Search

Background: Hairy cell leukemia-variant (HCL-v) is one type of chronic lymphocytic proliferative disorders which was classified into splenic B-cell lymphoma/leukemia, unclassifiable. Both clinical and laboratory characteristics and treatment strategy remain elusive due to the rarity of the disease. Here, we firstly presented the diseases features and efficacy of a variety of treatment options in a large cohort from China. Methods: Thirty-three patients were diagnosed with HCL-v in Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Science & Peking Union Medical College from June 1990 to February 2021. We analyzed the disease characteristics and treatment outcome, especially in terms of clinical manifestation, immunophenotypic and molecular evaluation and efficacy of multiple first-line treatment. Results: The median age of this cohort was 59 years (range, 34-79 years) at diagnosis, with 23 males and 10 females. Abdominal mass and relative signs (n=22) and abnormal complete blood count (n=9) were the most common chief complaints. Splenomegaly was present in 32 (97.0%) cases, among whom 26 (78.8%) cases were massive. Leukocytosis and leukopenia were presented in 23 (69.7%) patients and 5 (15.2%) patients, with a median white blood cell count of 21.58×10 9/L (range, 1.34-224.59×10 9/L). The median percentage of the leukemic cells in bone marrow tested by flow cytometry was 33% (range, 6.2%-96%), and immunophenotyping showed monoclonal B-cells positive for pan B-cell antigens (CD19, CD20, CD22, CD79b) in almost all patients (Table). CD11c was positive in all patients, and CD103 was positive in 20/26 (76.9%) patients. CD25 was negative in 30/33 (90.9%) patients, and CD23 was negative in 20/26 (76.9%) cases. CD200 was frequently expressed among patients (15/21, 71.4%). For the pathological pattern of bone marrow involvement, 20/27 (74.1%) patients showed a predominantly interstitial pattern. Moreover, Annexin A1 was negative in all (n=12) patients detected by immunohistochemistry. Conventional cytogenetic analysis showed abnormal karyotype in 10/24 (41.7%) patients, including 6 patients with complex karyotype. Fluorescence in situ hybridization analysis showed deletion of TP53 in 4/20 (20.0%) patients and IGH translocation in 5/16 (31.3%) patients. The BRAF V600E mutation was negative in all the patients (n=20). In 2 of 14 (16.7%) patients, TP53 mutation was detected by next-generation sequencing. Sixteen of nineteen (84.2%) patients showed monoclonal IGHV rearrangements, and the most common rearrangement fragment was VH4-34 (n=3, 18.8%). Six of seventeen (35.3%) patients presented with unmutated IGHV and patients with VH4-34 were all unmutated. 31 patients needed treatment finally. Treatment choices included interferon-α (IFN-α) in 11 patients, chlorambucil (CLB) in 5 patients, single purine nucleoside analogs (PNA) in 3 patients and PNA plus rituximab in 9 patients. Four patients who received IFN-α or CLB treatment also underwent splenectomy. The objective response rate was 100% for patients receiving PNA plus rituximab and 77% for cases receiving others. And people treated with PNA plus rituximab had a higher complete response rate compared to the other regimen (75% versus 12%, P = 0.004). During a median follow-up of 32 months (range, 3-207) for 29 patients, 14 (48.3%) patients experienced at least one relapse or progression, and 7 (24.1%) patients had 2 or more relapses. Of note, one case also underwent a diffuse large B-cell lymphoma transformation. The median PFS and OS were 31 months (95% CI 25.5-36.5) and 70 months (95% CI 50.5-89.5), respectively. PNA plus rituximab prolong PFS compared to the others (3-year PFS rate 80% [95%CI 20-97] versus 10% [95%CI 1-35], P = 0.012, Figure A). But no significant difference in 3-year OS rate was observed between two groups (100% [95%CI 100-100] versus 43% [95%CI 10-73], P = 0.128, Figure B). Conclusion: HCL-v is a rare disease with specific clinical and immunophenotypic features but may overlap with classic hairy cell leukemia or other splenic B-cell neoplasms. Overall, it is an indolent lymphoma with recurrent progression, and the use of PNA plus rituximab in first-line treatment can result in a deeper and longer remission. Figure 1 Figure 1. Disclosures Wang: AbbVie: Consultancy; Astellas Pharma, Inc.: Research Funding.

Background Hairy cell leukemia (HCL) variant (HCLv) is considered a separate, more aggressive entity compared to classic HCL. HCLv responds poorly to single-agent purine analog with complete response (CR) rates below 10% and overall response rates under 50%. Rituximab combined with purine-analog can improve response rate and duration, but long-term data have not been reported for HCLv, particularly regarding minimal residual disease (MRD). We therefore update the results of a phase II trial with cladribine and concurrent rituximab in patients with HCLv, previously reported for 10 of the 20 patients enrolled. Methods Patients with HCLv with 0 to 1 prior courses of cladribine, and/or 0-1 prior courses of rituximab, received cladribine (0.15 mg/kg days 1-5), with 8 weekly doses of rituximab (375 mg/m2) beginning day 1. The primary endpoint was to determine CR rate and secondary endpoints included evaluating minimal residual disease (MRD) by blood and bone marrow aspirate flow cytometry, and bone marrow biopsy immunohistochemistry. Patients were able to receive a 2nd course of rituximab ≥ 6 months after the first, if and when MRD was detected in blood. Results Twenty patients were enrolled. Median age was 67 (range: 42-86) years. No patients had prior concurrent cladribine-rituximab. Eight were previously untreated, 1 had only splenectomy, 6 had prior cladribine, 1 had prior cladribine and splenectomy, 1 had prior rituximab, 1 had prior rituximab and splenectomy, 1 had cladribine, rituximab, and splenectomy, and 1 had combination rituximab-containing chemotherapy followed by cladribine. Out of 20 patients receiving concurrent cladribine-rituximab (CDAR), the CR rate was 95% (95% CI: 75-100%). This CR rate was superior to a historical control group of 3 of 39 HCLv patients who achieved CR to cladribine alone (p Conclusion Concurrent cladribine with rituximab is highly effective in HCLv irrespective of prior purine-analog treatment or IGHV4-34 status and should replace purine analog monotherapy as treatment. Patients with long-term MRD-free CR are being followed to determine whether concurrent cladribine-rituximab as 1st-3rd line systemic therapy can permanently eradicate HCLv. Patients who progress have limited OS. This provides a rationale for the testing of higher intensity approaches up front and the identification of additional treatment options for HCLv. Disclosures Kreitman: Genentech: Research Funding. OffLabel Disclosure: Rituximab for hairy cell leukemia

