COMPANION MEETING LYMPHOMA

Transcription

1 Australasian Division of the International Academy of Pathology Limited 36 th Annual Scientific Meeting Sydney Convention & Exhibition Centre, Darling Harbour, NSW, Australia June 3 5, 2011 COMPANION MEETING LYMPHOMA Bayside Room 102 9:00 10:45 am Friday, June 3, 2011 Convenor: Dr Benhur Amanuel, PathWest, Perth, WA Case Presenters: 1. Dr Dominic Spagnolo, PathWest Laboratory Medicine, Perth, WA 2. Dr Debra Norris, QML, VMO Princess Alexandra Hospital, Brisbane, QLD 3. Dr Daniel Kearney, Surgical Pathology, SA Pathology/IMVS, Adelaide, SA 4. Dr Penny McKelvie, St Vincent s Hospital, Melbourne, VIC 5. Dr Benhur Amanuel, PathWest Laboratory Medicine, Perth, WA

2 Case 1 Presenter: Dr D Spagnolo, PathWest Laboratory Medicine, QEII Med. Centre, WA. (case courtesy of Dr M Havlat, St John of God Pathology, WA) Case History 83 yo female with 3 months' history of rapidly growing, solitary, painless, velvety skin nodule left chest wall. Clinically well without fever, sweats or weight loss. No medications. No significant past history. Her sister died of leukemia in 1989 (details not known). Skin biopsy performed (slide provided). Reviewed by clinical haematologist one month later. Four further small pink papules 5 15 mm in size found. These increased in number to 12 over one week, all limited to the trunk. There was no lymphadenopathy or hepatosplenomegaly. Peripheral blood examination was normal. Bone marrow had some increase in monocytoid cells but no myelodysplastic features, no increase in blasts or evidence of leukemic infiltration. Marrow cytogenetics showed a duplicate ring 1q chromosome of uncertain significance but thought likely to be associated with a dysplastic change. She was commenced on etoposide 50mg daily. The skin lesions resolved completely within 2 weeks. Pathological features A diffuse infiltrate of mononuclear cells fills and expands the dermis in a sheet-like manner though at the lateral margins shows a looser, dissecting pattern through dermal collagen. There is infiltration into subcutaneous fat both along expanded septa and interstitially within lobules. A narrow grenz zone separates the infiltrate from the epidermis. There is no epitheliotropism, angiocentricity or angioinvasion. The infiltrate is composed of medium sized and large cells (2 - >4x size of small lymphocytes) arranged dyscohesively. "Squaring off" of intercellular borders is evident. The cells have rounded, ovoid or reniform nuclei with stippled chromatin and one or more generally small nucleoli though macronucleoli are evident in some of the larger cells. The cytoplasm is moderately abundant and amphophilic to lightly basophilic with a suggestion of fine basophilic stippling in some cells at high power. On close scrutiny, only in very rare cells can fine eosinophilic granules be discerned in the cytoplasm. There are no mature granulocytes. Mitotic figures abound and there are at least 3 in most high power fields, including aberrant forms. Scattered apoptotic cells are seen throughout the infiltrate though coagulative tumour necrosis is lacking. Rarely, small mature lymphocytes may be found widely dispersed in the infiltrate. Immunohistochemistry: A broad panel of immunostaining was performed from the perspective of an "undifferentiated" haematolymphoid infiltrate of skin. The malignant cells stain as follows: positive: CD4, CD56 (both strong, diffuse), myeloperoxidase (heterogeneous, <50%), CD68 (KP1 clone; strong, diffuse), CD68R (PGM1 clone; fewer than KP1), CD163 (heterogeneous), CD15, CD43 (patchy), CD23, BCL2 (weak), MIB1 ~70%; negative: CD45RA, CD123, TCL1, CD303 (BDCA-2), CD2AP, BCL11A, CD3, CD2, CD5, CD7, CD8, CD57, TIA1, perforin, granzyme B, TdT, CD1a, CD99, CD34, CD117, ALK1, EMA, CD30, CD1A, langerin (CD203), S100, CD20, PAX5, CD138, CD10, cyclin D1, MUM-1, CD21, CD61, EBER. Interphase FISH: No RUNX1-RUNX1T1 fusion associated with t(8;21)(q22;q22); no CBFB inv(16)(p13.1q22); no PML-RARA fusion of t(15;17)(q22;q12); no MLL (11q23) abnormalities. There was no evidence of trisomy 8. DV Spagnolo IAP 2011 Lymphoma Club 1

3 Ultrastructure: Cells showing immature myeloid and monocytic features. DIAGNOSIS: Cutaneous myeloid sarcoma ( aleukemic myeloid leukemia cutis), consistent with acute myelomonocytic leukemia (AMML). DISCUSSION The diagnosis of myeloid leukemia cutis (MLC) in this case is not particularly problematic. The morphology and immunophenotype (MPO+, CD68+, CD163+, CD15+, other lineage markers negative) is definitive, though the strong and diffuse CD4 and C56 coexpression present a pitfall if it is not realised that these antigens may be expressed in MLC. The CD23 (low affinity Fc-epsilon Receptor II for IgE) positivity is unusual but may occur in a minority of AML cases. The case is presented (1) to emphasise the importance of considering MLC even when presenting as a solitary skin nodule in the absence of known leukemia, (2) to highlight the potential for misdiagnosis when considering a CD4+, CD56+ cutaneous infiltrate, and (3) to consider other differential diagnoses. Several recent useful reviews of leukemia cutis have been published (1-5). The classification of myeloid neoplasms is according to the latest WHO classification (2008) of acute myeloid leukemia (AML) and related precursor neoplasms (Table 1; Ref. 6). The incidence of specific subtypes in the skin according to this latest scheme is not clear as there are few published data (2) and early publications based on previous classifications do not translate accurately into the latest WHO classification. Nevertheless, the overall prevalence of cutaneous involvement in patients with myeloid neoplasms is estimated at about 3% (5), occurring most frequently in AMML and acute monoblastic/monocytic leukemia (AMoL) where as many as 50% of patients develop skin lesions. In the case presented, in the absence of fresh tissue for cytochemical and flow cytometry assessment the precise classification remains uncertain but the overall features are consistent with AMML. In any event, when confronted with such an infiltrate, urgent haematological opinion is indicated to determine the status of the blood and bone marrow and to enable precise classification of the process. WHO 2008 Classification of AML and related precursor neoplasms** AML with recurrent genetic abnormalities (7 entities, 2 provisional entities) AML with myelodysplasia-related changes Therapy-related myeloid neoplasms Acute myeloid leukemia, NOS AML with minimal differentiation AML without maturation AML with maturation Acute myelomonocytic leukemia Acute monoblastic/monocytic leukemia Acute erythroid leukemia (pure; or erythroid/myeloid) Acute megakaryoblastic leukemia Acute basophilic leukemia Acute panmyelosis with myelofibrosis Myeloid sarcoma Myeloid proliferations related to Down syndrome Blastic plasmacytoid dendritic cell neoplasm (**Abbreviated) Extramedullary myeloid sarcoma (MS): In a recent review of 92 cases of adult MS of any site (7), there was a slight male predominance, the median age was 55.8 yrs and the most frequent sites of involvement were skin (28%), lymph nodes (16%), testis (6.5%), intestine (6.5%), bone (3%) and DV Spagnolo IAP 2011 Lymphoma Club 2

4 CNS (3%). 27% presented with de novo MS, 35% had simultaneous AML, idiopathic myelofibrosis or myelodysplastic syndrome (MDS) and 38% had a previous history of AML, myeloproliferative disorder (MPD), mastocytosis or MDS. Notably, 40% of de novo cases had been submitted in consultation with alternative unrelated diagnoses. Most cases were of the "blastic" variant (now probably including the categories of AML with minimal differentiation or without maturation), followed by monoblastic and myelomonocytic categories. CD68(KP1) was the most frequently expressed marker, followed by MPO, CD117, CD99 and CD68(PG-M1). CD56 was positive in 13%, CD4 in only 1% and CD30 in 2%. Markers more specific for plasmacytoid dendritic cells (PDC) were not employed in this study. 89.5% died of disease at median F/U of 150 months. Patient outcome was not affected by age, sex, site, de novo disease or coexistence with AML, MDS or other MPD, morphology, immunophenotype or cytogenetic features. Patients treated aggressively as for AML and including allogeneic or autologous marrow transplantation, seemed to have a better outcome than those receiving less aggressive therapy. Myeloid leukemia cutis: Patients with acute (or less commonly, chronic) myeloid leukemias may develop cutaneous leukemic infiltrates. This typically occurs in the setting of known AMLs, particularly those showing monocytic differentiation. Chronic MPD or MDS cases with cutaneous involvement indicate blastic transformation to AML as there are no cutaneous phases of the chronic disorders per se (1). AML presenting ab initio in the skin is much less common and may occur in the absence of blood and marrow disease (aleukemic leukemia cutis) or it may occur simultaneously. Typically, there are multiple violaceous plaques or nodules, either localised or generalised. More rare, as in this case, is presentation as a solitary nodule. Other uncommon presentations mimic specific dermatoses such as viral or drug eruptions (8) or leukemic vasculitis (9). In most patients with a cutaneous presentation evolution to frank leukemia occurs usually within a year of presentation and the prognosis is poor. The largest series of MLC was recently published as a French multi-institutional retrospective study of 173 cases (1) classified according to the earlier FAB classification (M0 to M7). Males predominated (M:F = 1.4:1) and the median age was 62 yrs. The lesions were solitary in 23%, multiple in 77% and there was no site predilection. De novo presentation occurred in 7.5%, in 27% there was concurrence with a known myeloid disorder, in 61% the skin became involved subsequent to the diagnosis of a myeloid disorder and in 5% the chronology was not known. 65.3% of the cases (113) were AML and those showing monocytic predominance were overrepresented in skin compared with peripheral blood; 56% were FAB 4 or 5 (FAB4-AMML 13%, FAB 5-AMoL 43%), 21% were FAB 0-2. Similar distributions have been reported in smaller series (2,3). Medium sized cells predominated in most cases but nearly 20% had mixed medium and large cells. Mitoses typically were few. AMLs with monocytic differentiation (FAB 4,5) were significantly more likely to have a greater tumour cell density and to be composed of larger cells than FAB 1,2 cases. Antigen expression in the 173 MLC cases was as follows: CD68 (97%), CD163 (52%), CD14 (35%), CD4 (61%) these were considered as monocytic markers; MPO (63%), CD33 (93%), CD117 (30%) considered to be myeloid markers; and CD56 (19%), CD123 (9%), CD303(BDCA-2) (3%) considered to be plasmacytoid dendritic cell markers. In this series, de novo cases of MLC were more likely to have these characteristics: AML-FAB 4 or 5 (ie. with monocytic features) multiple rather than solitary nodules diffuse pattern of infiltration high tumour density large cell morphology, high mitotic index and apoptosis intense CD4 and CD56 expression, and widespread CD68 positivity. Antigen expression in MLC (1-3,6) The markers CD43 and CD68 (KP1) are expressed in >90% of all MLC cases DV Spagnolo IAP 2011 Lymphoma Club 3

