Flow cytometry immunophenotypic features of pure erythroid leukemia and the distinction from reactive erythroid precursors.

BACKGROUND: The immunophenotype of pure erythroid leukemia (PEL) as determined by flow cytometry immunophenotypic analysis is not well characterized. The immunophenotypic difference between PEL and reactive conditions is under-explored. METHODS: We assessed and compared the immunophenotype of 24 PEL cases and 28 reactive cases containing early erythroid precursors by flow cytometry. RESULTS: The neoplastic erythroid cells in all PEL cases were positive for CD36 and CD71. CD45 was also positive in all cases, but the expression level was often dimmer than granulocytes. CD117 expression ranged from partial to uniform, and CD235a was often only positive in the CD117-dim to negative cells, corresponding to more differentiated subset. PEL cases frequently (87%) showed decreased or negative CD38 expression, contrasting to reactive early erythroid precursors that showed bright CD38 (p < 0.0001). CD7 (25%) and CD13 (29%) aberrant expressions were only observed in PEL but not in the reactive erythroid cells. Normal early erythroid precursors in all reactive bone marrows showed partial expression of CD4; In contrast, aberrant CD4 expression was detected in 71% PEL cases, either uniformly positive (50%) or completely negative (21%). While normal/reactive bone marrows almost always contained a small subset of CD34-positive early erythroid precursors, the neoplastic pronormoblasts in all PEL cases were CD34 negative. Although not increased in number, CD34-positive myeloblasts were frequently detected in PEL and demonstrated an aberrant immunophenotype in 90% PEL cases. CONCLUSIONS: PEL shows a distinctive immunophenotype which can be distinguished from reactive erythroid precursors by flow cytometry immunophenotyping.

Breast T-Lymphoblastic Lymphoma Presenting as a Single Mass in a Young Female with Family History of Breast Cancer.

Breast involvement by lymphoma is rare, constituting ≤0.5% of all breast malignancies, with T-cell lymphomas, comprising 2.5 to 7.5% of all lymphomas involving breast. Several types of T-cell lymphomas have been reported in breast, including anaplastic large-cell lymphoma, breast implant associated anaplastic large cell lymphoma, peripheral T-cell lymphoma not otherwise specified, adult T-cell lymphoma/leukemia, NK/T-cell lymphoma, and T-lymphoblastic lymphoma. Breast involvement by T-lymphoblastic lymphoma is very unusual and when it is observed, it usually occurs as a secondary involvement by known lymphoma.We report the case of a 33-year-old woman with family history of breast cancer who presented with a single right breast mass which was diagnosed as T-lymphoblastic lymphoma. At presentation, the patient was feeling well and did not have any B symptoms or any other signs of lymphoma or leukemia. One month after diagnosis, the patient presented to the emergency room with chest pain and shortness of breath and was found to have a large mediastinal mass with both pleural and pericardial effusions. Subsequent evaluation of peripheral blood smear and bone marrow biopsy showed increased amount of blasts and involvement by T-lymphoblastic lymphoma. The patient was induced with cyclophosphamide, vincristine sulfate, doxorubicin hydrochloride, and dexamethasone chemotherapy. After two-cycles of chemotherapy, a computed tomography of the thorax showed marked interval decrease in size of anterior mediastinal mass, suggestive of positive treatment response.Here, we report the first well documented case of T-lymphoblastic lymphoma presented as a single breast mass without history of B symptoms and perform an extensive English language literature review.

Expression of BCL2 alternative proteins and association with outcome in CLL patients treated with venetoclax.

Venetoclax, a BCL-2 inhibitor, is highly effective for the treatment of patients with chronic lymphocytic leukemia (CLL) and dependence on alternative proteins may result in resistance to BCL-2 inhibition. Patients with CLL treated with venetoclax as monotherapy at MD Anderson Cancer Center between 05/2012 and 01/2016 were included and pretreatment bone marrow was analyzed by immunohistochemistry (IHC) for BCL-W, BCL-XL, BCL2-A1 and MCL-1. Twenty-seven patients were included. BCL-W + and BCL-2A1+ was found in 15% and 7% of the patients, respectively. Both BCL-XL and MCL-1 were negative in all samples. A higher CR and longer PFS rates were observed in patients with BCL-W+ (p = .60, p = .46), BCL-2A1+ (p = .60, p = .29), and either BCL-W + or BCL-2A1+ (p = .33, p = .20), though not statistically significant. Pretreatment IHC expression of BCL-2 alternative proteins does not predict response to venetoclax in CLL, but may be a surrogate for an indolent biology. Sensitive techniques are needed to explore anti-apoptotic pathways.