Introduction Splenic B-cell lymphoma/leukemia, unclassifiable is defined as being unable to be classified as any other B-cell neoplasm infiltrating the spleen (World Health Organization Classification of Tumors of Haematopoietic and Lymphoid Tissue 2017). In splenic B-cell lymphoma/leukemia, unclassifiable, splenic diffuse red pulp small cell lymphoma (SDRPL) and hairy cell leukemia variant (HCL-v) are provisionally defined. The incidence of these diseases is rare. It has been reported that they account for 9% of splenic B-cell lymphomas (Haematologica 2010;95:1122-1129). There are no reports of histopathological examinations or BRAF mutations (V600E) in Japan. It has been shown that BRAF mutations (V600E) are found in all patients with Hairy cell leukemia-classical (HCL-c) (N Engl J Med. 2011 364: 2305-2315). This study aimed to clarify the pathological features of splenic B-cell lymphoma/leukemia, unclassifiable. Methods We analyzed 5 cases of suspected splenic B-cell lymphoma/leukemia, unclassifiable in terms of splenectomy. Five cases include 0 cases of SDRPL, 2 cases of HCL-v, and 3 cases of splenic B-cell lymphoma/leukemia, unclassifiable not applicable to either case. We analyzed patients undergoing splenectomy who were pathologically diagnosed at the Kurume University Pathology Course between July 2012 and May 2017. Splenic B-cell lymphoma/leukemia, unclassifiable was suspected in 5 cases. From the findings of peripheral blood, bone marrow, and BRAF mutation (V600E) analyses, 2 patients were diagnosed with HCL-v. The other 3 patients were diagnosed with splenic B-cell lymphoma/leukemia, unclassifiable. Pathological examinations were performed in these cases. Marker expression analysis of the B-cell line was performed using immunohistochemical (IHC) staining and flow cytometry (FCM). IHC staining was performed on formalin-fixed paraffin-embedded samples. For FCM analysis, we confirmed the clonality of B cells and analyzed their expression. A positive judgment was made at 30% or greater. For BRAF mutation (V600E) analysis, deoxyribonucleic acid was extracted from formalin-fixed paraffin-embedded samples and performed using the Sanger sequencing method. Results The results of the analysis are shown in Table 1. HCL-v cases included 1 man and 1 woman, and the age was 62-71 years old. Splenic B-cell lymphoma/leukemia, unclassifiable cases included 2 men and 1 woman, and the age was 56-66 years. Splenic B-cell lymphoma/leukemia, unclassifiable cases involved bone marrow infiltration of B-cell lymphoma in 2 cases. In 3 cases of splenic B-cell lymphoma/leukemia, unclassifiable, there were no cases in which hairy cells were recognized in the peripheral blood. In all cases of splenic B-cell lymphoma/leukemia, unclassifiable, BRAF mutations (V600E) were not observed. All cases of splenic B-cell lymphoma/leukemia, unclassifiable and HCL-v tested positive for cluster of differentiation (CD)20, CD19, and B-cell lymphoma (Bcl-2) and tested negative for CD103 and CD10. All HCL-v cases and 2 of 3 cases of splenic B-cell lymphoma/leukemia, unclassifiable tested positive for cyclin D3. One case of splenic B-cell lymphoma/leukemia, unclassifiable tested positive for CD5, but negative for lymphoid enhancer-binding factor 1 (LEF1), cyclin D1, and Sox11. All other cases tested negative for CD5, LEF1, cyclin D1, and Sox11. One of two cases of HCL-v and 2 of 3 cases of splenic B-cell lymphoma/leukemia, unclassifiable tested positive for CD11c. All HCL-v cases tested negative and 1 of 3 cases of splenic B-cell lymphoma/leukemia, unclassifiable tested positive for Bcl-6. IHC staining was performed because FCM was not performed in 1 case of HCL-v. There was no obvious bias in κ and λ. One patient with HCL-v who underwent FCM was κ-positive. Splenic B-cell lymphoma/leukemia, unclassifiable was κ-positive in all cases. Conclusions In this analysis, splenic B-cell lymphoma/leukemia, unclassifiable did not involve BRAF mutations (V600E) in all cases. It has been reported that cyclin D3 is highly expressed in SDRPL. Splenic B-cell lymphoma/leukemia, unclassifiable and HCL-v confirmed the same tendency (Blood. 2017; 129:1042-1045). Further molecular biological analysis for splenic B-cell lymphoma/leukemia, unclassifiable is desired. Disclosures No relevant conflicts of interest to declare.