5 irrespective of their WHO classification and these constitute useful "screening" markers for the possibility of MLC, especially as ~50% of cases may be MPO negative. MPO positivity across all diagnostic categories ranges from 42 63% and depends on the degree of monocytic differentiation. MPO and CD33, often considered as markers of myeloid/granulocytic differentiation, lack specificity as they are frequently positive in cases showing monocytic differentiation. CD68 (KP1) shows high sensitivity for MLC across all categories but has low specificity in distinguishing between the subtypes. Note that CD68 (PGM-1 clone) has relatively greater specificity for the monocytic lineage. CD163 has lower sensitivity for MLC than CD68 across all diagnostic categories, and while it has greater specificity for monocytic differentiation, it may still be positive in 25% of AML (FAB 1,2) cases, whereas CD14 is far less likely (~10%) to be positive in AML-FAB 1,2 cases. Similarly, CD4 is reported in ~40% of AML-FAB 1,2 cases (and in up to 60% of all AML cases). Thus, none of these markers can be taken to be specific for distinguishing between myeloid (granulocytic) and monocytic differentiation. CD56 is positive in ~20-50% of all cases of MLC and is more often positive in AML- FAB 4,5 than AML-FAB 1,2 cases. CD4 is positive in approximately 60% of all MLC cases, though data are few. There is a strong correlation between CD56 and CD4 expression. In the series of Bénet et al (1), 85% of CD56+ cases were also CD4+ (most were AML; 2 were later reclassified as BPDCN). The stem cell marker CD34 is rarely positive in MLC (~5%) and CD117 is also uncommonly positive (~30%). There is frequent discordance between antigen expression by tissue IHC in cutaneous infiltrates compared with concurrent marrow infiltrates (flow cytometry), particularly for these antigens. Various explanations have been mooted to explain this discordance including treatment effects and differences in sensitivity between the testing modalities. Thus, these markers cannot be used to indicate the presence of blasts in skin. To a lesser extent, discordance may be found for other markers including CD56 and MPO. The PDC markers CD123 and CD303 are rarely expressed in AML-FAB 1,2 and are more likely to be positive in acute or chronic myelomonocytic cases. Cytogenetics: Data pertaining to MLC are few. Numerical abnormalities of chromosome 8 (trisomy mainly), a myeloid-associated but nonspecific aberration, appear to be significantly more common in AML patients with, than without cutaneous involvement, typically in AML-FAB 4,5 (2,4,5). The reasons for this are not known. Differential diagnosis: There are several differentials to be considered here, particularly in the context of an infiltrate which is strongly CD56+ and CD Blastic plasmacytoid dendritic cell neoplasm (BPDCN) 2. Lymphoblastic leukemia/lymphoma (precursor) 3. Non-Hodgkin lymphoma (mature; T or B) 4. Anaplastic myeloma 5. Mast cell sarcoma 6. Histiocytic/dendritic neoplasms 7. Non-haematolymphoid neoplasms, e.g. Merkel cell carcinoma; metastases. CD56+ cutaneous haematologic lesions: A multicentre study was recently conducted by the Cutaneous Lymphoma Task Force of the EORTC to define prognostic features and to establish diagnostic and therapeutic guidelines in 34 patients with CD56+ haematologic neoplasms presenting in the skin (10). Based on clinical, histological, immunophenotypic and molecular features, the cases were classified as follows: DV Spagnolo IAP 2011 Lymphoma Club 4

6 1. Blastic plasmacytoid dendritic cell neoplasms (20) 2. AML, CD56+ (4) 3. Extranodal NK/T-cell lymphoma, nasal type (5) 4. "Classical" primary cutaneous T-NHL, CD56+ (5). Of the AML cases (AMML) all were CD56, CD33, CD4, CD68 and MPO positive. The PDC markers CD123 and TCL-1 were positive in 1 of 2 cases, and in 2 of 3 cases studied, respectively. With the exception of the last category, all other cases had a very poor prognosis (93% died of disease, median survival of 11 months). Patients with T-NHL, nos, were all alive at last followup (median 62 months). While the study emphasises the importance of the correct categorisation of CD56+ haematological infiltrates of skin in respect of prognostication, it should be noted that the cases of T-NHL studied (lymphomatoid papulosis, mycosis fungoides, subcutaneous panniculitislike T-cell lymphoma) self-select for relatively indolent behaviour as this group did not include other aggressive forms of cutaneous T-NHL (e.g. gamma-delta T-NHL). Within the AML group, it has been previously recognised that CD56 expression may be associated with a more aggressive course and reduced survival compared with CD56 negative cases, and to have a greater propensity for extramedullary involvement at presentation, particularly involving skin and lymph nodes (10). Blastic plasmacytoid dendritic cell neoplasm (BPDCN) Given the CD4+ CD56+ phenotype, the main consideration is that of a BPDCN (reviewed in Ref. 11), which shares very similar clinicopathological features with MLC and which has far greater morphological and immunophenotypic heterogeneity than originally thought (12). Typically the cells of BPDCN are of medium size and the nuclear chromatin has a more blastoid quality but by morphology alone it is not be possible to make a distinction from MLC. Until recently the diagnosis depended on finding the CD4+ CD56+ phenotype (+/- CD45RA, CD68) in the absence of other lineage-specific markers. But none of these is specific for BPDCN and there is considerable overlap with myeloid neoplasms, particularly chronic forms. However, there are now relatively more specific markers for this entity (13) (paraffin-reactive, commercially available) - CD123, TCL1, CD303 (BDCA2), CD2AP, BCL11A, IRF8 - though it should be remembered that none of these has 100% specificity for BPDCN. While the myeloid marker CD33 may be expressed in these cases, myeloperoxidase is always negative, whilst CD7 may be positive in both AML and BPDCN. Some cases of BPDCN may evolve into, or be associated with various myeloid proliferations (11). In up to 20% of CMML cases there are tumoral accumulations of mature PDC and at least in some there are shared identical clonal cytogenetic aberrations. This underscores the relationship between these lineages (11). Lymphoblastic leukemia/lymphoma (LBL) Precursor LBL shows relatively frequent cutaneous involvement though precise data are few. Whilst in most cases there is known underlying LBL, in rare cases, even after thorough staging the skin is the sole site of involvement at presentation (more often in B-LBL than T-LBL) though evolution to frank leukemia typically ensues within weeks to months (14). The morphology can mimic BPDCN and MLC, but immunophenotyping should readily resolve the diagnosis (mixed lineage leukemias nothwithstanding). The precursor markers TdT, CD10, CD34 and CD99 are typically though variably expressed, in addition to B- or T-lineage markers. Note that TdT is not infrequently positive in BPDCN and may also be expressed in AML. Other considerations Primary or secondary cutaneous NHL (mature) should be readily resolved on the basis of the clinical features, morphology and an appropriate immunohistochemistry panel. In particular, NHL cases with high grade "blastoid" features might be confused with leukemia cutis (blastoid forms of mantle cell or follicular lymphoma, Burkitt lymphoma, DLBCL either DV Spagnolo IAP 2011 Lymphoma Club 5

7 conventional or showing features borderline with other aggressive lymphomas). Note that some MLC cases may express CD30 (potential for misdiagnosis of null-alcl). Similarly, various histiocytic/dendritic neoplasms (Langerhans cell histiocytosis, histiocytic sarcoma, indeterminate dendritic cell neoplasm, follicular dendritic cell tumour, interdigitating dendritic cell sarcoma) can be distinguished clinicopathologically and by including in the IHC panel, appropriate discriminating antibodies (S100, CD21/23/35, CD1A, Langerin/CD203, CD163). Exceptionally rare is dot-like keratin positivity which may lead to confusion with metastatic carcinoma or tumours in the "blue round cell" group such as Merkel cell carcinoma. Note that a number of haematolymphoid markers (TdT, CD56, CD99, CD117, CD10, BCL2) may be expressed in a range of malignant small blue cell tumours, hence errors may be made if only limited antibody panels are used in assessing these neoplasms. Suggested IHC panels for undifferentiated cutaneous haematolymphoid infiltrate: CD3, CD20 (include PAX5, CD79A if any prior Rituximab, or if plasmablastic is a consideration) for B- or T-NHL; CD43+, MPO, CD68 (KP1) for myeloid leukemia cutis; TdT, CD117, CD34, CD99 for lymphoblastic; CD4, CD56, CD123, TCL1 for BPDCN; S100, CD21, CD35, CD203/langerin, CD1A for other dendritic lineages; Additional - CD303/BDCA2, BCL11A, CD33, CD163, EBER; Other antibodies directed to identification of non-haematolymphoid neoplasms. SUMMARY The case illustrates the uncommon presentation as a solitary cutaneous nodule of aleukemic MLC (most likely AMML) strongly expressing CD4 and CD56 and thus necessitating distinction from other mimics, particularly BPDCN. Practice points to emphasise are: 1. ~50% of MLC cases are MPO negative, therefore one cannot rely on MPO negativity to exclude MLC; 2. CD43 and CD68 positivity in a lineage-negative infiltrate should suggest the possibility of MLC or BPDCN; 3. CD4 and CD56 are not infrequently expressed in MLC, especially cases showing monocytic differentiation; additional myeloid, PDC and lymphoid markers need to be applied to these cases to discriminate between MLC, BPDCN and NHL; 4. CD68 positivity in BPDCN is infrequent, usually present in scattered cells and typically in a dot-like Golgi pattern (unlike the strong and diffuse cytoplasmic staining in MLC); 5. Discordant immunophenotypes between marrow and cutaneous blasts is common in MLC, and there is frequently relative loss of CD34 and CD117 in the cutaneous infiltrates thus limiting the utility of these markers in the diagnosis of MLC. REFERENCES 1. Bénet C, Gomez A, Aguilar C, et al. Histologic and immunohistologic characterization of skin localization of myeloid disorders. A study of 173 cases. Am J Clin Pathol 2011; 135: Cronin DMP, George TI, Sundram UN. An updated approach to the diagnosis of myeloid leukemia cutis. Am J Clin Pathol 2009; 132: Cibull TL, Thomas AB, O'Malley DP, Billings SD. Myeloid leukemia cutis: a histologic and immunohistochemical review. J Cutan Pathol 2008; 35: Cho-Vega JH, Medeiros J, Prieto VG, Vega F. Leukemia cutis. Am J Clin Pathol 2008; 129: DV Spagnolo IAP 2011 Lymphoma Club 6

