fundamental importance to moni- follicles (Figures 4c-4e).87-90,93 These findings are tor minimal residual disease (MRD) following of value in recognizing extranodal MZLs. In the therapy: this goal can be easily achieved by the gastro-intestinal (GI) tract, the diagnosis made cheap immunohistochemical assay in paraffin on small biopsies performed during endoscopy sections. Indeed, patients treated with the more does not provide definite hints on the degree of recent approaches such as interferons, 2-chloro- GI wall infiltration, which should be assessed by deoxyadenosine or deoxycoformycin have been other means, such as echo-endoscopy. In the shown to retain isolated residual hairy cells stomach, where the tumor develops within the trapped within hyperplastic or fibrotic marrow, context of Helicobacter pylori+ (HP) chronic gas- the recognition of which is difficult or even tritis,94,95 Wotherspoon et al. proposed adopt- impossible at pure morphologic evaluation.76- ing a score system which highlights morpholog- 78,80,81 Molecular analysis shows a high rate of ically suspicious lesions (grades III and IV), somatic mutations.82 This finding does not com- which do not yet represent overt lymphomas, pletely clarify the exact position of HCL in B-cell but require careful follow-up with frequent biop- sies.94 MZL most often has a multicenter distri- development: it does, however, indicate that neo- 96 plastic cells originate from elements which have bution within the organ of origin, a fact that been exposed to the hypermutation mechanism should always be considered when taking ther- apeutic decisions (e.g. surgery vs. chemothera- and have thus passed through the germinal cen- 88-90 82 py). In case of dissemination to local nodes ter. Occasionally, genomic alterations have 97 been described in the form of t(2;8), p53 gene and spleen, the tumor frequently involves the deletion and trisomy 12.83,84 marginal zone, sometimes without total efface- ment of the normal structure.90 More rarely it Extranodal marginal zone B-cell lymphoma of colonizes other MALT sites (usually many years MALT type following the original diagnosis) or the bone This is a new category, which has been intro- marrow (reported incidence in different series: duced by the REAL Classification19 and main- 5-10% of cases).33 Recently, the ILSG members tained in the WHO scheme.19 Histogenetically, stated that the term MZL should be restricted to the tumor can be traced to elements of the mar- neoplasms consisting almost exclusively of small ginal zone surrounding the mantles of normal cells.19 However, at MALT sites large B-cell lym- follicles, which is scarcely perceptible in the phomas with or without residual MZL do also

Haematologica vol. 85(12):December 2000 1300 S. A. Pileri et al. occur, as do MZLs with a certain number of carboxyl terminal to CARD, which activates NF- blasts.98 The former situation should be diag- kappaB, but does not induce apoptosis.107,108 nosed as DLBCL (with residual MZL, if present), Mutant bcl-10 overexpression might have a two- while the latter is still matter of debate. Recent fold lymphomagenic function: loss of bcl-10 data from the International Extranodal Lymphoma pro-apoptotic effect may confer a survival Study Group (IELSG) (Ascona, February 25-26, advantage to MALT B-cells, and constitutive NF- 2000) suggest that a blastic component not kappaB activation may provide both anti-apop- exceeding 10% of the examined population does totic and proliferative signals mediated via its not affect the course of the disease. Further transcription factors.107,108 Therefore, great studies are needed to assess whether the cases emphasis was given to t(1;14) and bcl-10 over- with a higher number of blasts or clusters con- expression as major events in the development sisting of 20 or more blasts do actually run more of MALT lymphoma. Recent studies, however, aggressively – as proposed by Isaacson et al. – or suggest that bcl-10 mutations occur in a small- not. At phenotypic analysis (Table 3),18,33,88-90 er number of gastric MZLs than originally neoplastic cells are CD19-CD22+, CD79a+, thought, although they seem strictly related to a CD35+, CD11c+, CD68+/-, and DBA.44-/+. They more aggressive clinical course and unrespon- express monotypic Ig at the intracytoplasmic siveness to treatment.109-111 The (perinuclear) level, bearing µ or – more rarely – translocation (11;18) is detected in about half α heavy chain, but not δ chain. There is weak- gastric MALT lymphomas, while it is absent from moderate positivity at the determination of the nodal and splenic MZLs: it causes the expres- bcl-2 gene product. The