Introduction: Hairy cell leukemia-variant (HCL-V) is an uncommon disorder, accounting for approximately 0.4% of chronic lymphoid malignancies and 10% of all HCL cases, without sexual predominance. Here is an attempt to submit a rare case presentation of HCL-V. Case:A 66 year old female presented to the clinic after she was referred from her primary care for further evaluation of pancytopenia. Past medical history was significant for diabetes mellitus and hypertension. Splenomegaly was noted on examination. Initial bloodwork showed a leukocyte count of 4.9, hemoglobin level of 11.8, platelet count of 78. LDH was 166. CT scan showed significant splenomegaly along with retroperitoneal adenopathy. Peripheral blood flow cytometry demonstrated a CD11c positive monoclonal B cell population suggestive of Hairy Cell Leukemia variant, positive for CD19, CD20, CD22, CD45, CD52, and kappa (moderate-bright), negative for CD5, CD10, CD23, CD38, CD103, CD123, BRAF V600. Left shift myeloid elements were noted but no significant myeloid blasts were noticed. She was diagnosed HCL-V with 17p deletion and 90% marrow involvement. She was treated with 8 doses Rituximab. After 1 year, she relapsed and was started on Cladribine and Rituximab weekly for 8 doses. She relapsed 1 year after Cladribine and Rituximab with splenomegaly and cytopenias, and she was started on Ibrutinib. 4 months after treatment with Ibrutinib, her cytopenias improved with normalization of spleen size. She is tolerating off label medication Ibrutinib well and currently asymptomatic. Discussion: This case represents an extremely rare hematological malignancy a distinct case of HCL-v due to 17p deletion and refractory to multiple therapies. Patients with multiply relapsed HCL-v are also appropriate candidates for moxetumomab pasudotox because HCL-v retains a high level of expression of CD22. Hairy cell leukemia variant is a chronic lymphoproliferative disorder whose characteristics resemble the classic form and splenic marginal zone lymphoma, making differential diagnosis essential for therapeutic follow-up, as hairy cell leukemia variant has a decreased response to drugs prescribed for the classic form. Immunophenotyping is a key tool for diagnosis, as some markers are different between these neoplasms. Therefore, immunophenotyping, clinical, morphological, and immunohistochemical manifestations must be considered in the differential diagnosis. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Hairy cell leukemia (HCL) is an indolent B-cell neoplasm, strongly expressing CD20. Despite initial very high response rates following cladribine, many patients (pts) ultimately relapse. Having relapsed after prior treatment with cladribine, 24 HCL pts (21 male, 3 female) with a median age of 53.5 years were treated with rituximab at 375 mg/m2 intravenously weekly for 4 weeks. Of the pts, 3 (13%) achieved complete remissions and 3 (13%), partial responses. Thus, 6 (25%) of 24 pts achieved a response following rituximab. At a median follow-up of 14.6 months, 2 responders have relapsed; median time to relapse was not yet reached. The only grade III or IV toxicities demonstrated were culture-negative febrile neutropenia, transient and reversible disseminated intravascular coagulation related to rituximab administration, and a diverticular abscess, each in single patients. Of 18 nonresponders, 9 pts subsequently received other treatments; 5 pts were retreated with cladribine, 3 underwent splenectomy, and 1 received pentostatin. Follow-up data are available on 7 of these 9 patients; all 7 patients achieved improvements in hematologic parameters. Rituximab, administered at this dose and schedule, has only modest single-agent activity in cladribine-failed HCL patients when compared with other agents active in this disease.

Recently, the BRAF V600E mutation was reported in all cases of hairy cell leukemia (HCL) but not in other peripheral B-cell neoplasms. We wished to confirm these results and assess BRAF status in well-characterized cases of HCL associated with poor prognosis, including the immunophenotypically defined HCL variant (HCLv) and HCL expressing the IGHV4-34 immunoglobulin rearrangement. Fifty-three classic HCL (HCLc) and 16 HCLv cases were analyzed for BRAF, including 5 HCLc and 8 HCLv expressing IGHV4-34. BRAF was mutated in 42 (79%) HCLc, but wild-type in 11 (21%) HCLc and 16 (100%) HCLv. All 13 IGHV4-34+ HCLs were wild-type. IGHV gene usage in the 11 HCLc BRAF wild-type cases included 5 IGHV4-34, 5 other, and 1 unknown. Our results suggest that HCLv and IGHV4-34+ HCLs have a different pathogenesis than HCLc and that a significant minority of other HCLc are also wild-type for BRAF V600.

Hairy cell leukemia is a chronic B-cell disorder that follows an indolent, but progressive course. Cladribine (2-chlorodeoxyadenosine) induces complete remissions in the majority of patients after a single course. We report the long-term outcomes, including response rates and their duration; time-to-treatment failure (TTF) rates; retreatment results; toxicities; and survival rates of patients treated at Scripps Clinic (La Jolla, CA). A total of 358 patients with hairy cell leukemia were treated with cladribine at 0.087 or 0.1 mg/kg body weight per day by continuous intravenous infusion for 7 days. The expected number of second neoplasms was based on the National Cancer Institute’s Surveillance Epidemiology and End Results data. Of 349 evaluable patients, 319 (91%) achieved an initial complete response and 22 (7%) a partial response with an overall median duration of response follow-up of 52 months. Ninety patients (26%) had relapsed at a median of 29 months. The TTF rate for all 341 responders was 19% at 48 months, 16% for complete responders, and 54% for partial responders. Of 53 evaluable patients treated with second courses of cladribine at first relapse, 33 (62%) achieved complete responses and 14 (26%) partial responses. Twenty-seven patients (8%) developed second neoplasms (only 1 hematopoietic) with an observed-to-expected ratio of 1.88 (95% confidence interval, 1.24 to 2.74). The overall survival rate was 96% at 48 months. Single courses of cladribine induced long-lasting complete responses in the vast majority of patients. Relapse rates for complete responders were low. Patients who relapse can be successfully retreated with cladribine. Cladribine has high efficacy and a favorable acute and long-term toxicity profile when administered to patients with hairy cell leukemia. © 1998 by The American Society of Hematology.

[Figure][1] A 59-year-old female was diagnosed 13 years earlier with JAK2 617-positive polycythemia vera (PV) and treated with phlebotomies. Recently she developed progressive splenomegaly and decreased needs for phlebotomy. A diagnosis of post–PV-myelofibrosis was considered, but not