8 5. Agis H, Weltermann A, Fonatsch C, et al. A comparative study on demographic, hematological, and cytogenetic findings and prognosis in acute myeloid leukemia with and without leukemia cutis. Ann Hematol 2002; 81: Swerdlow SH, Campo E, Harris NL et al., Eds. WHO Classification of Tumours of Haemopoietic and Lymphoid Tissues. Lyon:IARC Press, 2008; Pileri SA, Ascani S, Cox MC, et al. Myeloid sarcoma: clinicopathologic, phenotypic and cytogenetic analysis of 92 adults. Leukemia 2007; 21: Cerroni L, Gatter K, Kerl H. An illustrated guide to skin lymphoma. 3d Ed. Wiley-Blackwell. 2009; Jones D, Doreman DM, Barnhill RL, Granter SR. Leukemic vasculitis: a feature of leukemia cutis in some patients. Am J Clin Pathol 1997;107: Assaf C, Gellrich S, Whittaker S, et al. CD56-positive haematological neoplasms of the skin: a multicentre study of the Cutaneous Lymphoma Project Group of the European Organisation for Research and Treatment of Cancer. J Clin Pathol 2007; 60: Jegalian AG, Facchetti F, Jaffe ES. Plasmacytoid dendritic cells. Physiologic roles and pathologic states. Adv Anat Pathol 2009; 16: Cota C, Vale E, Viana I, et al. Cutaneous manifestations of blastic plasmacytoid dendritic cell neoplasm morphologic and phenotypic variability in a series of 33 patients. Am J Surg Pathol 2010; 34: Marafioti T, Paterson JC, Reichard KK, et al. Novel markers of normal and neoplastic human plasmacytoid dendritic cells. Blood 2008; 111: Cerroni L, Gatter K, Kerl H. An illustrated guide to skin lymphoma. 3d Ed. Wiley-Blackwell. 2009; DV Spagnolo IAP 2011 Lymphoma Club 7

9 T-cell large granular leukemia and related proliferations Dr Debra Norris IAP Companion Meeting 2011

10 WHO recognises three distinct disorders of large granular lymphocytes: T-cell large granular lymphocytic leukemia (T-LGL) Chronic lymphoproliferative disorders of NK-cells (CLPD-NK provisional entity) Aggressive NK-cell leukemia Despite different cell of origin, overlap between T- LGL and CLPD-NK in presentation and therapy If treatment required, immunosuppressant therapy (low dose MTX or cyclosporin) Interest in agents which target dysregulated apoptotic pathways, characteristic of disease

11 Terminology of LGL T-LGL leukemia: clonal disorder of CD8+ lymphocytes T-LGL disease: reactive condition associated with proliferation of normal CD8+ cytotoxic T-lymphocytes, usu in association with autoimmune disorders (?potential precursor of T-LGL leukemia)? Borderline cases; T-cell clonopathy of undetermined significance Clonal disorders of LGL rare (<3% of small cases of SLL) and 2-5% of PTCL More rarely NK cells (CD3-/CD16+) WHO: chronic lymphoproliferative disorder of NK cells

12 T-LGL : Clinical Av age 60, though reported in all age groups including children; M=F More common in patients with autoimmune disorders, particularly rheumatoid arthritis (25-35% of T-LGL patients will have Rh Arthritis) Medial survival >10yrs Typical presentation: fever, recurrent bacterial infections, fatigue, wt loss; mouth ulcers; 30+% asymptomatic Mild to moderate splenomegaly (20-60%); hepatomegaly (<20%); lymphadenopathy rare Neutropenia often severe, most uniform feature (60-85%) Other rare associations: solid tumors, MGUS, neuropathy, endocrinopathy

13 Pathobiology Thought underlying initiator of T-LGL leukemia may be chronic immune stimulation with exogenous antigens or putative endogenous antigens (as in Rheumatoid arthritis) Malignant transformation via somatic mutation (?retrovirus initiators) T-LGL leukemic cells are resistant to Fas-mediated apoptosis; correlation exists between disease activity and circulating Fas-L levels, with reduction in treated patients Dysregulation of several intracellular signaling pathways, including FAS/FAS-L, P13K-AKT, and extracellular signal-regulated kinase (ERK) implicated in inhibition of apoptosis of leukemic LGLs in vitro

14 LGL Typical clinical presentation Cytopenias Unexplained lymphocytosis Unexplained neutropenia Lymphocytosis associated with autoimmune disorder Peripheral Blood smear Review Increased LGL Diagnosis : establish clonality Abnormal immunophenotype Abnormal cytogenetics (rare) Restricted Vβ expression Abnormal KIR expression Clonal TCR rearrangements

15 Lymphocytosis (typical range 2-20 X10 9 /l) cf normal LGL count in PB 0.1to 0.3 X10 9 Must be sustained for >6mths (unless obviously leukemic) Serologic abnormalities frequent Β2 microglobulin Rheumatoid factor ANA Increase LDH Aniplatelet and antineutrophil antibodies Positive Coombs MGUS Hyper or hypogammaglobulinemia

16 Hematologic disorders associated with LGL Leukemia ITP A-I hemolytic anemia Pure red cell aplasia Aplastic anemia PNH MDS HCL B-cell LPD Myeloma HL

17 Splenectomy Reactive increases in LGL HIV infection, other viral infections Allogeneic stem cell transplantation and solid organ transplantation Persistent clonal expansions of CD8+ T-cell subsets in the elderly Autoimmune disorders BUT : post organ transplantation and autoimmune disorders also described with LGL leukemia

18 PB and Histologic Findings PB characteristic but may not be appreciated if absolute lymphocyte count low Benign vs reactive vs neoplastic LGL not able to be distinguished on morphology Normally make up 5-15% of PB lymphocytes BM: subtle interstitial and intrasinusoidal infiltration Spleen: mild to moderate splenic enlargement; expansion of red pulp with minimal change in white pulp Lymph Node involvement : rare; sinuses and interfollicular area involvement Liver: few samples sinusoidal involvement, singly or in small clusters; portal triads may show infiltrates

19 Immunophenotype Normal LGL : CD8+, CD2+, cytcd3+, CD4-, CD5+, CD7+, CD8+, CD16-, CD56-, αβtcr+, γδtcr- Neoplastic LGL: 80% abnormal expression of 2 or more pan-t cell antigens CD5 and CD7 most common Abnormal expression of CD3 or CD2 seen rarely NK related antigens, CD16 (~80%) and CD57(~near 100%) Majority αβtcr+,controversy regarding inclusion of γδtcr+ cases

20 Molecular Genetics and Cytogenetics Clonal abnormalities described by routine cytogenetics rare only about 20 cases Recent studies: inversion of chromosome 14, inv7, deletion of 6q; +8, +14, t(11;12) no consistent chromosomal abnormality described TCR gene rearrangements

21 Variant forms of LGL Leukemia Rare and unclear whether distinct entities 1. CD4+ T-LGL leukemia 80% normal physical evaluation, rare splenomegaly, somewhat more fequent lymphadenopathy Lack neutropenia and anemia No apparent association with rheumatoid arthritis Association with other malignancies frequent (18% B-cell LPD) 2. CD56+ CD8+ T-LGL? More aggressive behaviour; younger age of onset 3. γδt-lgl cases Indolent disease, direct comparison with αβ T-LGL not been done

22 New Techniques: TCR V beta and Killer Cell Immunoglobulin-like receptors Broad array of antibodies directed against the variable (V) region of the T cell receptor (TCR) beta (V beta) have become available with flow cytometry In T-LGL leukemia, there is a dominant population expressing a single V beta pattern; considered clonal >50% of T cells express 1 V beta subtype and suggestive if 40-49% KIRs: in most cases of T-LGL single uniform intensity expression of KIR is seen

23 Differential Diagnosis T-PLL Hepatosplenic TCL Extranodal NK/T-cell lymphoma, nasal type Aggressive NK-cell leukemia Chronic NK lymphocytosis T-LGL disease Reactive

24 Liver lymphoma general usually secondary and represents advanced disease (30-50%) Primary lymphoma liver defined as extranodal lymphoma with main tumor burden within liver, and without lymphadenopathy or splenomegaly; normal FBC for at least 6 mths 0.016% of all NHL, and 0.4% of all extranodal lymphomas M:F 2:1; adolescents and adults; childhood rare Too rare to establish incidence DLBCL, extranodal MALT, follicular lymphoma Primary TCL more prevalent in Asia than western countries Data on coexisting chronic liver disease contradictory B-cell lymphomas usually exhibit characteristic infiltration patterns T-cell lymphomas more challenging and differentiation from viral infection including EBV more difficult

25 Liver and B-cell lymphomas 4 major infiltration patterns 1. diffuse nodular growth, with predominance of portal infiltrates: DLBCL, Burkitt lymphoma; B-CLL, FL, MZL, MCL 2. either dense sheets of neoplastic cells 3. or scattered neoplastic cells in background of reactive bystander cells: TCRBCL; HL 4. sinusoidal spread: myeloma (usu with accompanying EMH); HCL, ALL, DLBCL

26 T-PLL Adults (median 65yrs); Rapid onset and aggressive clinical course HSmegaly, generalised LN, skin infiltration 20% Lymphocytosis usu>100x10 9 /l Small to medium size lymphoid cells with nongranular basophilic cyt, variable nuclei with visible nucleolus Majority CD4+ or CD4+/CD8+; TCL1+ Cytogenetic abnormalities present in majority inv 14 with breakpoints in q11 and q32 (80%) Median survival <1yr

27 Hepatosplenic TCL Similar sites of involvement (PB, BM, Spleen, liver) More frequent in younger patients (median age 35yrs) presenting with severe thrombocytopenia, anemia, leukocytosis, marked hepatosplenomegaly Peripheral blood involvement uncommon 20% seen in setting of chronic immune suppression (inc post Tx) Immunophenotype: CD3+, CD56+/-, CD4-, CD8-, gamma delta, Cytogenetics : isochromosome 7q More aggressive clinical course; median survival <2yrs

28 Nasal type extranodal T/NK cell lymphoma Predominantly extranodal lymphoma, characterised by vascular damage, necrosis, cytotoxic phenotype and EBV association Sites: upper aerodigestive tract, other preferential sites: skin, soft tissue, GIT, testis. Immunophenotype: scd3-, ccd3ε+, CD56+, CD8-, EBER-ISH expression Cytogenetics: no specific translocation identified Prognosis: variable and improved in recent yrs; unfavourable factors include high stage, high IPI, extranasal location