A proposal for pathologic processing of breast implant capsules in patients with suspected breast implant anaplastic large cell lymphoma.

Breast implant anaplastic large cell lymphoma is an entity recently recognized by the World Health Organization. The tumor arises around textured-surface breast implants and is usually confined to the surrounding fibrous capsule. Currently, there are no recommendations for handling and sampling of capsules from patients with suspected breast implant anaplastic large cell lymphoma without a grossly identifiable tumor. We analyzed complete capsulectomies without distinct gross lesions from patients with breast implant anaplastic large cell lymphoma. The gross appearance of the capsules as well as the presence, extent and depth of tumor cells on the luminal side and number of sections involved by lymphoma were determined by review of routine stains and CD30 immunohistochemistry. We then used a mathematical model that included the extent of tumor cells and number of positive sections to calculate the minimum number of sections required to identify 95% of randomly distributed lesions. We identified 50 patients with breast implant anaplastic large cell lymphoma who had complete capsulectomies. The implants were textured in all 32 (100%) cases with available information. Anaplastic large cell lymphoma was found in 44/50 (88%) capsules; no tumor was found in six (12%) patients who had lymphoma cells only in the effusion. The median number of sections reviewed was 20 (range, 2-240), the median percentage of sections involved by tumor was 6% (range, 0-90%), and the median percentage of sections involved by lymphoma was 10% (range, 0-90%). Invasion deep into or through the capsule was identified in 18/50 (36%) patients. In patients with breast implant anaplastic large cell lymphoma without a grossly identifiable tumor we identified a spectrum of involvement and we propose a protocol for handling, sampling and reporting these cases. The number of sections to exclude the presence of lymphoma with more than 95% certainty was supported by a mathematic rationale.

Chronic lymphocytic leukemia with proliferation centers in bone marrow is associated with younger age at initial presentation, complex karyotype, and TP53 disruption.

The presence of expanded proliferation centers (PCs) in lymph nodes involved by chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma has been associated with adverse clinical outcomes, but the frequency and significance of PCs in bone marrow (BM) remain unclear. The study group included 36 patients with BM involvement by CLL in which PCs were present. We compared this group with 110 randomly selected BM samples involved by CLL without morphologically discernable PCs. Patients with PCs in BM were younger (median age, 53 years [range,18-71 years] versus 58 years [range, 31-82 years]; P = .007), more frequently experienced B symptoms (27.8% versus 8.2%, P = .0076), more often had Rai stage IV disease (30.6% versus 17.3%, P = .02) and higher serum lactate dehydrogenase (P = .0037) and ß2-microglobulin (P = .0001) levels, and lower hemoglobin (P = .026) and platelet counts (P = .0422). TP53 alterations were more common in patients with PCs in BM (45.4% versus 18.7%; P = .0049), as was a complex karyotype (26.4% versus 9%; P = .019). There were no significant differences in the frequency of ZAP70 or CD38 positivity or IGHV mutation status. The median time to first treatment was shorter in patients with PCs in BM (7 months versus 19 months, P = .047), and the frequency of Richter syndrome was higher (14% versus 4%, P = .041). Patients with PCs in BM had significantly shorter overall survival compared with the control group (median, 249.3 months versus undefined; P = .0241). These data suggest that identification of PCs in BM samples involved by CLL is associated with adverse prognostic features.

Myeloid neoplasms with t(16;21)(q24;q22)/RUNX1-RUNX1T3 mimics acute myeloid leukemia with RUNX1-RUNX1T1.