Hairy Cell Leukemia variant (HCL-V) is a rare disorder of small, mature, B-cell lymphocytes, and is characterized clinically by splenomegaly, an elevated white blood cell (WBC) count, and a hypercellular bone marrow. It accounts for 0.4% of chronic lymphoid and 10% of all cases of hairy cell leukemia (HCL) cases, respectively, and is now considered by the World Health Organization (WHO) to be a distinct provisional entity, biologically unrelated to classical HCL (HCL-C). The median survival is 9 years, with 42% of patients dying of unrelated causes. Transformation to large cell lymphoma is seen in 6% of patients. Whereas HCL-C patients are typically middle-aged males, who manifest lymphocytosis, splenomegaly, and cytopenias, with malignant lymphocytes that express CD103 and CD25, the median age of HCL-V patients is 71 years with no gender preference, and whose cells are characterized by expression of CD103, but lacking CD25. HCL-V also has a more aggressive clinical course, with far lower response rates to purine nucleoside analogs, but better responses to anti-CD20 and anti-CD22 immunotherapies. The BRAF-V600E mutation is considered the molecular hallmark of HCL-C, while 16% have recurrent inactivating mutations of the cell cycle inhibitor CDKN1B, making it the second most common mutation in HCL-C. In contrast, while del17p13 and mutations of the TP53 and MAP2K1 genes are frequent in HCL-V, there are no defining chromosomal or genetic abnormalities, and little is known about the genetic underpinnings of this rare condition. We evaluated a 50 year-old African-American male who was referred to us for an incidental asymptomatic lymphocytosis of 11,990/uL. Flow cytometry showed these monoclonal B lymphocytes to express CD103, CD11c (bright), CD20, CD22, and CD52, but they did not express CD5, CD10, or CD25, with a chronic lymphocytic leukemia (CLL) immunophenotypic (Matutes) score of 0, consistent with HCL-V. A CCND/IgVH translocation was seen in 5% of these cells, and the IgVH gene was mutated at a rate of 7.2%. The patient had a normal hemoglobin and platelet count, and a computed tomography (CT) scan with contrast showed no adenopathy or splenomegaly. Foundation One' Heme Next Generation Sequencing (NGS) was performed on this patient's peripheral blood lymphocytes, and revealed several variants of unknown significance (VUS) including ALK P1599H, BRD4 Q969_L9, CARD11 D764G, CBL L43_S44, CHEK2 D438Y, FANCC Q465R, FANCL S82R, FOX03 P138L, GNAS T398M, GSK3B R396Q, HDAC7 T161M, MET V919I, PIM1 E142D, SOX2 T232A, SYK Y348C, TRAF2 R372H, and YY1AP1 S47P. Notably, no abnormalities in the BRAF, TP53, MAP2K1, and CCND family of genes were observed. A search of multiple literature and genetic databases for the gene sequence variants listed above was performed to assess for their known correlations to lymphoproliferative malignancies, especially HCL-V and HCL-C. These databases included PubMed (http://www.ncbi.nlm.nih.gov/pubmed), Genetics Home Reference (https://ghr.nlm.nih.gov/) Cancer Genetics Web (http://www.cancer-genetics.org/), and Gene Cards (http://www.genecards.org/). Mutations or translocations in the genes ALK, CARD11, HDAC7, MET, leukemia, Burkitt's lymphoma and Hodgkin's disease leukemia, Burkitt's lymphoma and Hodgkin's disease leukemia, Burkitt's lymphoma and Hodgkin's diseasePIM1, SOX2, and SYK, were found to have clear associations with various lymphoid malignancies, including large B cell lymphoma (CARD11, PIM1), Burkitt lymphoma (HDAC7, MET), anaplastic large cell lymphoma (ALK, SOX2), Hodgkin lymphoma (MET), mantle cell lymphoma (SOX2), pro-B cell ALL (HDAC7), and rare peripheral T cell lymphomas (SYK). Alterations in CARD11 have also been associated with congenital B cell lymphocytosis. However, none of the specific variants/mutations identified by NGS of our patient's HCL-V cells have previously been described to be associated with either HCL-V or HCL-C. These results may thus help to elucidate the molecular pathogenesis of this rare hematologic malignancy, and emphasize the need to apply this technology to a greater number of HCL-V cases to assess for recurring genetic variations that may distinguish this unique disorder. Disclosures No relevant conflicts of interest to declare.

Hairy cell leukemia (HCL) is a B-cell chronic lymphoproliferative disorder in which the pathologic cells show a strong expression of CD25 (interleukin-2 [IL-2] receptor alpha chain or p55). “Variant” cases of HCL, characterized by hyperleukocytosis, neoplastic elements with a prominent nucleolus and a higher nucleo/cytoplasmic ratio, and an easily obtained bone marrow aspirate, lack surface CD25 determinants. Limited information is available on the expression of the IL-2 receptor beta chain (p75) on normal and neoplastic B cells. In this study, we have assessed by immunofluorescence and mRNA analysis the presence of the IL-2 receptor alpha and beta chains on 12 cases of classic HCL, as well as on 3 variant cases. The results obtained show that, while the alpha chain of the IL-2 receptor is present only on classic HCL, the IL- 2 receptor beta chain (p75) is expressed on both the classic and variant form. Unlike hairy cells, only 8 of the 15 B-cell chronic lymphocytic leukemia cases tested showed a weak expression of the p75 antigen on a small proportion of cells. Purified B lymphocytes from normal healthy controls, as well as Epstein-Barr virus-transformed lymphoblastoid cell lines, showed a weak staining for the p75 determinant, while being CD25-. The results of this study suggest that the expression of the alpha and beta chains of the IL-2 receptor appears to be upregulated or downregulated during the process of B-cell- lineage activation and differentiation.

Hairy cell leukemia variant (HCLv) presents with high disease burden, lack of typical antigens like CD25, and poor response to standard treatments like cladribine. Occasionally, patients with classic HCL respond poorly. Clinical and molecular features of HCL and HCLv has not been compared. Rearrangements expressing immunoglobulin VH chain were sequenced, including 22 from 20 patients with HCLv and 63 from 62 patients with classic HCL. Most patients were seeking relapsed/refractory trials, representing a poor-prognosis population. VH4-34, a gene commonly used in autoimmune disorders, was observed in 8 (40%) HCLv and 6 (10%) classic (P = .004) HCL patients. Compared with 71 VH4-34− rearrangements, 14 VH4-34+ rearrangements were more frequently (P < .001) unmutated, defined as greater than 98% homologous to germline sequence. VH4-34+ patients had greater white blood cell counts at diagnosis (P = .002), lower response rate (P < .001) and progression-free survival (P = .007) after initial cladribine, and shorter overall survival from diagnosis (P < .001). Response and survival were more closely related to VH4-34 status than to whether or not patients had HCLv. VH4-34+ HCL is an important disorder that only partly overlaps with the previously described HCLv. Response to initial single-agent cladribine therapy is suboptimal; these patients should be considered for alternative approaches, including antibody-related therapy.