29 Chronic NK cell lymphocytosis Rare; indolent course Some cases, complete lack of symptoms Cytopenias less common and less severe; systemic features eg Hsmegaly uncommon Increase in PB NK cells (usu >2x10 9 /l) with no identifiable cause scd3-, ccd3ε+, CD16+, weak CD56+, cytotoxic markers +, aberrant co-expression CD5, CD8 Germline TCR Abnormal KIRs expression used as surrogate marker of clonality Karyotype usually normal

30 Aggressive NK-cell leukemia More common in Asian countries and nearly always associated with EBV Geographic distribution reflecting both genetic and environmental factors Almost exclusively young adults M>F Clin: rapidly progressive B symptoms, jaundice, LN, HSmegaly, circulating leukemic cells and cytopenias, +/- DIC, liver dysfunction and multiorgan failure; skin involvement rare Median SR <2mths despite treatment

31 Aggressive NK-cell leukemia CD2-, scd3-, CD3ε+, CD56+, CD57-, CD16+ Germine TCR Clonal integration of EBV Cytogenetic abnormalities: del 6q, del 21q and del 17p

32 LGL and treatment 50% may require no therapy Treatment indicated for symptomatic cytopenias (anemia <9g/dl; neutropenia <0.5 x 10 9 /l; platelets <50 x 10 9 /l); or recurrent infections, massive splenomegaly; systemic features Objective of treatment to alleviate cytopenias rather than eliminate malignant clone Immunosuppressive agents: low dose MTX or cyclosporin Purine analogues (eg fludarabine, cadribine) Monoclonal antibodies (high CD52 expression targeted)

33 1/05/11! Lymphoma Companion Meeting 2011 IAP! Clinical History! Clinical History"! 68 year old Aboriginal man from Central Australia.! Past history of alcohol abuse, diabetes, ischaemic heart disease, Hepatitis B and liver failure.! Daniel Kearney, IMVS / SA Pathology! Clinical History! He presented to a local hospital with bilateral arm and back pain associated with dyspnoea, lethargy, malaise and abdominal pain.! He was found to have widespread bulky lymphadenopathy (mediastinal, axilla, abdominal, pelvic) and hepatosplenomegaly.! Clinical History! Streptococcus viridans was isolated from blood cultures and IV antibiotics were commenced.! He was transferred to Adelaide for diagnostic work up and treatment.! Clinical History! A CBE revealed:! Haemoglobin 77 g/l ( )! Platelets 41 ( )! WCC 44 (4-11)! 98% lymphocytes with malignant features! Clinical History! An axillary lymph node core biopsy and bone marrow biopsy were performed, confirming the presence of a peripheral T- cell lymphoma (CD 2, CD 3, CD 4 and CD 25 +; CD 5, CD 7 and CD 8 -).! 1!

34 1/05/11! Clinical History! Clinical History! HTLV-1 testing revealed a positive result.! Strongyloides serology was negative! Rapidly progressive liver failure prevented chemotherapeutic treatment, and the patient died from fulminant liver failure 17 days after admission.! Blood Film! 2!

35 1/05/11! Core Biopsy! CD 2! CD 4! CD 3! CD 5! CD 8! CD 30! CD 20! MIB-1! Bone Marrow Biopsy! CD 3! 3!

36 1/05/11! Diagnosis! Adult T-cell leukaemia / lymphoma (ATL)! ATL! A cytologically or histologically confirmed peripheral T-cell malignancy associated with Human T-cell lymphotropic virus type 1! Retrovirus that infects 20 million people world wide! First retrovirus to be shown to cause a human cancer! ATL / HTLV-1! HTLV-1! ATL first described in 1977 in Japan, HTLV-1 discovered in 1980's.! Vertically transmitted in neonatal period (breastfeeding), sexually transmitted, and infective via parenteral means! Endemic in Japan, Africa, Caribbean, and South America! Also seen in Melanesia, PNG, Solomon Islands and Australian Aborigines (infection rates of up to 14%)! HTLV-1! Rare in North Americans and Europeans! First case in an Australian aboriginal documented in 1991! 4!

37 1/05/11! HTLV-1! HTLV-1 is only infective when intracellular! The viral regulatory protein Tax has been identified as having oncogenic potential! HTLV-1! Tax influences cells via:! transcriptional deregulation,! activation of signal transduction! deregulation of cell cycle control! induction of genetic instability! HTLV-1! Infection is also a risk factor for complicated Strongyloides stercoralis infection! Also associated with:! HTLV-1 associated myelopathy / tropical spastic paraparesis,! HTLV-1 associated uveitis and infective dermatitis! HTLV-1! The long latency period (4 decades or longer) between infection and malignancy supports a multistep carcinogenesis model! The lifetime risk of developing ATL is approximately 5% for an infected individual! 4 clinical subtypes! smouldering! chronic! lymphoma! acute! ATL! ATL! Clinical findings include:! generalised lymphadenopathy, hepatomegaly, splenomegaly, skin involvement, hypercalcaemia, and organ infiltration! 5!

38 1/05/11! ATL! Symptoms and signs include:! abdominal pain, diarrhoea, ascites, pleural effusion, cough and chest x-ray abnormalities! lytic bone lesions not uncommon! Pathology! The neoplastic cells vary greatly in appearance, ranging from small convoluted cells (flower cells) seen in the peripheral blood, to large blasts with prominent nucleoli! Immunophenotype! CD 4+ T-cells which strongly express IL-2 receptor (CD 25)! Usually express CD 3, +/- CD 2 and CD 5! CD 7 / 8 are usually negative! Immunophenotype! The cell of origin is likely to be a regulatory T-cell, as FoxP3 is commonly positive, which in combination with CD 4 and CD 25 is characteristic! This provides a potential mechanism for the immunodeficiency commonly seen in ATL! 6!

39 1/05/11! Prognosis! The acute and lymphoma subtypes are aggressive diseases that respond poorly to conventional treatment! The 4 year survival rate is 5%! The acute type has a median survival of 6 months! Adult T-cell leukaemia / lymphoma! Tempting to view as an exotic disease unlikely to be encountered! HTLV-1 infection is endemic in some Australian populations, and diseases related to infection will be encountered! 7!

40 Case 4 Submitted by Dr Penny McKelvie, St Vincent s Hospital, Melbourne Diagnosis:EBV-positive diffuse large B-cell lymphoma of the elderly Case history. This 74 year old Croatian lady first presented in December 2009 to an Oncologist with a two month history of worsening nocturnal cough, night sweats and 3kg weight loss. Her medical history included herpes zoster (shingles), right total hip replacement, splenomegaly and thrombocytopenia in 2006 with no cause identified on bone marrow examination. Investigations included CT chest/ abdomen/ pelvis which showed a 5cm mass behind the left heart border, splenomegaly and 3cm lesion in the liver. She did not have a bone marrow performed on this admission due to anxiety issues. PET showed widespread lymphadenopathy in the left lower cervical, subcarinal, hilar and para-aortic lymph nodes. Flow cytometry of the peripheral blood was negative. She was admitted to St Vincent s Hospital in March 2010 for investigation of painless jaundice, 15 kg weight loss and drenching night sweats since December She had recently been treated for E. coli UTI at another hospital. Investigations showed negative hepatitis B and C serology, microcytic anaemia of 95g/L, abnormal liver function tests with elevated ALP (173 U/L), ALT (62U/L), hyperbilirubinaemia of 212 (normal <21μmol/ L). CT showed extensive lymphadenopathy in the chest and abdomen, with many nodes having a necrotic appearance, and low density lesions in the spleen and liver. She underwent core biopsy of liver on 24 th March. She became hypotensive with acute renal failure thought to be secondary to E coli UTI and hypovolaemia. She failed to respond to 40 g IV dexamethasone daily and she became increasingly confused. Her condition was too poor to start chemotherapy and she died on 31 st march, only two weeks after her final admission. DISCUSSION: EBV-positive diffuse large B-cell lymphoma of the elderly was introduced as a new entity in WHO classification in This is defined as an EBV+ clonal B-cell lymphoid proliferation that occurs in patients > 50 years and without any known immunodeficiency or prior lymphoma (Nakamura et al). The first description from the Nagoya group in Japan appeared in 2003 (Oyama et al, 2003) and they have expanded this initial series of 22 cases in subsequent publications (Oyama et al 2007, Asano et al, 2009; Shimoyama et al, 2006, Shimoyama et al, 2008; Shimoyama et al, 2009). One point of interest in view of later publications by other groups from Asia (Wada et al) and the West (Hoeller et al, Castillo et al, Gibson and Hsi, etc) is that the Nagoya group did not indicate a cut-off point for positivity for the EBER ISH reaction in early articles, with the comment that the majority of the large cells were reactive, but adopted >50% in later papers (Oyama et al, 2007; Shimoyama et al, 2008; Shimoyama et al, 2009). These authors estimate that age-related EBV-related diffuse large B-cell lymphoma account for about 8-9% of all DLBCL among eastern Asian patients without underlying immunodeficiency (Shimoyama et al, 2009). 1

41 EBER ISH criteria for positive diagnosis: Wada et al, 2011, using cases from the Osaka Lymphoma Study Group in Japan, in an attempt to explain the discrepancy between the rate of 9% of Epstein-Barr virus positive diffuse large B-cell lymphoma of elderly in East Asia and the very low rate in the West demonstrated that establishment of definite criteria for EBV-positivity (either >20% positively stained tumour cells, or >50% positively stained tumour cells) showed a similar incidence of 3.3% or 1.0% respectively to those reported in Western cases. Examination of the cutoff points for EBER ISH positivity adopted by different groups is interesting. Examination of the table below also highlights that the percentage of EBER+ve diffuse large B-cell lymphomas in the literature has been expressed as a percentage of the total number of DLBCL in immunocompetent patients in some papers, rather than just in the elderly group. In some articles, there is sufficient data to extract the elderly group which I have done for Shimoyama et al, 2008 and Hoeller et al, but there again, there is discrepancy in the definition of age-related from > 40years, >50 years, > 60 years or >60 years. Therefore, these factors of an absence of agreement on definitions of: 1) percentage of EBER ISH positivity in tumour; 2) the actual age group studied; preclude exact comparison of data from different geographical locations. Authors Origin EBER % % cases +ve No of cases Age cutoff Gibson, Hsi, USA All 80 - NA 5 cases in >60y % series Hoeller et al Switzerland >10% = 3.2% of >50y 243 > 50yrs >50y 2010 Italy >20% = 2.9% of >50y 258 in series Moraeles et Peru cases Any signal 18.4% of >60y 54 > 60yrs >60y al, 2010 USA Park et al Korea >20% 14.4% of >60y 139 > 60yrs >60y % of <60y 241 < 60yrs Oyama et al, Japan >50% NA 96 >40yr >40y 2007 (Nagoya) 79 >60yr Shimoyama Japan No 12.1% of >60y 1028 >60yr >60y et al, 2008 (Nagoya) definition 4.7% of <60y 652 < 60yr Wada et al, 2011 Japan (5 centres) >10% >20% >50% 4.3% of all 3.3% of all 1.0% of all 484 but only 89.7% > 50y 74% >60 yr Ages 12-95y Median 68y CLINICAL: The disease was described first in 22 Japanese patients > 60 years, who had no evidence of immunodeficiency (Oyama et al, 2003). Constitutional symptoms of fever, malaise and weight loss were observed in 50% of patients. Although 64% of cases presented with peripheral lymphadenopathy, extranodal involvement, particularly skin and gastrointestinal tract was common, occurring in 82% of patients. 2