search for CD5, CD10, sion of a chimeric transcript fusing 5’ API2 on CD23, bcl-1, and bcl-6 turns out to be negative chromosome 11 to 3’ MTL on chromosome (Figure 4f). The content of Ki-67+ cells is low. 18.116-118 Finally, p53 and/or c-myc mutations IRF4 and BSAP are detected in a proportion of would correspond to the final phase of blastic the neoplastic cells.50,51 T-cell markers reveal the transformation.99-101 presence of a high content of reactive T-lym- Among MZLs, the gastric forms have gained phocytes, mainly of the CD4+ type (Figure 4g).87- special interest both at the clinical and patho- 90 The application of anti-cytokeratin antibodies logic level, because of their clear-cut patho- allows easy identification of LELs, which appear genetic correlations with HP infection.94,95,119 In as negative images (Figure 4d).33,84 In case of particular, the infective agent causes the devel- blastic transformation, the Ki-67 index is opment of acquired MALT within the stomach increased; overexpression of p53 and/or c-myc and sustains a state of chronic inflammation, may also be seen.99,100 Molecular studies show which shows varying degrees of activity regular clonal rearrangements of Ig-encoding – expressed by the amount of granulocytes – and genes: in particular, the occurrence of numer- persists until HP eradication.120 The prolonged ous somatic mutations and the possible detec- antigenic stimulation facilitates the outgrowth tion of ongoing mutations assign MZL to the of clones, which produce autoantibodies against group of post-germinal center cell derived lym- structural components of the gastric mucosa phomas.101-103 Peng et al. have reported the and contribute to the maintenance of lymphoid occurrence of a replication error (RER) pheno- proliferation.121-130 Within this context, granulo- type in 50% of cases, which might facilitate the cytes are responsible for oxidative phenomena, onset of tumor.100 However, this finding has not which cause DNA instability/damage and might been confirmed by others, who found no RER herald the appearance of clones with RER phe- phenotype in gastric MALT lymphomas.104-106 notype.90,99,100 The occurrence of chromosomal While t(11;14) and t(14;18) are absent (with alterations leads to the selection of more resis- only a few exceptions, which are matter of tant clones (oligoclonal phase), one of which debate), there is a series of recurrent aberrations gains advantage over the others and produces which are thought to play a role in process of an overt small cell MZL (monoclonal phase).90 At lymphomagenesis and include t(1;14)(p22;q32), this time, however, the tumor persists only in the trisomy 3, and t(11;18) (q21;q21).107-118 In par- presence of HP infection and by the co-operation ticular, t(1;14) causes overexpression of bcl-10, of CD4+ T-cells via a CD40/CD40L mecha- a novel apoptotic signaling gene that encodes nism.122 Furthermore, it tends to remain local- an amino-terminal caspase recruitment domain ized at the primary site for a long time – with (CARD), homologous to that found in several possible local diffusion to the regional nodes – apoptotic molecules.107,108 Wild-type bcl-10 acti- because of the peculiar circulation pathway of vates NF-kappaB but induces apoptosis, as MALT elements, controlled by the adhesion mol- shown in MCF7 or 293 cells. Bcl-10 expressed by ecule MAdCAM-1 and homing receptor α4b7.126 lymphoma cells carrying t(1;14) exhibits a frame Further late genomic alterations can make the shift mutation resulting in truncation either in or growth independent of the micro-environment

Haematologica vol. 85(12):December 2000 Pathologist’s view point: indolent lymphomas 1301 and p53 and/or c-myc mutations can finally spleen.18 Although it is now quoted as an accept- cause its transformation into a DLBCL.90 Based ed entity in the WHO scheme,19 its histogenesis on this model, it becomes understandable why: remains controversial. Several immunologic and a) HP eradication produces tumor regression in molecular data suggest in fact that the tumor about 70% of cases (i.e. in cases which still may be unrelated to splenic marginal zone B- depend on local antigenic stimulation and have cells.131,132 appropriate histologic grade and stage90,125,126) SMZL has rather different features from those and b) untreated gastric MZL needs several years of the two varieties of B-cell marginal zone lym- to transform into a DLBCL.90,98 The time interval phoma quoted above.18 In particular, it most between HP eradication and lymphoma regres- often displays: a) dissemination, with intrasinu- sion is highly variable (from 4 weeks to 14 soidal