We describe the clinical and laboratory features of 17 adult patients with a variant form of hairy cell leukemia (HCL-V) studied over the last 7 years. The main findings were: splenomegaly, moderate anemia, thrombocytopenia, and a raised white blood cell count (median 116 x 10(9)/L; range 15 to 482). The circulating lymphoid cells had abundant villous cytoplasm and a round, occasionally bilobed nucleus, with a prominent nucleolus. Monocytopenia, a feature of typical HCL, was not seen; neither was tartrate-resistant acid phosphatase demonstrated in eight cases tested. HCL-V cells had a mature B-cell phenotype: CD19+, CD20+, CD22+, FMC7+, CD11c+, CD10-, CD5-, with light chain isotope restriction in 15 cases. In contrast to typical hairy cells, HCL-V cells were negative with the monoclonal antibodies anti-HC2 and anti- TAC (CD25). Immunoglobulin (Ig) was not detected in two cases and IgG was expressed in the cell membrane of 73% of cases. Bone marrow histology was different from HCL, showing interstitial infiltration by cells clumped together and a moderate amount of reticulin, but the spleen showed the typical red pulp expansion of HCL. HCL-V patients did not respond to splenectomy (5 of 7) or alpha-interferon (7 of 7); 2 of 3 patients had a partial response to 2′deoxycoformycin. The clinical course was benign with 15 patients alive with a median survival greater than 4 years. We confirm that HCL-V is a distinct clinico-pathologic entity with intermediate features between HCL and B-prolymphocytic leukemia.

The presence of circulating villous lymphocytes (VLs) in lymphoma patients usually points to splenic marginal zone B-cell lymphoma (SMZL), even if the VLs can be found occasionally in other small B-cell lymphomas. However, those cells are variably described, and detailed cytologic characterization is often lacking. We identified lymphoma cases with numerous basophilic VLs among the large group of splenic lymphoma with VLs, and for further delineation, 37 cases with this particular cytology were analyzed. Patients, predominantly older men, presented with moderate lymphocytosis and splenomegaly without pancytopenia. The monoclonal B cells expressed IgM + D, IgM + G, IgM or IgG, as well as CD76 and CD11c, frequently CD103, and rarely CD123. Spleen sections were peculiar, with atrophic white pulp and a monomorphic diffuse lymphoma infiltration in a congested red pulp. Bone marrow infiltration was interstitial and intrasinusoidal without extensive fibrosis. Cytogenetic analysis showed a frequent absence of clonal aberrations (68%). Most cases (79%) were IgH mutated, with an overrepresentation of VH3 and VH4 gene families. These results, as well as the clinical evolution, show that those lymphoma cases represent a homogeneous group distinct from SMZL and reminiscent of hairy cell leukemia variant, perhaps corresponding to a separate lymphoma entity.

Background: EZH2, amember of the polycomb protein group, is an important methyltransferase that is over-expressed in various carcinomas, some B and T cell lymphomas, as well as myeloid disorders. We investigated EZH2 expression in the range of low and high grade B cell neoplasms and correlated its expression with that of p-ERK, MYC, and p-STAT3, potential regulators of EZH2 expression, in high grade B cell lymphomas. Methods: Immunohistochemical staining (IHC) for EZH2 was performed on a total of 162 low and high grade B cell non-Hodgkin lymphomas, using formalin fixed, paraffin-embedded tissue [Table 1]. We subsequently performed IHC for p-ERK, p-STAT3, and MYC on high grade B cell lymphomas. Cases were scored for percentage positivity of neoplastic cells using the above antibodies. Table 1. EZH2 expression in low grade and high grade B cell lymphomas Lymphoma type Cases (POS/Total) EZH2 positive % low grade MM 10/12 10 LPL 9/12 5-10 CLL/SLL 15/18 10--15 MCL 8/9 30 MZL 20/21 15-20 FL(1-2) 8/10 20 HCL 13/14 5-10 HCL-V 9/10 30-40 High grade DLBCL 29/33 90 BL 19/19 100 CLL-Richter 5/5 80-90 FL-3 6/6 80 DHL 22/22 90-100 PMLBCL 19/19 60-80 B-ALL 11/11 95-100 Abbreviations: MM - multiple myeloma; LPL - lymphoplasmacytic lymphoma; CLL/SLL - chronic lymphocytic leukemia/small lymphocytic lymphoma; MCL - mantle cell lymphoma; MZL - marginal zone lymphoma; FL(1-2) - follicular lymphoma grade 1-2/3; HCL - hairy cell leukemia; HCL-V - hairy cell leukemia-variant; DLBCL - diffuse large B cell lymphoma; BL - Burkitt lymphoma; CLL-Richter transformation of CLL; FL -3 - follicular lymphoma grade 3/3; DHL - double hit lymphoma; PMLBCL - primary mediastinal large B cell lymphoma; B-ALL - B lymphoblastic leukemia/lymphoma. Results: In low grade lymphomas, 5-40% of neoplastic cells were positive for EZH2, with variable intensity of staining. Of note, there was a significant difference in EZH2 expression in hairy cell leukemia versus hairy cell leukemia-variant (p Conclusion: EZH2 expression correlates with tumor grade in B cell neoplasms, and the high level of EZH2 expression in high grade B cell lymphomas suggests that this molecule may function as an oncogenic protein in these neoplasms. Furthermore, our findings show there are different signaling cascades in the regulation of EZH2 expression in different types of high grade B cell lymphomas. The p-ERK signaling cascade, but not MYC expression, plays an important role in high EZH2 expression in DLBCL; while in BL and DHL, high MYC expression, but not p-ERK expression, is associated with increased EZH2 expression, possibly through miRNA regulation. These findings suggested that EZH2 and specific disease-related signaling cascades may serve as therapeutic targets for the treatment of high grade B cell lymphomas. Disclosures No relevant conflicts of interest to declare.