42 PATHOLOGY: Two histological patterns were recognized : 1) polymorphous lymphoproliferative disorder comprising a polymorphous infiltrate of large atypical lymphoid cells and background of inflammatory cells; 2) large cell lymphoma, monomorphic type. Necrosis was a striking feature in 77% of cases with angiodestructive or angiocentric infiltrate in over 50%. The polymorphous variant comprised large atypical lymphoid cells, with features of immunoblasts but also more pleomorphic cells and giant cells with Reed- Sternberg-like features were present. The background cells included small lymphocytes, histiocytes, eosinophils and even neutrophils. The monomorphic variant had sheets of large cells like DLBCL, but these large cells were often quite polymorphic. Immunohistochemistry shows that the large cells are reactive for LCA, B-cell markers including CD20, CD79a and PAX-5. Cases with plasmablastic features may be CD20 negative (Gibson & Hsi). CD30 is positive in about 50% but CD15 is negative. About equal numbers of germinal centre versus activated B-cell type phenotype have been identified in those studies using appropriate panels (Hoeller et al, 2010). Background lymphocytes were to be predominantly CD3+, CD4+ T-cells with smaller numbers of CD8+ T-cells (Oyama et al, 2003). Later studies showed that 65% of cases showed over 30% cytotoxic T-cells in the infiltrate (Asano et al, 2009). Ki67 proliferation is usually high (>70%). FISH studies showed gains of BCL-6 in 2/4 cases and ¼ cases in one series (Hoeller et al, 2010). IgH monoclonality was found in 9/12 cases studied (75%) of the first series in 2003 (Oyama et al). Criteria for positive diagnosis of EBER ISH reactivity varies according to series, as in table and discussed above. LMP was found in 29/29 Japanese patients of age-related polymorphous lymphoproliferative disease (Shimoyama et al, 2009). COMPARISON of clinical and pathological features with patients with EBV+ CHL. Japanese authors led by the Nagoya group have studied the difference between age-related EBV-related B-cell lymphoproliferative disorder (aebvlpd) (34 cases) and EBV-positive classical Hodgkin lymphoma (108 cases), in patients aged 50 years and older, publishing the same data in two articles in 2009 (Asano et al, 2009, Shimoyama et al, 2009). The aebvlpd group was more closely associated with aggressive clinical parameters than EBV+ CHL, with higher age at onset (71 vs 63 years); lower male predominance (M:F ratio of 1.4: 3.3) and higher rate of involvement of skin (18% vs 2%), gastrointestinal tract (15% vs 4%) and lung (12% vs 2%). The aebvlpd group histology was characterized by greater incidence of geographic necrosis, greater increase (>30%) of cytotoxic T-cells in background lymphocytes and higher rate of CD20-reactivity (100% vs 19% with >30-50% cells positive). CD15 was negative in aebvlpd but positive in 60% of EBV+ CHL group. The aebvlpd group had a significantly poorer prognosis than EBV+ CHL (Asano et al, 2009). These authors also showed reduced overall survival for the aebvlpd group compared with EBVnegative DLBCL (median survival 24 months versus not reached) (Shimoyama et al, 2009). There has been one transitional case with histology in a 71 year old Japanese patient with the postulated diagnosis of age-related EBV-associated B-cell lymphoproliferative disorder metachronously showing two distinct morphological patterns resembling classical Hodgkin lymphoma (CHL) and DLBCL. Initial biopsy of an axillary lymph node in October 1999 showed features interpreted as CHL. He received six cycles of ABVD with complete 3

43 remission achieved by CT and MRI. In October 2000, one year later, he suffered recurrent lymphadenopathy and a biopsy of cervical soft tissue showed features of DLBCL. He received IMVP-16 chemotherapy with no response but underwent complete remission with IV bleomycin. He subsequently developed acute myeloid leukaemia 2 years later which was treated with DA and CAG regimens, but the leukaemia ultimately became refractory and finally 6 years after the initial diagnosis, he suffered recurrent lymphadenopathy in the right supraclavicular node. Biopsy showed again features resembling CHL (Murase et al, 2009). In this patient the Reed-Sternberg like cells in the CHL like pattern were CD30+, CD15+, Oct2+, BOB.1+, CD20+ (90%), EBER ISH+. These authors suggest that their CHL-like histology was in fact the polymorphous variant of age-related EBV-associated B-cell lymphoproliferative disorder, despite the CD15-positivity that has not been reported in any other series. However, one also has to consider the WHO definition of diagnosis of EBV+ DLBCL of the elderly states that there must be no prior lymphoma. OUTCOME of DLBCL +/- EBV-positivity. Other authors have examined the effect of EBV status on outcome of DLBCL regardless of age (Park et al, Moraeles et al). The Korean Data showed that EBV+ DLBCL (EBER >20%) was significantly associated with age > 60 years, more advanced stage, more than one extranodal site, higher IPI risk group, presence of B-symptoms and poorer outcome to initial therapy (Park et al). The EBER+ group had significantly poorer overall survival (35.8months with median not reached (Park et al). Only 16% of the entire study group (6% of the EBER ISH-positive group and 18% of the EBER ISH-negative cases) received rituximab. Moraeles et al studied 74 patients with nodal DLBCL of all ages (only 73% >60 years) and showed that positive EBV status (any signal on EBER ISH) had a negative impact on survival with a hazard ratio of 3.1. However, none of the patients in this study received rituximab. Yoshino et al showed that patients with EBV+ DLBCL (defined as EBER ISH >50% +ve cells) of the stomach (4/50 or 8%, all of whom were over 60 years) showed resistance to standard chemoradiotherapy (not including rituximab) compared with the EBV-negative group. EBV in non-immunocompromised hosts. A recent international meeting clarified the status of Epstein-Barr virus associated lymphoproliferative disease in non-immunocompromised hosts (Cohen et al, 2009). This classified the types into seven categories, three of B-cells and four of T/NK cells: 1. Chronic active EBV infection (CAEBV) B-cell type 2. EBV+ large B-cell lymphoma of the elderly 3. Lymphomatoid granulomatosis 4. Hydroa vacciniforme (HV) 5. HV-like lymphoma 6. Severe mosquito bite allergy 7. Systemic EBV-positive T-cell lymphoproliferative disorder of childhood. EBV LATENCY. The majority of the world s population is infected by EBV (Cohen et al, 2008). EBV has a unique set of growth activating genes that are used to establish a latent infection of B- lymphocytes. The growth of B-cells latently infected with EBV is normally controlled by the host immune response, particularly virus-specific T-cells. The great majority of people carry latent EBV all their lives without any symptoms, but in certain situations, latent EBV infection is associated with EBV+ malignancies including Burkitt lymphoma, B-cell lymphoproliferative disorders, Hodgkin lymphoma and T-cell lymphomas. 4

44 Four types of EBV latency patterns are recognized classified as 0 III (Thompson & Kurzrock). Type 0 in healthy individuals shows expression of only one of over 100 viral proteins, LMP-2. Latency I shows expression of EBNA-1, BARF0 and the EBERs. Latency I is usually associated with Burkitt lymphoma. Latency II has been associated with Hodgkin lymphoma, peripheral T-cell lymphoma and nasopharyngeal carcinoma and gene expression includes EBNA-1. EBERs. LMP-1, LMP-2A and LMP-2B. The final pattern of gene expression (latency III) occurs mainly in immunocompromised patients with post-transplantand AIDS- associated lymphoproliferative disorders. Latency III usually involves the unrestricted expression of all EBNAs, EBERs and LMPs. Data to date suggest that the EBV+ large cell lymphoma of the elderly is related to latency II. THERAPEUTIC OPTIONS for immunocompetent patients with type II latency EBVassociated lymphomas. Chemotherapy is still first choice for immunocompetent patients with EBV-associated lymphomas. These malignancies are relatively radiosensitive and chemosensitive in early stages, but less so in later stages or following relapse (Cohen et al, 2008). EBV-associated malignancies in immunocompetent patients present a challenge for immunotherapy, as they do not express many of the immunodominant viral antigens. However, T-cells specific for the weakly immunogenic LMP2 protein have been isolated and expanded from these patients. After adoptive transfer, these LMP2-specific CTLs augmented T-cell responses and promoted tumour regression in a subset of patients with Hodgkin lymphoma. In the pre-clinical state, the combination of valproic acid with its histone deacetylating properties and ganciclovir have shown efficacy on depleting EBV-transformed cells with compared with chemotherapy alone (Jones et al). Bortezomib, a proteasome inhibitor approved for treatment of multiple myeloma, blocks NF-kappaB, which has been associated with EBV-associated B-cell transformation. Bortezomib in combination with other chemotherapy may prove of value in therapy of EBV+ lymphomas. A series of mammalian targets of rapamycin inhibitors may also be of value in treating EBV+ DLBCL (Castillo et al, 2011). REFERENCES: 1. Asano N, Yamamoto K, Tamaru J-I et al. Age-related EBV+ B-cell lymphoproliferative disorders: comparison with EBV-positive classic Hodgkin lymphoma in elderly patients. Blood 2009; 113: Castillo JJ, Beltran BE, Miranda RN et al. Epstein-Barr virus-positive diffuse large B-cell lymphoma of the elderly: what we know so far. The Oncologist 2011; 16: Cohen JI, Kimura H, Nakamura S, Ko Y-H, Jaffe ES. Epstein-Barr virus associated lymphoproliferative disease in non-immunocompromised hosts: a status report and summary of an international meeting, 8-9 September, Ann Oncol 2009; 20: Cohen JI, Bollard CM, Khanna R, Pittaluga S. Current understanding of the role of the Epstein-Barr virus (EBV) in lymphomagenesis and therapeutic approaches to EBV-associated lymphomas. Leuk Lymphoma 2008; 49(Suppl 1): Gibson SE, HSI ED. Epstein-Barr virus positive B-cell lymphoma of the elderly at a United States tertiary medical center: an uncommon aggressive lymphoma with a nongerminal centre phenotype. Hum Pathol 2009; 40:

