ALBERT

Description: Patients with chronic lymphocytic leukemia (CLL) may develop other B cell malignancies in their clinical course including aggressive diffuse large B-cell lymphomas and rarely myelomas. In a large proportion of cases, the secondary B cell malignancies reflected the emergence of immunophenotypically and genetically different clones. An immature type plasma cell myeloma developed in a 73-year-old female patient in whom CLL was diagnosed four years previously. The CLL expressed CD5, CD19, CD23, CD38 and surface kappa light chain, but were negative for ZAP-70. Trisomy 12 was detected by FISH analysis. PCR analysis of the peripheral blood for immunoglobulin heavy chain genes demonstrated two sharp bands that were initially interpreted as biallelic heavy chain gene rearrangements. Myeloma cells were CD38 and CD138 positive, CD19 negative and expressed cytoplasmic kappa light chain, but not heavy chains. In order to investigate the clonal relationship between these B cell malignancies, a detailed analysis of VHDJH and VκJκ gene rearrangements in individually sorted CD5 and CD19 double-positive CLL cells and also in CD38-positive and CD19-negative myeloma cells by single cell PCR of genomic DNA and direct sequencing was carried out. This technique permitted identification and pairing of both the heavy and light chain immunoglobulin genes from the same individually sorted cell. A total of 17 individual CLL and 23 myeloma cells were successfully analyzed. Our analysis demonstrated (a) the presence of two discrete clones of CLL, one with usage of [VH1-2*04/D3-3*01/J3*02]-[Vκ2-28*01/J1*01] without VH and Vκ hypermutation and the other with usage of [VH1-2*04/D4-11*01/J6*02]-[Vκ1-5*03/J1*01] with VH and Vκhypermutation; (b) no clonal relationship between the CLL and myeloma cells that utilized different VHDJH and VκJκ rearrangements [VH3-66*02/3-10*01/J4*03]-[Vκ1-33*01/J2*02] with VH and Vκ hypermutation. To our knowledge, this is the first demonstration of a biclonal CLL with mutated and unmutated clones in the same patient along with a third clonally unrelated B cell malignancy. This result suggests that single cell analysis may be necessary to detect subtle biclonality of CLL that might be associated with a more aggressive phenotype.

Description: Cytogenetic abnormalities detected by fluorescent in situ hybridization (FISH) can predict differences in survival in patients with CLL. Patients (pts) with the 13q14 deletion experience a longer median survival that those with the 17p13.1 deletion. Overexpression of ZAP-70 and CD38 correlate with a poor prognosis. Data on 44 pts currently enrolled on a prospective CLL prognostic study were analyzed for cytogenetic abnormalities by FISH and CD38 by flow. Pt age ranged from 25 to 85 (median 59). 25/44 patients (57%) were followed without any therapy. Zap-70 expression was measured by immunohistochemistry using a monoclonal antibody to ZAP-70 (clone 2F3.2). Results: 22/44 (50%) pts were found to have abnormal cytogenetics. Of the 22 abnormalities, 17 had a 13q14 deletion (including one with an additional 11q22.31 deletion, and two with nullisomy 13q), five patients had trisomy 12, one patient a 17 p13.1 deletion. 8/44 patients overexpressed CD38. Of the pts with an isolated 13q deletion, 12/16 (75%) were CD38 negative and 10/17 (59%) were followed without any therapy, compared to 20/27 (74%) and 14/27 (52%) in those with other cytogenetic features respectively. Of those with other abnormal FISH findings, 4/5 pts (90%) with trisomy 12 were CD38 negative, a patient with 17p13.1 deletion and another patient with both 13q14 and 11q deletion were also CD38 positive. Of the patients with normal FISH results 19/21 (90%) were CD38 negative. ZAP-70 overexpression was noted in 22/26 pts with available data including 8/10 (80%) patients with and 13/26 (50%) patients without the 13q del. Conclusion: The presence of the 13q14 deletion appeared to be independent of CD38 expression. Further studies to determine the association of 13q14 deletion with ZAP-70 expression are warranted, as is the correlation with clinical data.