bone marrow infiltration (Figures 5a and months)90,127,128 and the histologic regression is 5b);133 b) presence of a leukemic component usually associated with phenomena of sclerosis (with a “villous” appearance in about 50% of and hyalinosis of the mucosa. Prolonged follow- cases) (Figure 5c);134 c) splenic involvement, up studies have revealed that most patients who both ring-like around Malpighian follicles and have experienced tumor regression remain in plurifocally in the red pulp (Figure 5d);135 d) a complete remission some years following the very indolent clinical course and favorable completion of the antibiotic treatment.128 His- response to splenectomy.136,137 In particular, at tology seems to represent the best indicator for microscopic examination, the tumor typically judging the achievement and maintenance of shows dimorphic cytology with an inner core of complete remission, since it has been shown that small cells and a peripheral rim of medium-sized polymerase chain reaction (PCR) may display a clear elements, with a few blasts intermingled clonal band even 2 years following therapy, (Figure 5e). Features of plasmacellular differen- which does not predict relapse of the disease and tiation may occasionally be seen. These findings will disappear in the long run.129 All these factors along with the phenotypic profile (B-cell mark- are relevant for the therapeutic strategy which, er+, CD5–, CD10–, CD23–, bcl-2+weak, bcl-6-, at least initially, can be rather conservative, as BSAP+, IRF4–, IgM+, IgD+, DBA.44+/-, and well as for patients’ management, implying indef- CD68+/-) (Figures 5b and 5f) allows the distinc- inite follow-up of the apparently cured cases. tion of SMZL from HCL (which primarily Patients who do not obtain tumor regression or involves the red pulp) as well as from other B-cell display aggressive histology or disseminated dis- lymphomas (such as B-CLL, LPL, MCL and ease should be treated according to convention- FCL), which may involve the spleen also pro- al strategies, which include total gastrectomy ducing a marginal zone pattern and imply dif- and/or , depending on individual ferent therapeutic strategies.18,49-53,135 On mole- risk factors (degree of stomach wall infiltration, cular grounds, neoplastic cells display IgV(H) systemic diffusion, etc.).33,90,130 gene mutations, consistent with a post-germi- nal center cell derivation.132 In some cases, Nodal marginal zone B-cell lymphoma ongoing mutations have been observed in ele- (± monocytoid B-cells) ments obtained from the spleen: this finding Since the cytological, architectural and phe- apparently contrasts with a study showing that notypic features of the nodal variety do not dif- blood-borne tumor cells from patients with cir- fer from those of the extranodal form, the dif- culating villous lymphocytes do not show signs ferential diagnosis must be made by the exclu- of ongoing mutations.132 However, it is possible sion of an evident MALT lymphoma in any of its that ongoing mutations are acquired in the characteristic sites.18,19 The nodal form appears splenic microenvironment. No examples of to have a higher rate of early relapse than the SMZL showing the t(11;14) and t(14;18) other marginal zone B-cell lymphomas and an translocations have been described in the liter- overall survival similar to that of FCL.20 ature; by contrast, recurrent abnormalities of chromosomes 1, 3, 7 and 8 are detected in more Splenic marginal zone B-cell lymphoma 131 (± villous lymphocytes) than half the cases. Finally, a recent report has Splenic marginal zone lymphoma (SMZL) was proposed the existence of an aggressive variant included in the REAL Classification as a provi- of SMZL, characterized by an increased number of blasts and frequent 7q loss and/or p53 inac- sional entity, since the authors felt that further tivation: this variant should require a different studies were needed to shed light on its histo- clinical management of the patients.138 genesis and in particular whether it was derived from the marginal zone alone or tended to Follicular lymphoma reproduce all the B-cell maturation steps physi- This category – termed follicular center lymphoma ologically occurring in the white pulp of the in the REAL Classification18 and follicular lym-