45 6. Hoeller S, Tzankov A, Pileri SA et al. Epstein-Barr virus positive diffuse large B-cell lymphoma in elderly patients in rare in Western populations. Hum Pathol 2010; 41: Jones K, Nourse J, Corbett G et al. Sodium valproate in combination with ganciclovir induces lysis of EBV-infected lymphoma cells without impairing EBV-specific T-cell immunity. Int J Lab Hematol 2010; 32:e169-e Morales D, Beltran B, Hurtado de Mendoza F et al. Epstein-Barr virus as a prognostic factor in de novo nodal diffuse large B-cell lymphoma. Leuk Lymphoma 2010; 51: Murase T, Fujita A, Ueno H et al. A case of age-related EBV+ B-cell lymphoproliferative disorder metachronously showing two distinct morphologic appearances, one of a polymorphic disease resembling classical Hodgkin lymphoma and the other of a large-cell lymphoma. Int J Hematol 2009; 89: Nakamura S, Jaffe ES, Swerdlow S. EBV positive diffuse large B-cell lymphoma of the elderly. In WHO classification of tumours of the haematopoietic and Lymphoid Tisssues. Eds Swerdlow SH, Campo E, Harris NL et al. Lyon P Oyama T, Ichimura K, Suzuki R et al. Senile EBV+ B-cell lymphoproliferative disorders. A clinicopathologic study of 22 patients. Am J Surg Pathol 2003; 27: Oyama T, Yamamoto K, Asano N et al. Age-related EBV-associated B-cell lymphoproliferative disorders constitute a distinct clinicopathologic group: a study of 96 patients. Clin Canc Res 2007; 17: Park S, Lee J, Ko YH et al. The impact of Epstein-Barr virus status on clinical outcome in diffuse large B-cell lymphoma. Blood 2007; 110: Shimoyama Y, Oyama T, Asano N et al. Senile EBV-associated B-cell lymphoproliferative disorders: a mini review. J Clin Exp Hematopathol 2006; 46: Shimoyama Y, Yamamoto K, Asano N et al. Age-related EBV-associated B-cell lymphoproliferative disorders: special references to lymphomas surrounding this newly recognized clinicopathologic disease. Cancer Sci 2008; 99: Shimoyama Y, Asano N, Kojima M et al. Age-related EBV-associated B-cell lymphoproliferative disorders: diagnostic approach to a newly recognized clinical entity. Path Intern 2009; 59: Thompson MP, Kurzrock R. Epstein-Barr Virus and cancer. Clin Care Res 2004; 10: Wada N, Ikeda J, Hori Y et al. Epstein-Barr virus in diffuse large B-cell lymphoma in immunocompetent patients in Japan is as low as in Western countries. J Med Virol 2011; 83: Wong HH, Wang J. Epstein-Barr virus positive diffuse large B-cell lymphoma of the elderly. Leuk Lymphoma 2009;50: Yoshino T, Nakamura S, Matsuno Y et al. Epstein-Barr virus involvement is a predictive factor for the resistance to chemoradiotherapy of gastric diffuse large B- cell lymphoma. Cancer Sci 2006; 97:

46 IAP Lymphoma Club 2011 Case Contributor: Dr Benhur Amanuel, PathWest Laboratory Medicine, Nedlands, WA. CASE NUMBER 5 Case History A 30 year old well and fit male patient presented with right sided lymphadenopathy. Initial FNA suggested a lymphoma/large cell malignancy. Following this, an open biopsy was performed but was non-diagnostic. Recent PET scan showed PET avidity in the suprarenal gland and left external iliac lymph nodes. There is no past medical history of lymphoma or leukaemia and the patient has no B symptoms. An open cervical lymph node biopsy and removal of right level I and II lymph nodes was performed. Pathological features Microscopic features The lymph node architecture is largely effaced though there is a rim of residual reactive lymphoid tissue is seen towards the periphery of the lymph node. There is a diffuse infiltration of the nodal parenchyma by a large cell lymphoma composed of highly pleomorphic cells with round nuclei and conspicuous nucleoli. On low power microscopy, the tumour is pale staining and there is clear cut demarcation between the infiltrate and surrounding residual lymphoid tissue. There is a proliferation of cohesive nests and sheets of cells, in many areas divided by delicate vascular network which appear rimmed by residual lymphoid tissue and aggregates of foamy histiocytes. A close inspection of these cohesive nests and sheets of lymphoid cells shows a sinusoidal distribution of cells that also extend beyond the sinuses to infiltrate the nodal parenchyma. Most of the neoplastic cells have an immunoblastic appearance in that the cytoplasm appears basophilic and the nuclei possess macronucleoli. Scattered binucleated cells, some even resembling Reed-Sternberg cells, are apparent. Unlike conventional anaplastic large cell lymphoma (ALCL), cells with horseshoe-shape nuclei are not prominent and hallmark cells are not identified. Besides, scattered multinucleated tumour cells are discernible and areas of necrosis are visible. Mitotic activity is brisk. Apoptotic bodies as well as collections of pyknotic cells some even within macrophages are observed. Immunohistochemistry: Several B-cell lineage markers i.e., CD20, CD79A and PAX5 are negative on tissue immunophenotyping. There is also lack of CD45 expression but patchy membranous staining for EMA is observed. In addition to this, there is no expression of Pan-T-cell markers including CD3, CD2, CD5 and CD7. Both CD4 and CD8 are also negative in tumour cells, although background non-neoplastic T-cells that express both CD4

47 and CD8 are apparent. Nuclear BCL6 expression is seen in majority of tumour cells and there is also focal labelling for MUM1. The B-cell transcription factors, BOB1 and OCT2 are positively expressed and there is convincing, weak to focally moderate granular cytoplasmic ALK-1 expression with focal membranous condensation. Strong, cytoplasmic kappa expression is seen. There is also focal IgA, diffuse intracytoplasmic IgG and possibly IgD staining in tumour cells. There is no expression of CD57, CD68, CD163, CD30, CD138, HHV8, lambda, CD45RO, OCT4, PLAP, B-HCG, CD117, myogenin, pan-cytokeratin and the melanoma markers S100, HMB-45 and Melan-A. Molecular studies 1. Interphase FISH: The interphase fluorescence in-situ hybridisation (FISH) assay revealed the presence of ALK-1 breakapart in 75 of 200 cells examined (see Fig 1). 2.Capillary Electrophoresis Clonality Assay: DNA was extracted from Formal Fixed Paraffin Embedded (FFPE) tissue and clonality assay performed using BioMed-2 primers. The PCR products were run by capillary electrophoresis (ABI 3100) and electropherograms viewed by gene scan. There was unequivocal immunoglobulin heavy chain gene rearrangement using the FR1 consensus primers. A monoclonal, 355 nucleotide in size, product was identified

48 (Fig 1). There were also monoclonal kappa light chain gene rearrangement using the VK-J consensus primers. Two monoclonal peaks, and nucleotides in size were detected (Fig 2). Polyclonal TCR-gamma and -beta products were also detected (Fig 3). Q11M048A_IGH: Lymph node: R11B470L Tube A Fr nt nt IGH Tube B Fr2A polyclonal nt Tube C Fr3A polyclonal nt 2011pcr052 Fig 1. A monoclonal, 355 nucleotide in size product was detected in Tube A Lymph node: R11B470L 1:100 dilution Tube A N.S nt nt nt nt nt IGK nt Tube B polyclonal nt nt 2011pcr053 Fig 2.Two monoclonal IgK peaks, and nucleotides in size were detected in tube A.

49 Q11M48A_TCRB_G: Lymph node: R11B470L Tube A Insufficient products nt TCRB Tube B polyclonal nt Tube C polyclonal DB nt DB nt TCRG Tube A Tube B polyclonal polyclonal nt nt 2011pcr050_051 Fig 3. Polyclonal products were detected for TCR-gamma and -beta in all assays. 3. RT-PCR for NPM ALK fusion RNA was extracted from FFPE tissue. 15 μm sections of tumour were taken, each using a new, sterilized blade on a microtome cleaned with ethanol. Sections were placed in a sterile 1.5 ml microcentrifuge tube and RNA extracted using the High Pure RNA Extraction Kit (Roche). Reverse transcription cdna synthesis: Approximately 2 μg of RNA was added to a master mix containing 1 x reverse transcriptase (RT) buffer (TaqMan, ABI), 5.5 mm MgCl 2, 500 μm of each dntp, 2.5 μm of random hexamers, 0.4 U/μl of RNase Inhibitor and 1.25 U/μl of Multiscribe Reverse Transcriptase (ABI) for reverse transcription by incubating at 25 o C for 10min followed by 48 o C for 30 min and termination of the reaction at 95 o C for 5min. PCR: The resulting cdna was amplified in a single tube containing a multiplex reaction containing a master mix of NPM ALK -specific primers. The housekeeping gene phosphoglycerokinase (PGK) was amplified in a separate reaction from each sample as a check for amplifiable RNA using appropriate primers. Results:

50 Amplification of cdna from NPM ALK fusion transcripts resulted in the production of an amplicon 178bp in size. A slightly larger (191 bp) product was amplified from PGK transcripts. Whilst all controls showed expected results, no product could be detected for the test case (Lanes 1 and 2) indicating absence of NPM ALK fusion transcript (Fig 4). NPM ALK fusion bp 200 bp 191 bp 100 bp 1. Q11M048A; IAP case 5 2. Q11M048A; IAP case 5 3. Karpas-299 (+ve control) 4. Q09M070D; (-ve control) 5. H 2 0 water control NPM/ALK PGK 6. Q11M048A; IAP case 5 7. Q11M048A; IAP case 5 8. Karpas-299 (+ve control) 9. Q09M070D; (-ve control) 10. H 2 0 water control Fig 4. NPM-ALK fusion transcript could not be detected by RT-PCR (lanes 1 and 2). Amplifiable RNA was obtained as demonstrated by PGK products (lanes 6 and 7). DIAGNOSIS ALK-positive, diffuse large B-cell lymphoma. DISCUSSION The morphological and immunophenotypic features of the tumour are consistent with ALK positive, (CD20 and CD30 negative) diffuse large B-cell lymphoma. FISH revealed the underlying genetic footprint of an ALK gene (2p23) rearrangement t although a fusion partner has not been determined. The case is presented here: 1. To discuss the approach to distinguishing this lymphoma from several haematolymphoid mimics, metastatic nonhaematopoietic malignancies and to elucidate the potential pitfalls in diagnosis. 2. To emphasize the recognition of this rare entity and to distinguish it from other haematopoietic mimics that have overlapping immunohistochemical features (e.g. null or T-cell ALCL, plasmablastic lymphoma and immunoblastic DLBCL). 3. To discuss its relationship to a number of haematopoietic and nonhaematopoietic malignancies which in common share ALK gene abnormalities.