Leukemic B-cell chronic lymphoproliferative disorders (B-CLPD) encompass a heterogeneous group of defined disease entities. However, around 10% of the cases cannot be reliably classified based on the current criteria and are considered B-CLPD not otherwise specified (B-CLPD NOS). Few recurrent mutations and genetic alterations have been reported in some entities, but none is specific. We performed gene expression profiling (GEP) to develop a robust GEP-molecular classifier for leukemic B-CLPD. We analyzed purified blood of 189 B-CLPD, including 54 chronic lymphocytic leukemia (CLL), 54 mantle cell lymphoma (MCL), 12 follicular lymphoma (FL), 23 splenic marginal zone lymphoma (SMZL), 4 splenic diffuse red pulp lymphoma (SDRPL), 4 hairy cell leukemia (HCL), 4 HCL-variant (HCL-v), 6 lymphoplasmacytic lymphoma (LPL) and 28 B-CLPD NOS. We used a multiple step approach to build a GEP-array classifier and then analyzed an additional series as a validation cohort by quantitative PCR (qPCR). Mutational analysis of BRAF, MAP2K1, MYD88, NOTCH1, NOTCH2, SF3B1 and TP53 and copy number alterations were studied. In the training set, a supervised analysis clustering revealed that each B-CLPD entity has a specific expression profile. By a multi-step GEP classifier using 43 genes ( Table 1 ) we could classify CLL, SOX11-positive MCL (MCLc), HCL, FL, SOX11-negative MCL (MCLi) and HCL-v cases in six successive stages. However, we were not able to clearly identify distinct signatures for LPL, SMZL and SDRPL. Furthermore, we could classify 36% of B-CLPD NOS cases. Interestingly, the 43-gene signature identified in leukemic samples could also classify the 28 tumor splenic biopsies. Finally, we built a simple 8-gene predictive model using qPCR data ( including: FMOD, KSR2, SOX11, MYOF, MME, CCND1, CXCR4, and CAMSAP2 ) that was used in an independent validation cohort and classified 14% B-CLPD NOS cases. The classification yield increased to 61% and 50%, for GEP-array and qPCR, respectively, when additional morphological, molecular and genetic features were considered. Our findings support the use in a routine base of a simple test as a diagnostic tool that can be applied to help multiparameter interpretation in the classification of leukemic B-CLPD and specially B-CLPD NOS. | Step model | B-CLPD | Specific signatures | | ---------- | ----------------------------------- | ------------------------------------------------------------------------------------------------------------------ | | 1 | CLL | FMOD, KSR2 , ADTRP, CLNK, LEF1, FILIP1L, CTLA4, IGSF3, EBF1 | | 2 | MCLc | SOX11 , PLEKHG4B, HDGFRP3, CNN3, PON2, SH3BP4, FCGBP, STMN1, FARP1, DBN1, NREP, NINL, MARCKSL1, MEX3D, CRIM1, KAZN | | 3 | HCL | HPGDS, IL1R2, TJP1, PLOD2, EMP1, NOVA1 | | 4 | FL | MME , SLC2A5, SMAD1, PRDM15 | | 5 | MCLi | CCND1 | | 6 | HCL-v | TUSC1, LRP1B, KCNJ3, NRCAM, MS4A14, FAM129C, CXCR4 | | 7 | Miscellaneous LPL-SDRPL-SMZL | No specific genes | Table 1. Gene signatures (43 genes) and steps used for the GEP-array model. CLL: chronic lymphocytic leukemia; FL: follicular lymphoma, HCL: hairy cell leukemia; HCL-v: hairy cell leukemia variant; LPL, lymphoplasmacytic lymphoma; MCLc: conventional SOX11-positive mantle cell lymphoma; MCLi: indolent SOX11-negative mantle cell lymphoma; SDRPL, splenic diffuse red pulp lymphoma; SMZL, splenic marginal zone lymphoma. Bold letters indicate some of the genes included in the simple qPCR model. Disclosures Lopez-Guillermo: Roche, Celgene, Mundipharma, Gilead, Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding.

The purpose of this study was to investigate the efficacy and safety of the monoclonal antibody, rituximab, in relapsed or refractory hairy cell leukemia (HCL). Fifteen patients with relapsed or primary refractory HCL after nucleoside analogs received rituximab 375 mg/m2 weekly for a total of 8 planned doses. An additional 4 doses could be administered to responders who had not achieved complete response (CR). The overall response rate was 80%. Eight patients (53%) achieved CR, 2 (13%) attained CR by hematologic parameters with residual marrow disease (1% to 5% marrow hairy cells), and 2 (13%) had a partial response. Of the 12 responders followed for a median of 32 months (range, 8 to 45+ months), 5 patients (42%) had progression of disease 8, 12, 18, 23, and 39 months from the start of therapy. Three patients failed to respond (after 4, 6, or 8 doses). Reductions in serum interleukin-2 receptor (sIL-2R) levels correlated with response. Toxicity was minimal, and no infectious episodes were observed. Rituximab has significant activity and minimal toxicity in HCL and warrants further study. Rituximab should be explored further in HCL with regard to eradication of minimal residual disease and in combination with nucleoside analogs.