Chromosome translocation t(16;21)(q24;q22)/RUNX1-RUNX1T3 is an infrequent but recurrent chromosomal abnormality identified in myeloid neoplasms, with only 25 cases have been reported to date. Here, we report eight cases (six women and two men) of myeloid neoplasms associated with t(16;21)(q24;q22): five with therapy-related myeloid neoplasms, two with relapsed acute myeloid leukemia (AML), and one with blast phase of chronic myeloid leukemia. Morphologic and immunophenotypic features include granulocytic dysplasia, blasts with prominent perinuclear hof, large orange-pink granules, long and slim Auer rods, and aberrant expression of CD19. Six patients received AML-based regimens, and five achieved complete remission after initial induction therapy. Our study suggests that myeloid neoplasm with t(16;21)/RUNX1-RUNX1T1 resembles AML with t(8;21)(q22;q22)/RUNX1-RUNX1T1, in regard to morphology, immunophenotype, and response to therapy. Therefore, the clinical management of AML with t(8;21) may provide the best model for patients with myeloid neoplasms with t(16;21).

CD200 expression in mantle cell lymphoma identifies a unique subgroup of patients with frequent IGHV mutations, absence of SOX11 expression, and an indolent clinical course.

CD200, a marker currently utilized in the diagnosis of B-cell lymphoma, is uniformly positive in chronic lymphocytic leukemia/chronic lymphocytic leukemia, and is usually absent in mantle cell lymphoma. Over a 6 year-period, of 668 mantle cell lymphoma assessed by flow cytometry, CD200 expression was detected in 25 patients (~4%). All 25 patients had bone marrow involvement; however, 11 (44%) patients had no nodal or extranodal disease and belonged to non-nodal leukemic variant mantle cell lymphoma. Morphologically, bone marrow showed an unusual interstitial infiltrative pattern in 14/25 (56%) and small round cells resembling chronic lymphocytic leukemia in 9/25 (36%). CD23 was positive in 19/25 (76%) patients; and SOX11 was only positive in 5/21(24%). All 4 patients tested showed IGHV mutations. With a median follow-up of 23 months, 12/24 (50%) patients were not treated. These clinicopathological features were significantly different from 154 randomly chosen CD200-negative mantle cell lymphoma patients, in SOX11 positivity (24% versus 74%, P<0.0001), CD23 expression (76% versus 8%, P<0.0001), a non-nodal leukemic presentation (44% versus 2%, P<0.001), and therapy requirement (50% versus 92%, P<0.0001). This is the first study to show that CD200 expression in mantle cell lymphoma, though uncommon, identifies a subgroup of mantle cell lymphoma patients with characteristic pathological features, frequent non-nodal leukemic variant, and an indolent clinical course.

Correlation between FDG/PET, histology, characteristics, and survival in 332 patients with chronic lymphoid leukemia.

Richter syndrome (RS) is associated with poor outcome. The prognosis of patients with histologically aggressive chronic lymphocytic leukemia (CLL), or HAC, has not been studied. We aimed to correlate 2-deoxy-2-[(18)F]fluoroglucose/positron emission tomography (FDG/PET) data, histological diagnosis, clinical characteristics, and survival in patients with CLL. A total of 332 patients with CLL were histologically classified as: 95 RS, 117 HAC, and 120 histologically indolent CLL (HIC). HAC and RS patients had higher maximum standardized uptake value (SUVmax), more frequent constitutional symptoms, poorer performance status (PS), lower hemoglobin and platelets, and higher lactate dehydrogenase and ß-2-microglobulin. An SUVmax ≥10 strongly correlated with mortality (overall survival [OS], 56.7 vs 6.9 months in patients with SUVmax <10 vs ≥10). Survival of patients with RS and HAC was similar among patients with SUVmax <10 or ≥10. SUVmax ≥10, PS ≥2, bulky disease, and age ≥65 were independently associated with shorter OS. In patients undergoing both fine-needle aspiration and biopsy, the former proved diagnostically inadequate in 23%, 29%, and 53% of HIC, HAC, and RS, respectively. FDG/PET is a useful diagnostic tool in patients with CLL and suspected transformation. Patients with HAC show different characteristics and worse prognosis compared with those with HIC. Patients with different CLL phases, but similar SUVmax have similar outcome. Tissue biopsy should be preferred for diagnosing RS.

Expression of serine 194-phosphorylated Fas-associated death domain protein correlates with proliferation in B-cell non-Hodgkin lymphomas.