51 How to distinguish ALK+ve DLBCL from other mimics? Despite the absence of pan-b- or T-cell marker expression by the lymphoma cells, there is compelling corroborating evidence that we are dealing with a B-cell lymphoma. The B-cell transcription factor OCT2 and its co-activator BOB1 were expressed, there was strong, cytoplasmic kappa light chain restriction accompanied by diffuse IgG and focal IgA positivity, and monoclonal rearrangements of the IgH and kappa genes, all consistent with B-cell lymphoma. Of key interest in the case therefore was the finding of convincing, weak to focally moderate ALK-1 expression in a granular cytoplasmic pattern with focal membranous condensation, and interphase FISH evidence of ALK-1 rearrangement. The features therefore are those of an ALK-positive diffuse large B-cell lymphoma. ALK positivity in this case raised the possibility of a T-cell or null-cell ALCL. Typically in these one would expect t(2;5) or other variant forms of ALK translocations, leading to either nuclear, or nuclear and cytoplasmic ALK-1 expression in tumour cells. This classic cytogenetic abnormality is characteristic of the vast majority of T-cell or null-cell ALCL, whilst the majority of ALK-positive DLBCL show a variant translocation, i.e. t(2:17), which corresponds to Clathrin-ALK rearrangement (discussed further below). Whilst we have been able to demonstrate chromosomal breakpoint at ALK gene locus (2p23) in our case, we are unable to show the fusion partner by FISH due to lack of appropriate probes for this. However, reverse transcriptase PCR for t(2:5) was negative indicating that the fusion partner is not NPM (nucleophosmin). In addition to this, lack of expression of CD30, CD3, CD2, CD4, TIA-1, granzyme B, and germ line configuration of T-cell receptor gamma and beta genes aid in excluding ALCL of T- or null-cell types. A number of other high grade haematolymphoid malignancies with sinusoidal pattern of growth such as plasmablastic lymphoma, primary effusion lymphoma, monocytic leukaemia, histiocytic sarcoma and even Hodgkin lymphoma can closely mimic ALK+ DLBCL and enter into the differential diagnosis. Other non-haematopoietic malignancies such as metastatic anaplastic carcinoma, malignant germ cell tumours, rhabdomyosarcoma and melanoma also need to be excluded. Some of the salient features of the most important mimics are discussed below. Plasmablastic lymphoma. Plasmablastic lymphoma is a serious consideration in the differential diagnosis in this case as it has considerable immunophenotypic overlap with ALK+ DLBCL. Plasmablastic lymphoma is an aggressive B-cell lymphoma that reveals cytomorphologic and immunophenotypic features reminiscent of plasmablastic plasma cell myeloma. In the WHO classification, the tumour is classified as a variant of DLBCL. Morphologically, plasmablastic lymphoma is manifest by cells with basophilic cytoplasm, paranuclear hof and prominent nucleoli, features that are often seen in ALK+ DLBCL. Presence of bi-or multinucleate tumour cells and cells resembling Reed Sternberg cells as seen in this case are common features in both

52 tumours. With the exception of EBER and ALK-1 expression, both plasmablastic lymphoma and ALK+ DLBCL have overlapping immunophenotypic and morphological features and both are often negative for CD45, CD20, CD79a, PAX-5 and CD30. Moreover, monotypic light chain and EMA expression can be seen in both entities. Similarly, in view of the post germinal centre B-cell derivation of both lymphomas, MUM-1 can be expressed by both and is therefore not a useful discriminator between these two entities. Interestingly enough, both entities often express CD138, indicating that ALK +ve DLBCL may be clonally related to the terminal plasma cell stage of B-cell differentiation. The case presented here can be distinguished from plasmablastic lymphoma on the basis of granular cytoplasmic ALK-1 expression by the tumour cells, demonstration of ALK-1 breakapart by FISH and lack of CD138 expression. Anaplastic Large Cell Lymphoma (ALCL) ALCL was first described by Stein et al (10) in 1985 as a pleomorphic large cell, Ki-1 (CD30/Ber-H2) expressing non-hodgkin lymphoma (NHL). It is characterised by a proliferation of large pleomorphic cells with irregularly shaped ('bean or horse shoe') nuclei and abundant cytoplasm, growing in sheets and often in a sinusoidal distribution, making morphological distinction from ALK+ DLBCL difficult. Typically, ALCL is CD30 positive, shows positivity for cytotoxic associated antigens such as TIA-1, granzyme and perforin and in the majority of cases either nuclear and/or cytoplasmic ALK-1 expression can be demonstrated. Those that show T-cell lineage differentiation label for T-cell associated markers, in particularly CD2, CD5 and CD4, whilst CD3 is often negative. The null-cell type lacks T-cell lineage antigens but otherwise has a similar immunoprofile to classical ALCL. Many of these immunophenotypic features were absent in our case and although there was evidence of ALK breakapart by FISH, unlike the majority of classical ALCL, a t(2;5) translocation could not be identified by our RT-PCR indicating that the fusion partner was not NPM. The association of ALCL with a characteristic t(2;5)(p23;q35) cytogenetic abnormality was first described in 1989 by several investigators (12-14, 19, 28, 22). Prior to this, in 1985, for the first time Kristofferson et al. and Morgan R et al. independently described the t(2;5) cytogenetic abnormalities in large cell non- Hodgkin lymphomas that were described as malignant histiocytosis (16, 17). Kaneko et al in 1989 published in Blood their findings of a t(2;5)(p23;q35) and its variant t(2;5;13)(p23;q35;q12)- in three paediatric patients diagnosed to have peripheral T- cell lymphoma (20). All three patients had undergone complete remission without treatment. A review of the morphological features of the cases showed a large cell Ki-1 (CD30) and EMA positive lymphoma with T-cell immunophenotype akin to what was then known as malignant histiocytosis. It took 5 years to clone the translocation breakpoints and confirm the discovery that the chromosomal rearrangement fuses part of the nucleophosmin (NPM) gene on chromosome 5q35 to a portion of the anaplastic lymphoma kinase (ALK) gene on

53 chromosome 2p23, generating a chimeric mrna molecule and a unique 80-kD NPM- ALK fusion protein (also referred to as p80) (18,19). Nowadays, ALCL is defined by the WHO classification as a subtype of a non- Hodgkin lymphoma with either T-cell or null cell immunophenotype, expression of CD30, cytotoxic granule proteins and ALK-1 protein as well as distinct genetic alterations i.e., t(2;5)(p23;q35), the latter being found in about ~ 70% of cases (11a). It should be noted that ALK protein is not usually expressed in normal lymphoid cells and that NPM is the most frequently implicated fusion partner, accounting for ~70% of classic ALCL. Several variant translocation partners that appear also to activate ALK (51-53) have been described (see Figure 8). Immunoblastic DLBCL This is a morphological variant of DLBCL characterised by large cells with prominent eosinophilic nucleoli and abundant basophilic cytoplasm. Like conventional DLBCL, the tumour cells express B-cell associated antigens. The tumour discussed here did not express B-cell lineage markers and lacked CD45 expression and instead showed granular cytoplasmic ALK-1 expression, thus effectively excluding an immunoblastic DLBCL. Anaplastic carcinoma, melanoma and metastatic germ cell tumours The tumour cells were negative for pancytokeratin, S-100, melan-a and HMB-45 therefore metastatic carcinoma and melanoma can be confidently ruled out and there were no immunophenotypic features that would indicate metastatic high grade sarcoma or leukemic nodal infiltrate, as the cells were negative for a number of mesenchymal markers including myogenin as well as myeloid markers such as myeloperoxidase (MPO). Absence of CD68, S100 and CD163 militate against a histiocytic sarcoma. It is worth mentioning that there was patchy EMA expression by the tumour cells but no high or low molecular weight cytokeratins could be detected. Also of interest is the weak, or at times absent CD45 labelling of tumour cells which, together with EMA expression, as seen in our case here can lead to an erroneous diagnosis of metastatic anaplastic carcinoma, in particular if a limited panel of immnunohistochemical stains were to be performed. This is further compounded by the fact that some cases of ALK+ DLBCL can show expression of cytokeratins. In such instances the use of a comprehensive panel of immunohistochemical stains is necessary to exclude non-haematopoietic malignancies. Germ cell tumours such as metastatic seminoma (plasmacytoid variant) and embryonal carcinoma have been excluded on the basis of absence of cytokeratin, CD117, CD30, OCT-4 and PLAP. IHC PANEL ALK+VE DLBCL ALCL PLASMOBLASTIC LYMPHOMA ANAPLASTIC CARCINOMA MELANOMA IMMUNOBLASTIC DLBCL

54 LCA CD30 + +/- +/- ALK +CYTO +NUC +CYTO CD20 + CD /- +/- CD3 +/- CD4 +/- TIA + GRAN + KERATIN + S100 + EMA + - +/- +/- - - Table 1. Summary of IHC features of main differential diagnosis. ALK+ve Diffuse Large B-cell lymphoma Anaplastic lymphoma kinase-positive large B-cell lymphoma (ALK+ DLBCL) is a rare neoplasm recognised as a separate entity by the latest (2008) WHO classification of hematologic malignancies (11b). So far, less than 40 cases of ALK+ DLBCL have been reported in the English literature (2-5, 59). The lymphoma spans all age groups, ranging from 9-85 years with an average age of 37 years and has a male predominance (M:F ratio, 4:1). Mediastinal and nodal involvement are the most common presentations although extranodal sites can also be involved (55, 56). The lymphoma is seen mostly in adult patients. It has been reported in only < 8% of patients aged <15 years (15) and frequently presents with extensive disease. ALK+ DLBCL lymphoma displays a characteristic immunoblastic/plasmablastic morphology and a distinct immunophenotype i.e., ALK+, CD3, CD20, CD138+, and as described above mimics a number of haematological and non-haematological malignancies. The tumour lacks expression of cytotoxic granule associated proteins and T-cell associated antigens, with the exception of CD4, a marker that is not restricted to T-cells but can also be expressed in monocytes and histiocytes. Granular intracytoplasmic ALK expression is typical of ALK+ve DLBCL. Significant anaplasia and extensive zones of necrosis are commonly seen in such tumours as seen in the case discussed here and the initial differential diagnosis often is broad, ranging from plasmablastic lymphoma, metastatic carcinoma, melanoma to sarcoma. Morphologically, ALK+ DLBCL is characterised by a proliferation of large immunoblast-like cells with prominent nucleoli and extensive sinusoidal distribution of tumour cells akin to lymph node metastasis of anaplastic carcinoma, malignant germ cell tumours and melanoma. In the case illustrated here, the sinusoidal distribution is subtle and this is due to the fact that the infiltrate is bulky and over-runs the sinuses to extend into the nodal parenchyma. The entity was for the first time described in 1997 by Delsol et al. (1) as a distinct type of DLBCL expressing the ALK protein. The group described 7 cases such cases with plasmablastic/immunoblastic morphology, having ALCL like features but lacked CD30. These lymphomas expressed EMA and intracytoplasmic IgA (five cases) as well as intracytoplasmic ALK-1 indicating that they express the 'full-length form of ALK receptor kinase'. The cases lacked lineage associated leukocyte antigens with the exception of CD4 but labelled for antibodies directed against the intracytoplasmic and extracellular regions of the ALK-1 which was the basis for the authors to suggest that these cases express the 'full length' form of this molecule. At