Haematologica vol. 85(12):December 2000 1302 S. A. Pileri et al. phoma in the WHO scheme19 – comprises both ularly negative for CD5, DBA.44 and CD68.18 the centroblastic/centrocytic and centroblastic Monotypic surface and cytoplasmic Ig can be follicular forms of the UKC.9,10 Their inclusion as detected only in a minority of cases, even apply- a single group is justified by their shared histo- ing the more sensitive antigen retrieval tech- genesis (from follicular center cells), phenotype niques. On the other hand, in more than 95% of and chromosomal abnormalities.18 Follicular cases neoplastic follicles carry the bcl-2 gene center lymphoma (FCL) is usually characterized product (Figure 6f): this finding, which is relat- by the formation of neoplastic follicles (Figure ed to the occurrence of t(14;18) or bcl-2 gene 6a), which – in the lymph node – affect the cor- rearrangements, is of practical relevance, as it tex, paracortex and medulla.18 Conversely to nor- causes protection of neoplastic cells against mal follicles, neoplastic follicles are quite homo- apoptosis and – along with the above mentioned geneous in size and shape, tend to grow back-to- morphologic criteria – contributes to the dis- back (compressing the interfollicular areas) and tinction of FCL from florid follicular hyperpla- lack well-developed mantles.7,10,18 In less than 5% sia.18,148-152 The latter is in fact characterized by of cases, the tumor is purely diffuse: the pres- bcl-2 negativity, since the clonal selection occur- ence of diffuse areas should always be reported ring within the follicles produces elimination of and quantified because of its impact on prog- unsorted follicular center cells via apoptosis. The nosis.18 On cytological grounds, FCL consists of amount of lymphomatous elements expressing centrocytes and centroblasts in different pro- the proliferation-associated nuclear antigen Ki- portions (Figures 6a and 6b), which are ran- 67 varies from case to case and even from one domly distributed within the follicles without the follicle to the other within the same case: on the typical zoning pattern observed in normal ger- whole, the higher the number of centroblasts, minal centers and corresponding to clonal mat- the higher the content of Ki-67+ cells (Figure uration and selection.7,10,18 In view of the variable 6g).153 In general, FCL is characterized by a low ratios of centroblasts and centrocytes and, as a proliferative capacity and a strong protection consequence, of the different clinical behavior against apoptosis, a combination which justifies (grade III FCL will be included among aggressive on the one hand the resistance of the tumor to lymphomas) a grading system has been pro- conventional , and on the other posed, according to the Berard cell-counting the application of novel strategies, such as anti- method [grade I: 0-5 centroblasts/high power CD20 antibodies and vaccines.45 As to other bio- field (hpf); grade II: 6-15 centroblasts/hpf; grade logical markers, FCL is negative at the determi- III: > 15 centroblasts/hpf].18,19,139 Some centro- nation of IRF4, with the exception of rare cases cytes are always comprised within the interfollicu- with plasmacellular differentiation, which show lar areas, a finding never observed under physio- positivity limited to the plasma cell component logic conditions. Occasionally, centrocytes acquire (Figures 6h and 6i).50 BSAP is strongly expressed a signet-ring-like appearance (Figure 6c), possibly by neoplastic cells: the detection of this mole- engendering a misdiagnosis of metastatic adeno- cule, along with bcl-6 and CD10, allows easy carcinoma.140 In other instances, the tumor identification of interfollicular tumoral compo- exhibits plasmacellular or marginal zone differ- nents, thus contributing to the diagnostic entiation at the periphery of neoplastic follicles process (Figure 6j).51 Finally, like normal cen- and/or in the interfollicular areas: these findings troblasts and centrocytes, lymphomatous ele- compel the differential diagnosis vs. extra- ments are accompanied by follicular dendritic medullary plasmacytoma and marginal zone cells: these are observed in variable amounts and lymphoma.141,142 Phenomena of sclerosis (more can be easily detected by the application of the frequent in retroperitoneal neoplasms) and following markers: CD21, CD23, CD35, and necrosis may be seen.143,144 In particular, in the R4/23.18 From 80% to 90% of FCLs cases carry presence of a fully necrotic node the pathologist the t(14;18)(q32;q21) translocation, which should examine silver impregnated slides care- involves the bcl-2 gene.149-152 The translocation is fully, since this stain can reveal an otherwise not exclusive to FCL, since it also occurs in a pro- undetectable follicular pattern.7,10 The latter is portion of DLBCLs (see Part II). In particular, it quite characteristic of a lymph node involved by joins the bcl-2 gene at its 3’-untranslated region FCL, which has undergone massive infarction to IgH sequences, resulting