Abstract 2679 Background: The BRAF V600E mutation has recently been discovered in nearly all cases of hairy cell leukemia (HCL), but not in cases of HCL-variant (HCL-v). However, this perfect correlation has been challenged by studies reporting HCL cases without BRAF V600E. Interestingly, the immunoglobulin heavy chain variable region gene IGHV4–34, which has been associated with poor prognosis in HCL, appeared exclusively and to a high percentage in these BRAF V600E-negative cases of classic HCL and also in HCL-v (Xi et al., Blood, 2011). Further, splenic marginal zone lymphoma (SMZL) is a disease closely related to HCL and HCL-v and BRAF has been shown to be unmutated in this entity. Aims: 1. To characterize our cohorts of HCL, HCL-v and SMZL for the presence of BRAF V600E and to correlate the results with IGHV gene usage. 2. We hypothesized that other genes of the RAF/RAS pathway might be affected. Thus we analysed NRAS, and KRAS in addition to BRAF for mutations in all three entities. Methods: We analyzed the bone marrow or peripheral blood of 314 cases (182 cases with HCL, 49 cases with HCL-v, and 83 cases with SMZL) at diagnosis as confirmed by multiparameter flow cytometry and cytomorphology. The BRAF V600E mutation was analyzed by an mRNA-based reverse transcription allele-specific real-time quantification (RQ-PCR) assay. The BRAF V600E expression was calculated as %BRAF V600E/BRAF wt. NRAS and KRAS were analyzed by melting curve analysis and subsequent Sanger sequencing. IGHV genes and mutation status were analyzed by the use of Biomed-2 primers. An identity of ≥98% of the analyzed IGHV sequence compared to published germline sequences was considered an unmutated IGHV status. Results: In our cohort the median percent leukemic cells was 16% (range 0.2–74%) for HCL, 33% (range 5–59%) for HCL-v and 29% (range: 1–84%) for SMZL as determined by multiparameter flow cytometry. The BRAF V600E mutation was detected in 178/182 (97.8%) of HCL cases, whereas 0/49 of HCL-v and 0/83 SMZL were positive. Thus, the BRAF V600E mutation is 100% specific for HCL regarding these three entities. The median BRAF V600E expression ratio of positive cases was 14.2 (range 0.22 – 280.3). After normalization to % pathological cells as assessed by multiparameter flow cytometry the median ratio was 173 (range:22–1,788). However, in 4 cases with 4%, 8%, 28% and 66% percent leukemic cells by multiparameter flow cytometry, which is within the clone size that can be clearly detected by the BRAF V600E-specific RQ-PCR assay, no mutation was detected. Thus, BRAF V600E detection used for the identification of HCL has a sensitivity of 97.8%. Further, NRAS and KRAS mutation screening in all cases with HCL, HCL-v, and SMZL did not detect any mutation except for one case with SMZL that harboured an NRAS Gly12Asp mutation. This case was found to have an MDS in parallel and thus the mutation more likely belongs to the MDS clone. Thus, analysis of NRAS and KRAS mutations does not further improve diagnostics in these diseases. Further, we analyzed the IGHV usage in all 4 BRAF unmutated HCL and in additional 60 cases (total n=64) with HCL and 41 cases with HCL-v. IGHV4–34 usage was very frequent in HCL-v with 14/41 (34.1%). In contrast, it was never detected in HCL including the BRAF wildtype cases. Thus, we were not able to confirm the usage of the IGHV4–34 gene, which was previously suggested for BRAF V600E negative HCL. On the other hand IGHV5–51 was most frequently found in HCL (9/64, 14.1%) but never detected in HCL-v. We detected an unmutated IGHV status in 12/62 (19.4%) of HCL, which was less frequent compared to 14/40 (35.0%) in HCL-v (p = 0.095). The IGHV mutation status was unmutated in 9/11 (81.8%) IGHV4–34 cases (100% identity to germline each). The four cases of HCL, which lacked BRAF V600E mutation, expressed the IGHV genes IGHV1–3*01 (96.5% identity), IGHV1–69*02 (94.0% identity), IGHV3–9*01 (96.9% identity) and IGHV6-1*01 (99.0% identity), which were also expressed by various BRAF V600E positive HCL cases in our cohort. Conclusions: 1) In our cohort of 314 cases with HCL, HCL-v, and SMZL we confirm a high specificity (100%) and sensitivity (97.8%) for BRAF V600E mutations to detect HCL. 2) Other RAS pathway mutations (NRAS, KRAS) were not detected in any of the three analysed entities. 3) In the 4 rare cases of HCL with BRAF wt we were not able to confirm the previously postulated IGHV4–34 usage. 4) IGHV4–34 further delineates classic HCL from HCL-v. Disclosures: Schnittger: MLL Munich Leukemia Laboratory: Equity Ownership. Dicker:MLL Munich Leukemia Laboratory: Employment. Eder:MLL Munich Leukemia Laboratory: Employment. Jeromin:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Equity Ownership.

Abstract 1595 Background: Immunohistochemistry for Cyclin D1 (CCND1) expression is routine in cases suggestive of mantle cell lymphoma (MCL). Most MCL are t(11;14)/IGH-CCND1-positive by FISH. PCR based detection of the fusion transcript is hampered by widespread breakpoints. Only few data is available on quantitative real-time PCR (RQ-PCR) for CCND1 expression measurement. Aims: To assess CCND1 mRNA expression and correlate it with t(11;14) in mature B-cell neoplasms. Methods: We established a RQ-PCR assay for CCND1 mRNA measurement and investigated 451 cases: 142 MCL (in all cases IGH-CCND1 confirmed by FISH), 76 chronic lymphocytic leukemia (43 typical CLL, 33 CLL/PL), 20 hairy cell leukemia (HCL), 13 hairy cell leukemia-variant (HCL-v), 20 splenic marginal zone lymphoma (SMZL), 91 other mature B-cell neoplasms. CCND1 background expression was assessed in 29 pts with other hematological neoplasms and 60 healthy individuals. FISH and/or chromosome banding analysis for the t(11;14) was available in 364 pts. Bone marrow (BM, n=267) or peripheral blood (PB; n=184) samples were analyzed by cytomorphology, multiparameter flow cytometry (MFC), FISH, and RQ-PCR. CCND1 mRNA expression was given by RQ-PCR in comparison to ABL1 mRNA expression (%CCND1/ABL1). Limited dilution of high expressers into cDNA of healthy controls revealed a sensitivity of the assay of up to 0.1 %. Results: IGH-CCND1 translocation carriers had higher %CCND1/ABL1 than those without which hold true in the total cohort (mean±SD, 420.4±740.3 vs 17.8±128.3; p Conclusions: CCND1 expression is highly correlated with t(11;14)/IGH-CCND1 and significantly increased in MCL as compared to other B-cell lymphoma subtypes. Increased CCND1 expression can also be found in other lymphoma subtypes. CCND1 expression measurement by RQ-PCR may be considered a useful adjunct for diagnosis in pts with suspected MCL or in lymphoma pts with suspected t(11;14)/IGH-CCND1 but ambiguous results by other techniques. This technique may also be useful to monitor therapy response. Disclosures: Bacher: MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Alpermann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership.