55 the time, this notion was confirmed by Western Blotting in one of the cases tested and also by PCR amplification of mrna encoding full length ALK sequences. Whilst for a while the expression of ALK fusion proteins in lymphoma had been thought to be restricted to ALK-positive ALCL, in 2003 Gascoyne et al. (2) and others independently described the expression of either CLTC ALK or occasionally NPM ALK in a rare form of B-cell NHL (3-6). This subset of B-cell lymphoma was comprised of poor-prognosis tumours, with an inferior clinical outcome following therapy compared with typical ALK-positive ALCL. It is worth noting that although Delsol et al. had shown that these lymphomas expressed the full-length ALK receptor in fact, one of the cases included in the 2003 study from Gascoyne et al. was also included in the original paper by Delsol et al. in 1997 (1). Gascoyne et al revealed the presence of t(2;17) (p23;q23) and CLTC ALK mrna in the neoplastic cells of 2 of 6 cases by FISH and RT-PCR studies including in one of the original Delsol cases from 1997, indicating that the previous hypotheses suggesting that ALK+ DLBCL arises by different mechanisms that leads to over expression of the full-length ALK protein may not be true in most cases. Nevertheless, in 2 of the 6 cases studied by Gascoyne et al full-length ALK transcripts were demonstrated by RT-PCR but the significance of these results remains largely unexplained. Results of the subsequent studies suggest a prominent role for CLTC-ALK fusion in the pathogenesis of ALK+ DLBCL but leave open the role of full-length ALK protein. The absence of nuclear ALK labelling in an ALK+ DLBCL indicates the expression of one of the variant ALK fusions in the tumour cells. ALK fusion proteins not only self-associate, they also hetero-associate with the normal counterpart of their partner proteins; consequently, the localization of ALK fusions in tumour cells is determined largely by the normal subcellular distribution of their partner proteins. CLTC ALK exhibits a granular cytoplasmic staining due to its hetero-association with the normal clathrin protein present in coated vesicles. Occasional cases of ALK+ DLBCL that harbour a t(2;5) or express full-length ALK protein have also been reported in the literature. Onciu et al (5) and Adam et al. (l7) reported a few cases of ALK+ DLBCL associated with t(2;5)(p23; q35) and expressing the NPM-ALK fusion protein as seen in T-cell/null ALCL. These lymphomas at an immunophenotypic level frequently lack expression of CD20 and CD79a, which is unique for B-cell lymphomas. Unlike conventional ALCL, they lack CD30 and apart for CD4, they are often negative for T-cell associated antigens. Some points on Anaplastic Lymphoma Kinase Anaplastic lymphoma kinase (ALK or CD246), a receptor tyrosine kinase in the insulin receptor superfamily of Receptor Tyrosine Kinases (RTKs), was initially identified in constitutively activated oncogenic fusion forms as a result of the cloning of the nucleolar protein nucleophosmin (NPM). ALK is encoded by the ALK gene, a genomic locus found at the chromosomal band 2p23 in the human (18,19) (Fig 5).

56 Fig 5. Schematic diagram of ALK gene. ALK plays considerable role in the development of normal brain and exerts its effects in specific neurons of the central nervous system. ALK protein in adult human tissues was confirmed by anti-alk immunocytochemical studies, in which only rare scattered neural cells, pericytes and endothelial cells in the brain were shown to be immunoreactive (25, 26). ALK knockout mice demonstrate enhanced performance in novel object recognition/location tests, as well as increased struggle time in tail suspension and Porsolt swim tests. Neurochemical analysis of the knockout mice revealed increased basal dopaminergic signaling tone selectively within the prefrontal cortex. Collectively, these data suggest antagonism of ALK as a potential new approach for the therapeutic management of cognitive and/or mood disorders (54). Within the context of these findings, it is also interesting to note that certain polymorphisms within the ALK gene locus have been reported to segregate with the development of schizophrenia in some families (27). ALK rearrangements, chimeric proteins and ligands As described above an association between ALCL and the t(2;5)(p23;q35) chromosomal rearrangement was reported in the late 1980s (12-14). The genes involved in this translocation were identified in 1994 as those encoding NPM at 5q35 and the novel ALK RTK at 2p23 (19). Since then, at least 15 translocations involving the ALK gene have been identified in ALCL (50, 54) and also see Fig 6 below. Subsequent studies have identified ALK fusions in DLBCL, systemic histiocytosis, inflammatory myofibroblastic tumours, oesophageal squamous cell carcinomas and non-small-cell lung carcinomas (51). In addition, at least seven variant translocations involving the ALK gene have been identified in inflammatory myofibroblastic tumour (IMT); see Fig 8, (51). As with other RTKs, ALK possesses an extracellular ligand-binding region, a transmembrane spanning domain and a cytoplasmic kinase catalytic region (Figure 7). ALK shows significant homology to leucocyte tyrosine kinase (LTK), another RTK in the IR superfamily that is, to date, of uncertain normal or pathologic function (25). ALK is a single-chain transmembrane protein of 1620 amino acids (aa) in the human (25) with 1030-aa extracellular domain of human ALK containing several motifs (19).

57 The expression of ALK predominately in the nervous system together with the observation that the distribution of ALK mrna and protein partially overlaps with that reported for members of the neurotrophin RTKs both suggest that ALK could serve as a receptor for a neurotrophic factor(s) (28). Fig 6, Schematic representation of ALK receptor. Recently, the related proteins pleiotrophin (PTN) and midkine (MK) have been reported as being ligands for ALK in mouse and man. PTN, MK and ALK appear to play roles in the regulation of cell survival. PTN and MK themselves were implicated in the regulation of apoptosis some time ago and more recent studies by Bowden et al. suggested that this activity is regulated, at least in part, by ALK (29). One mechanism by which ALK transduces survival signals probably involves PTN stimulated phosphorylation and activation of the serine/threonine kinases, PI3-kinase and protein kinase B (PKB/Akt), which are anti-apoptotic proteins (31). A role for PTN (and MK) in angiogenesis has also been reported and Stoica et al. postulated that ALK may mediate this process, documenting ALK mrna and protein expression in umbilical endothelial cells (31, 32). The kinase domain (KD) of ALK includes a three-tyrosinecontaining motif (tyrosines 1278, 1282 and 1283) within its activation loop. As with other IR superfamily RTKs, these tyrosine residues represent major autophosphorylation sites that regulate the activation loop during the kinase-activation process following phosphorylation of the triplet tyrosines (22, 23, 25). Fusion of the chromosome 5 gene encoding NPM to the chromosome 2 gene encoding ALK generates the chimeric tyrosine kinase, NPM ALK (Fig 8). The entire intracytoplasmic portion of ALK, exclusive of the Trans Membrane portion (TM), is incorporated into the NPM ALK and all other ALK chimeric proteins (Fig 7).

58 Chimeric ALK proteins have only been reported in lymphomas and not in leukaemias or myeloproliferative disorders. Figure 7. (A) ALK receptor tyrosine kinase and the NPM ALK fusion protein created by t(2;5). (B) Representative ALK fusion proteins and chromosomal rearrangements in various tumours; Adopted from Webb et al. So far reported ALK fusions include: NPM ALK, TPM3 ALK RANBP2 ALK, TPM4 ALK, CLTC ALK ALO17 ALK, CARS ALK, MYH9 ALK, SEC31L1 ALK, ATIC-ALK, TFGL-ALK, TFGS-ALK, MSN-ALK, EML4-ALK and TFGXL ALK. ALK fusions identified in ALCL (T/null cell type) are; NPM ALK, TPM3 ALK, TPM4 ALK and CLTC ALK, MSN-ALK, TFGL-ALK, TFGS-ALK, TPM3 ALK, CLTC ALK, ATIC-ALK ALK fusions identified in inflammatory myofibroblastic (IMT) tumour include: o o CARS ALK, CLTC ALK, RANBP2 ALK, SEC31L1 ALK, TPM3 ALK and TPM4 ALK. CARS-ALK, RANBP2 ALK and SEC31L1 ALK occur exclusively in IMT

59 ALK fusions identified in ALK+ve Diffuse Large B-cell lymphoma are: o CLTC ALK 70%, NPM1-ALK (10%) ALK fusions identified in non-small cell lung cancer o EML4-ALK; multiple variants (V1 to V7) have been identified (see Fig 8). Fig 8. Schematic representation of EML4-ALK translocations in non small-cell lung cancer. The role of ALK in non haematopoietic tumours Interest in ALK small-molecule inhibitor development has heightened the role of ALK in a number of human malignancies. More recently, genomic DNA amplification and protein over-expression (34), as well as activating point mutations of ALK and effects of levels of PTN and MK, have been described in neuroblastomas (38, 39, 40, 45). In addition to those cancers for which a causative role for aberrant ALK activity is well established, more circumstantial links implicate the full-length, normal ALK receptor in the genesis of other malignancies including glioblastoma and breast cancer (36), via a mechanism of receptor activation involving autocrine and/or paracrine growth loops with ALK ligands such as PTN and MK. Data suggestive of such a mechanism have been generated in studies of glioblastoma (31, 51) but this has not yet been unequivocally proven for any tumour type. Several tumour types are known to express the full-length ALK-RTK. For example, in cell lines and/or primary specimens representing a variety of tumours including neuroblastomas (37), neuroectodermal tumours (32), glioblastomas (33) and