Fas-associated death domain protein is a key component of the extrinsic apoptotic pathway. In addition, in animal models, Fas-associated death domain protein phosphorylation at serine 194 has been shown to affect cell proliferation, especially in T lymphocytes. The importance of Fas-associated death domain protein phosphorylation at serine 194 for the proliferation of B lymphocytes, however, is uncertain. Here we show in reactive lymph nodes that serine 194 phosphorylated Fas-associated death domain protein is expressed predominantly in the dark (proliferative) zone of germinal centers. In B-cell non-Hodgkin lymphoma cell lines, serine 194 phosphorylated Fas-associated death domain protein levels are substantially higher in highly proliferating cells and lower in serum-starved cells. We also used immunohistochemical analysis to assess Fas-associated death domain protein phosphorylation at serine 194 expression in 122 B-cell non-Hodgkin-type lymphomas. The mean percentage of serine 194 phosphorylated Fas-associated death domain protein positive tumor cells was 81% in Burkitt lymphoma, 41% in diffuse large B-cell lymphoma, 18% in follicular lymphoma, 18% in plasma cell myeloma, 12% in extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue, 11% in mantle cell lymphoma, and 2% in chronic lymphocytic leukemia/small lymphocytic lymphoma (P < .0001, Kruskal-Wallis test). Furthermore, in chronic lymphocytic leukemia/small lymphocytic lymphoma, serine 194 phosphorylated Fas-associated death domain protein was detected predominantly in proliferation centers. In the entire study group, the percentage of cells positive for serine 194 phosphorylated Fas-associated death domain protein correlated significantly with the proliferation index Ki-67 (Spearman R = 0.9, P < .0001). These data provide evidence that serine 194 phosphorylated Fas-associated death domain protein is involved in the proliferation of normal and neoplastic B cells and has features of a novel proliferation marker.

Expression and effects of inhibition of type I insulin-like growth factor receptor tyrosine kinase in mantle cell lymphoma.

BACKGROUND: Type I insulin-like growth factor receptor (IGF-IR) tyrosine kinase induces significant oncogenic effects. Strategies to block IGF-IR signaling are being tested in clinical trials that include patients with aggressive solid malignancies. Mantle cell lymphoma is a B-cell neoplasm with poor prognosis and a tendency to develop resistance. The expression and potential significance of IGF-IR in mantle cell lymphoma are not known. DESIGN AND METHODS: We used reverse transcriptase polymerase chain reaction, quantitative real-time polymerase chain reaction, immunoprecipitation, western blotting, flow cytometry, and immunohistochemistry to analyze the expression of IGF-IR mRNA, and IGF-IR and pIGF-IR proteins in mantle cell lymphoma cell lines and patients' specimens. Selective and specific blockade of IGF-IR was achieved using picropodophyllin and short-interfering RNA, respectively. Cell viability, apoptosis, cell cycle, cellular morphology, cell proliferation, and target proteins were then analyzed. RESULTS: We detected the expression of IGF-IR and pIGF-IR in mantle cell lymphoma cell lines. Notably, IGF-IR molecules/cell were markedly increased in mantle cell lymphoma cell lines compared with human B-lymphocytes. IGF-IR and pIGF-IR were also detected in 78% and 74%, respectively, of 23 primary mantle cell lymphoma specimens. Treatment of serum-deprived mantle cell lymphoma cell lines with IGF-I salvaged these cells from apoptosis. Selective inhibition of IGF-IR by picropodophyllin decreased the viability and proliferation of mantle cell lymphoma cell lines, and induced apoptosis and cell cycle arrest. Selective inhibition of IGF-IR was associated with caspase-3, caspase-8, caspase-9, and PARP cleavage, cytochrome c release, up-regulation of cyclin B1, and down-regulation of cyclin D1, pCdc2, pIRS-1, pAkt, and pJnk. Similar results were obtained by using IGF-IR short-interfering RNA. In addition, picropodophyllin decreased the viability and proliferation of primary mantle cell lymphoma cells that expressed IGF-IR. CONCLUSIONS: IGF-IR is up-regulated and frequently activated in mantle cell lymphoma. Our data suggest that IGF-IR could be a molecular target for the treatment of mantle cell lymphoma.

mTOR signaling is activated by FLT3 kinase and promotes survival of FLT3-mutated acute myeloid leukemia cells.