Description: Langerhans cell histocytosis (LCH) is a neoplasm of histiocytic cells believed to be derived from cells of the dendritic system, but the disease pathogenesis is unknown. FMS-like tyrosine kinase 3 (FLT3) is a type III receptor tyrosine kinase that is expressed on the surface of hematopoietic stem cells and plays an important role in normal hematopoiesis. Recently, FLT3 has been shown to play a role in regulating dendritic cell development and elevated levels FLT3-ligand was found in the serum of LCH patients. Therefore we evaluated 14 cases of LCH for FLT3 mutations by PCR analysis of genomic DNA, obtained from formalin-fixed paraffin-embedded tissue samples, followed by PAGE for deletions or internal tandem duplications using a commercially available kit (InVivoScrib Technologies, San Diego, CA). There were 7 males and 7 females with a mean age of 25.1. The tissues involved included bones, lymph nodes, bone marrow, breast, lungs and various soft tissues. No mutations in FLT3 were identified in any of the 14 patient samples. In the same patient population we also looked at the JAK2 (V617F) mutation, which has been found in patients with myeloproliferative disorders other than chronic myeloid leukemia and which has not yet been evaluated in LCH by a single strand conformation polymorphism assay using a commercially available kit (Ipsogen, New Haven, CT). There was no JAK2 V617F mutation in any of the 14 cases. These results suggest that FLT3 and JAK2 V617F mutations do not play a role in the pathogenesis of LCH.

Description: ZAP-70 is a tyrosine kinase protein involved in T cell signaling, overexpression of which is associated with a poor prognosis in CLL. Clinical information on 32 pts with CLL, and 6 pts with prolymphocytic transformation (PLT) followed at our institution was retrospectively reviewed for Rai stage, ZAP-70, CD38, FISH and response to treatment to determine the prognostic association among these factors. Ages ranged from 37 to 78 (median 59). 5/32 pts with CLL had received prior initial therapy, while 17/19 pts had not. They eventually were all treated with fludarabine based regimens either as salvage or upfront. The remaining 8/32 had not required therapy. Response to therapy was correlated with ZAP-70 expression as measured by immunohistochemistry using a monoclonal antibody to ZAP-70 (clone 2F3.2).17/32 patients (53%) were ZAP-70 positive, as were 3/6 (50%) of the patients with PLT. Of the patients with CLL who were ZAP-70 positive, 5 (30%) were stage 0-II, and 12 (70%) were stage III/IV, while18% did not require therapy. Of the 13 who were treated there were 4 (31%) CR, and 5 (38%) PR. Of the patients who were ZAP-70 negative, 50% (7/14) had stages 0-II and 50% had stage III/IV disease. 5/14(36%) were not treated. Of the 9 who were treated there were 2 CR 22%, and 7 PR 78% (p=0.01). 3 PLT patients were previously treated for CLL with fludarabine-based combinations. When they required therapy for PLT, two achieved a CR (one to rituximab/fludarabine/cytoxan_ZAP-70 positive patient, and one to R-CHOP combination_ZAP-70 negative), and another a PR (to R-EPOCH combination_ZAP-70 negative patient). Of the previously untreated PLT patients, two ZAP-70 positive patients achieved a CR (one to rituximab/fludarabine/cyclophosphamide, and one to R-CHOP). One ZAP-70 negative patient was refractory to fludarabine/rituximab combination. Conclusion: ZAP-70 positive CLL is more likely to be refractory to treatment, while ZAP-70 negative pts achieved a higher ORR (p=0.01). Although pts who were ZAP-70 positive were more likely to have advanced stage, 30% of these pts had early stage disease. As such, ZAP-70 did not closely correlate with stage, and may be an independent prognostic factor in these pts. Response to treatment could also not be predicted by ZAP-70 expression. ZAP-70 Number of Pts Previous Treatment Number of Pts Response Positive 16 No 10 3CR, 4PR Yes 3 1CR, 1PR Negative 14 No 7 2CR, 5PR Yes 2 2PR