Abstract 3467 Poster Board III-355 Hairy cell leukemia (HCL) is an uncommon B-cell malignancy that shares some features with a variant form, HCL-variant, and splenic marginal zone lymphoma (SMZL). The distinction between these disorders can be difficult, but correct diagnosis is important for patient management and clinical outcome. The recent WHO classification has recognized SMZL as a distinct entity and HCL-variant was included in a provisional category of unclassifiable splenic lymphomas involving the splenic red pulp that also includes splenic diffuse red pulp small B-cell lymphoma (SDSL). The molecular features of these disorders are still largely uncharacterized and genomic studies have been limited due to their rarity. We have used Affymetrix gene expression microarrays to compare the transcription profiles of 31 HCL, 24 HCL-variant, 44 SMZL and 5 cases of SDSL with the aim of elucidating the relationships between these disorders and to identify novel potential therapeutic and disease-specific diagnostic targets. Total RNA was extracted from PBMC samples enriched for tumor cells (median 89%, range 70-98%) and the expression of approximately 18,000 genes was interrogated using the Affymetrix Human Exon 1.0 ST Array. Data were normalized and analyzed using Partek Genomics Suite® and differentially expressed transcripts between the disorders were identified by ANOVA. Unsupervised hierarchical clustering revealed disease-related patterns within the transcription profiles. Strikingly, the HCL-variant, SMZL and SDSL profiles clustered together and separately from those of typical HCL. Using a false discovery rate threshold of 2 we identified 366 transcripts that distinguished HCL from HCL-variant, SMZL and SDSL. Using these same criteria, and even those less stringent, we could not identify a gene expression signature that could distinguish between HCL-variant, SMZL and SDSL. Biological interpretation of the data revealed many of the differentially expressed transcripts linked to immune response and signal transduction processes, including key oncogenes and tumor suppressor genes. Transcripts with higher expression in HCL-variant, SMZL and SDSL relative to HCL were associated with lymphocyte activation and proliferation, NFκB signaling and integrin signaling. Conversely, transcripts with higher expression in HCL relative to HCL-variant, SMZL and SDSL, comprised amongst others, pathways of antigen processing, MAP kinase signaling, JAK-STAT signaling, and FLT3 signaling. Comparison of the transcription signatures of HCL, HCL-variant and SMZL provides insight into their relationships and for the first time provides strong evidence that HCL-variant and SDSL may be variant forms of SMZL and that HCL remains as a distinct disease entity characterized by different signaling pathways. These data further strengthen our previous observation that showed that the immunoglobulin heavy chain gene (IGH) repertoire of HCL-variant is more similar to SMZL than to HCL and that, in contrast to HCL, both HCL-variant and SMZL contain subsets of cases with unmutated IGHV. Interestingly, the expression profiles of both HCL-variant and SMZL could not be discriminated based on their IGHV mutation status. We are currently performing further analyses of these data, including comparison with the expression profiles of normal B cells, with the aim of gaining further insight into the clonal history and the normal B-cell counterpart of these disorders. Disclosures No relevant conflicts of interest to declare.

Hairy cell leukemia (HCL) and its variant form (HCL-v) are rare B-cell disorders, with different and distinct morphology, immunophenotype, clinical behaviour and response to treatment. HCL-v patients do not respond to purine analogues and have a median survival of seven years compared to over 20 years in HCL, a disease potentially curable with purine analogues in up to 80% of cases. Despite these differences, the resemblance between these two malignancies and splenic marginal zone lymphoma (SMZL) has led to the suggestion that they all may share a common clonal progenitor that homes within the marginal zone of the splenic white pulp. Since the study of immunoglobulin heavy chain gene (IGH) rearrangements provides crucial information regarding the differentiation stages at which particular B-cells are transformed, we have investigated the configuration of the IGH genes in a series of well defined HCL (n=17), HCL-v (n=28) and SMZL (n=82) samples. VDJH rearrangements were PCR-amplified and sequenced using an automated 3130xL sequencer (ABI). The sequences obtained were compared to the closest germline segments using the IMGT database (http://imgt.cines.fr) in order to study the level of somatic hypermutation as well as gene segment usage and CDR3 composition in IGH rearrangements. We were able to amplify a functional VDJH rearrangement in all 127 cases. A significant proportion of SMZL (31/82; 38%) and HCL-v (7/28; 25%) cases were found to be unmutated (range: 0–1.8%). In contrast, all but one HCL cases 16/17 (94%) showed more than 2% deviation from the germline sequence. This suggests that HCL-v is more similar to SMZL than HCL with respect to IGH mutational status. There was a significant over-representation of the VH1 family in the SMZL cases (38%; p CONCLUSIONS: Our findings show that in SMZL specific VDJH stereotypes can be identified, likely as a consequence of antigen interaction, which are not present in HCL or HCL-v. Furthermore, HCL-v shares more similarities with SMZL, in terms of IGH rearrangement and somatic hypermutation patterns, than with typical HCL and suggest that different processes occur in the pathogenesis of these three disorders. The clinical implications of these findings are being addressed.

Hairy cell leukemia (HCL) is a rare B-cell disorder, with a variant form, which differs from the former in clinical behaviour, morphology and immunophenotype. The resemblance of these malignancies to splenic marginal zone lymphoma (SMZL), has led to the suggestion that they may share a common clonal progenitor that homes within the marginal zone of the splenic germinal centres. The immunophenotype and recent global gene expression results offer some support for this view, with a similar profile seen in HCL, and a subset of memory B-cells that are localized to the marginal zone. However, studies of the mutational status of the Ig genes are controversial. The majority of HCL cases are mutated, whereas up to 30% of SMZL cases are unmutated. Further investigation to understand these differences and to identify the cell of origin in these disorders is needed. In addition, there are no data on gene expression or VH mutation in HCL-variant (HCL-v), an entity with a poorer outcome and for which there is as yet no effective therapy. We have analysed a series of 13 HCL and 23 HCL-v, for the configuration of the Ig heavy chain rearrangements. VDJH rearrangements were PCR-amplified and sequenced using an automated 3130xL sequencer (Applied Biosystems, Warrington, UK). The sequences obtained were compared to the closest germline segments using V-base (CRI, Cambridge, UK) in order to study the level of somatic hypermutation as well as gene segment usage in IgH rearrangements. The majority of the HCL (92%) showed more than 2% deviation from the closest germline VH gene segment, compared to only 17/23 (74%) in the HCL-v cases. In the mutated cases, the percentage of mutation was similar for HCL and HCL-v (mean 5.8% and 5.9%, respectively), and none of the cases appeared to show ongoing hypermutation. Within the HCL-v group, 4 out of the 6 unmutated cases used VH4-34 segment, and the remaining 2 used VH1-03. Both genes are known to be associated with autoimmune diseases, and they are excluded from the normal and malignant memory plasma cell repertoires. Also, JH5 segment was found in 47% of the HCL-v, while it was absent in the HCL repertoire. This finding is particularly interesting, as JH5 segment is normally under-represented in most normal and malignant B-cells, except for a characteristic group of IgG-expressing chronic lymphocytic leukemias. Our data suggest that HCL-v is, unlike HCL, heterogeneous regarding IgVH mutations, but has restricted IgH repertoire that, at least in some cases, might be derived from activated, self-reactive B-lymphocytes that have not experienced the germinal-centre reaction.