Activating mutations of the FLT3 gene mediate leukemogenesis, at least in part, through activation of PI3K/AKT. The mammalian target of rapamycin (mTOR)-Raptor signaling pathway is known to act downstream of AKT. Here we show that the mTOR effectors, 4EBP1, p70S6K and rpS6, are highly activated in cultured and primary FLT3-mutated acute myeloid leukemia (AML) cells. Introduction of FLT3-ITD expressing constitutively activated FLT3 kinase further activates mTOR and its downstream effectors in BaF3 cells. We also found that mTOR signaling contributes to tumor cell survival, as demonstrated by pharmacologic inhibition of PI3K/AKT/mTOR, or total silencing of the mTOR gene. Furthermore, inhibition of FLT3 kinase results in downregulation of mTOR signaling associated with decreased survival of FLT3-mutated AML cells. These findings suggest that mTOR signaling operates downstream of activated FLT3 kinase thus contributing to tumor cell survival, and may represent a promising therapeutic target for AML patients with mutated-FLT3.

Immunohistochemical detection of ZAP70 in chronic lymphocytic leukemia predicts immunoglobulin heavy chain gene mutation status and time to progression.

Zeta-associated protein-70 (ZAP70) expression measured by flow cytometry has been proposed as a surrogate marker of the somatic mutation status of the immunoglobulin heavy chain variable region (IGHV) genes in chronic lymphocytic leukemia. However, attempts to implement this approach in clinical flow cytometry laboratories have been problematic; many commercially available antibodies give unreliable results. Assessment of ZAP70 protein expression by immunohistochemistry in chronic lymphocytic leukemia tissue sections is an easy, alternative approach, although lack of quantitation and subjective interpretation of results are potential pitfalls. In this study, we correlated ZAP70 protein expression, assessed by immunohistochemistry, with ZAP70 messenger RNA (mRNA) transcript expression, assessed by semi-quantitative real-time reverse transcriptase-polymerase chain reaction assay, with the somatic mutation status of the IGHV genes in previously untreated patients with chronic lymphocytic leukemia. Expression of ZAP70 protein and mRNA transcripts correlated strongly (P=8.238 × 10(-12)). Expression of ZAP70 protein and mRNA transcripts also correlated strongly with the somatic mutation status of the IGHV genes (P=0.000071 and P=0.00076, respectively). Further, ZAP70 positivity by immunohistochemistry was associated with an increased risk of progression to therapy requirement (3-year risk 83% vs 31% for ZAP70 negative by immunohistochemistry, P=0.03). These results show that ZAP70 expression assessed by immunohistochemistry is a reliable surrogate marker of the somatic mutation status of the IGHV genes, and predicts time to progression.

p53 expression by immunohistochemistry is an important determinant of survival in patients with chronic lymphocytic leukemia receiving frontline chemo-immunotherapy.

Although chromosome 17p abnormalities and TP53 mutations have been reported as poor prognostic indicators in chronic lymphocytic leukemia (CLL), the impact of aberrant p53 expression as assessed by immunohistochemistry (p53-IHC) has not been defined in patients with CLL treated with chemoimmunotherapy, particularly in the context of other novel markers such as ZAP-70 expression and IgVH mutation status (IgVH MS). We assessed p53-IHC in 222 bone marrow (BM) specimens from patients with CLL enrolled in a phase II trial with fludarabine, cyclophosphamide, and rituximab (FCR). ZAP70 expression and IgVH MS were assessed in 208 and 108 patients, respectively. One hundred sixty-eight patients had concurrent classical cytogenetic analysis. p53-IHC correlated with abnormal karyotype (p = 0.002) and adversely affected overall survival independent of ZAP70 expression and IgVH MS (p < 0.001). Patients with p53-IHC(+) CLL were less likely to achieve complete remission, but patients who did achieve complete remission showed a durable response. In this patient cohort, p53-IHC is an important determinant of complete remission and overall survival, but not remission duration, in patients with CLL receiving FCR.

Whole-genome scanning by array comparative genomic hybridization as a clinical tool for risk assessment in chronic lymphocytic leukemia.