in deregulation of because of blood vessel infiltration. At pheno- bcl-2 expression. In about 70% of cases the typic analysis, lymphomatous cells carry B-cell breakpoints on chromosome 18 are clustered markers, along with CD10 and bcl-6 gene prod- within a major breakpoint region (MBR), while uct, i.e. molecules regularly found in normal fol- in the remaining ones they occur in the more dis- licular center elements.18,49,68,145-147 CD20 is tant minor cluster region (mcr). The role of bcl- strongly expressed, while the staining for CD79a 2 deregulation in the development of FCL is mat- is weak-moderate (Figures 6d and 6e). FCL is reg- ter of debate. It is likely that bcl-2 activation is

Haematologica vol. 85(12):December 2000 Pathologist’s view point: indolent lymphomas 1303 not enough for the onset of the tumor, which in’s lymphomas (based on the Updated Kiel Classifi- should require the occurrence of other genetic cation). Berlin: Springer-Verlag 1992. 11. Kay HE. Letter: Classification of non-Hodgkin’s lym- lesions or host factors, such as chronic antigen phomas. Lancet 1974; 2:586. stimulation and selection. Deletion of chromo- 12. Lennert K. Comments by ‘expert’ pathologists. Hema- some 6 is seen in approximately 20% of cases.154 tol Oncol 1983; 1:95-6. Accumulation of p53 mutations, rearrange- 13. Lukes RJ. Comments by ‘expert’ pathologists. Hema- tol Oncol 1983; 1:96-7. ments of c-myc or inactivation of p16 are fre- 14. Classification of non-Hodgkin’s lymphomas. Repro- quently observed in case of progression to a ducibility of major classification systems. NCI non- DLBCL.155,156 Hodgkin’s Classification Project Writing Committee. Cancer 1985; 55:91-5. Contributions and Acknowledgments 15. Nathwani BN, Metter GE, Miller TP, et al. What SAP was responsible for the conception and design of should be the morphologic criteria for the subdivision this review. SA and ES were responsible for drafting the of follicular lymphomas? Blood 1986; 68:837-45. 16. Hastrup N, Hamilton-Dutoit S, Ralfkiaer E, Pallesen article. GFO was responsible for the analysis and inter- G. Peripheral T-cell lymphomas: an evaluation of pretation of morphologic data. SP and MP were respon- reproducibility of the updated Kiel classification. sible for analysis and interpretation of phenotypic data. Histopathology 1991; 18:99-105. PPP was responsible for analysis and interpretation of 17. Banks PM, Chan J, Cleary ML, et al. Mantle cell lym- phoma. A proposal for unification of morphologic, clinical data. BG was responsible for analysis and inter- immunologic, and molecular data. Am J Surg Pathol pretation of molecular data. PLZ and LL were respon- 1992; 16:637-40. sible for revising the article critically. BF approved the 18. Harris NL, Jaffe E, Stein H, et al. A revised European- final version of the paper. The criteria for the order of American classification of lymphoid neoplasms: a pro- names were: involvement in design and organization of posal from the International Lymphoma Study Group. Histopathology 1994; 25:517-36. the paper, laboratory research, analysis of clinical data, 19. Harris NL, Jaffe ES, Diebold J, Flandrin G, Muller-Her- and reviewing the paper. The order of the names was melink HK, Vardiman J. Lymphoma classification – decided on the basis of each individual contribution to from controversy to consensus: the R.E.A.L. and WHO the above criteria. The authors thank Ms. Federica San- classification of lymphoid neoplasms. Ann Oncol 2000; 11:3-10. dri and Mr. Luigi Chilli for their skillful technical assis- 20. A clinical evaluation of the International Lymphoma tance. Study Group classification of non-Hodgkin’s lym- phoma. The Non-Hodgkin’s Lymphoma Classification Funding Project. Blood 1997; 89:3909-18. This paper was supported by grants from AIRC 21. Longo DL. Biologic agents and approaches in the (Milan), MURST (Rome) and ABSTE (Bologna), Italy. management of patients with lymphoma. A critical appraisal. Hematol Oncol Clin N 1991; 5:1067-87. 22. Longo DL. The REAL classification of lymphoid neo- plasms: one clinician’s view. In: PPO Updates. Rosen- References berg S, Ed. Philadelphia: Lippincott 1995; 9:1-12. 23. Gerdes J, Stein H, Pileri S, et al. Prognostic relevance 1. Rappaport H. Tumors of the Hematopoietic System. of tumour-cell growth fraction in malignant non- In: Atlas of Tumor Pathology, Sect. 3, Fasc. 8. Wash- Hodgkin's lymphomas. Lancet 1987; 2:448-9. ington, D.C.: Armed Forces Institute of Pathology, 24. Gerdes J, Sabattini E, Pileri S. Immunohistochemical 1966. determination of the proliferative capacity of malig- 2. 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