Array-based comparative genomic hybridization (array CGH) provides a powerful method for simultaneous genome-wide scanning and prognostic marker assessment in chronic lymphocytic leukemia (CLL). In the current study, commercially available bacterial artificial chromosome and oligonucleotide array CGH platforms were used to identify chromosomal alterations of prognostic significance in 174 CLL cases. Tumor genomes were initially analyzed by bacterial artificial chromosome array CGH followed by confirmation and breakpoint mapping using oligonucleotide arrays. Genomic changes involving loci currently interrogated by fluorescence in situ hybridization (FISH) panels were detected in 155 cases (89%) at expected frequencies: 13q14 loss (47%), trisomy 12 (13%), 11q loss (11%), 6q loss (7.5%), and 17p loss (4.6%). Genomic instability was the second most commonly identified alteration of prognostic significance with three or more alterations involving loci not interrogated by FISH panels identified in 37 CLL cases (21%). A subset of 48 CLL cases analyzed by six-probe FISH panels (288 total hybridizations) was concordant with array CGH results for 275 hybridizations (95.5%); 13 hybridizations (4.5%) were discordant because of clonal populations that comprised less than 30% of the sample. Array CGH is a powerful, cost-effective tool for genome-wide risk assessment in the clinical evaluation of CLL.

Bone marrow involvement by marginal zone B-cell lymphomas of different types.

We compared the morphologic findings of different types of marginal zone B-cell lymphoma (MZL) involving the bone marrow (BM), including 18 splenic (SMZL), 6 extranodal (mucosa-associated lymphoid tissue lymphoma), and 6 nodal cases. The median percentage of BM involvement was 15%, and multiple overlapping patterns of infiltration were observed in all MZL types. The most frequent patterns were nodular (87%) and interstitial (63%). A focal sinusoidal pattern of involvement was found in one third of SMZLs and rarely in MALT lymphoma. Germinal centers (GCs) were uncommon in routinely stained BM biopsy sections and were observed only in SMZL. However, antibodies specific for CD21 and CD23 highlighted follicular dendritic cells in most MZLs of all types. MZLs cannot be distinguished from each other by examining BM sections alone. However, a sinusoidal pattern or presence of GCs is suggestive of SMZL. Furthermore, correlation with the CBC count can further enhance the reliability of diagnosing SMZL.

Lenalidomide induces complete and partial remissions in patients with relapsed and refractory chronic lymphocytic leukemia.

This study investigated the activity of lenalidomide in patients with relapsed/refractory chronic lymphocytic leukemia (CLL). Lenalidomide was given at 10 mg daily with dose escalation up to 25 mg daily. Three patients (7%) achieved a complete response (CR), one a nodular partial remission, and 10 patients a partial remission (PR), for an overall response (OR) rate of 32%. Treatment with lenalidomide was associated with an OR rate of 31% in patients with 11q or 17p deletion, of 24% in patients with unmutated V(H), and of 25% in patients with fludarabine-refractory disease. The most common toxicity was myelosuppression, and the median daily dose of lenalidomide tolerated was 10 mg. Plasma levels of angiogenic factors, inflammatory cytokines, and cytokine receptors were measured at baseline, day 7, and day 28. There was a dramatic increase in median interleukin (IL)-6, IL-10, IL-2, and tumor necrosis factor receptor-1 levels on day 7, whereas no changes were observed in median vascular endothelial growth factor levels (20 patients studied). According to our experience, lenalidomide given as a continuous treatment has antitumor activity in heavily pretreated patients with CLL.

Fine-needle aspiration biopsy findings in patients with small lymphocytic lymphoma transformed to hodgkin lymphoma.

Although small lymphocytic lymphoma (SLL) is an indolent lymphoma, approximately 5% of cases can transform to a higher-grade lymphoma, rarely Hodgkin lymphoma (HL). We report the fine-needle aspiration (FNA) results of 6 cases of SLL/chronic lymphocytic leukemia (CLL) that transformed to HL. FNA findings were correlated with the histologic features and clinical follow-up. The patients included 5 men and 1 woman, ranging in age from 49 to 72 years at the time of SLL/CLL diagnosis with time for development of HL ranging from 0 to 95 months (mean, 49.3 months). The FNA diagnoses were SLL with HL transformation (2 cases), SLL with large atypical cells (1 case), and atypical lymphoid proliferation with large atypical cells (3 cases). Flow cytometry performed in 5 cases (2 FNA specimens) demonstrated a monoclonal B-cell population with CD19/CD5 coexpression. The presence of large atypical mononucleated and binucleated cells in lymph node FNA specimens from patients with SLL/CLL with progressive adenopathy should raise the possibility of transformation to HL. In these cases, histologic confirmation is always recommended, not only to differentiate HL transformation from other entities but also for subclassification of HL.