Immunophenotype Of Chronic Lymphocytic Leukemia Research Articles

BCL3 rearrangements in B-cell lymphoid neoplasms occur in two breakpoint clusters associated with different diseases.

The t(14;19)(q32;q13) often juxtaposes BCL3 with immunoglobulin heavy chain (IGH) resulting in overexpression of the gene. In contrast to other oncogenic translocations, BCL3 rearrangement (BCL3-R) has been associated with a broad spectrum of lymphoid neoplasms. Here we report an integrative whole-genome sequence, transcriptomic, and DNA methylation analysis of 13 lymphoid neoplasms with BCL3-R. The resolution of the breakpoints at single base-pair revealed that they occur in two clusters at 5' (n=9) and 3' (n=4) regions of BCL3 associated with two different biological and clinical entities. Both breakpoints were mediated by aberrant class switch recombination of the IGH locus. However, the 5' breakpoints (upstream) juxtaposed BCL3 next to an IGH enhancer leading to overexpression of the gene whereas the 3' breakpoints (downstream) positioned BCL3 outside the influence of the IGH and were not associated with its expression. Upstream BCL3-R tumors had unmutated IGHV, trisomy 12, and mutated genes frequently seen in chronic lymphocytic leukemia (CLL) but had an atypical CLL morphology, immunophenotype, DNA methylome, and expression profile that differ from conventional CLL. In contrast, downstream BCL3-R neoplasms were atypical splenic or nodal marginal zone lymphomas (MZL) with mutated IGHV, complex karyotypes and mutated genes typical of MZL. Two of the latter four tumors transformed to a large B-cell lymphoma. We designed a novel fluorescence in situ hybridization assay that recognizes the two different breakpoints and validated these findings in 17 independent tumors. Overall, upstream or downstream breakpoints of BCL3-R are mainly associated with two subtypes of lymphoid neoplasms with different (epi)genomic, expression, and clinicopathological features resembling atypical CLL and MZL, respectively.

Germline CHEK2 and ATM Variants in Myeloid and Other Hematopoietic Malignancies

An intact DNA damage response is crucial to preventing cancer development, including in myeloid and lymphoid malignancies. Deficiencies in the homologous recombination (HR) pathway can lead to defective DNA damage responses, and this can occur through inherited germline mutations in HR pathway genes, such as CHEK2 and ATM. We now understand that germline mutations can be identified frequently (~ 5-10%) in patients with myeloid and lymphoid malignancies, and among the most common of these are CHEK2 and ATM. We review the role that deleterious germline CHEK2 and ATM variants play in the development of hematopoietic malignancies, and how this influences clinical practice, including cancer screening, hematopoietic stem cell transplantation, and therapy choice. In recent large cohorts of patients diagnosed with myeloid or lymphoid malignancies, deleterious germline loss of function variants in CHEK2 and ATM are among the most common identified. Germline CHEK2 variants predispose to a range of myeloid malignancies, most prominently myeloproliferative neoplasms and myelodysplastic syndromes (odds ratio range: 2.1-12.3), and chronic lymphocytic leukemia (odds ratio 14.83). Deleterious germline ATM variants have been shown to predispose to chronic lymphocytic leukemia (odds ratio range: 1.7-10.1), although additional studies are needed to demonstrate the risk they confer for myeloid malignancies. Early studies suggest there may also be associations between deleterious germline CHEK2 and ATM variants and development of clonal hematopoiesis. Identifying CHEK2 and ATM variants is crucial for the optimal management of patients and families affected by hematopoietic malignancies. OPENING CLINICAL CASE: "A 45year-old woman presents to your clinic with a history of triple-negative breast cancer diagnosed five years ago, treated with surgery, radiation, and chemotherapy. About six months ago, she developed cervical lymphadenopathy, and a biopsy demonstrated small lymphocytic leukemia. Peripheral blood shows a small population of lymphocytes with a chronic lymphocytic leukemia immunophenotype, and FISH demonstrates a complex karyotype: gain of one to two copies of IGH and FGFR3; gain of two copies of CDKN2C at 1p32.3; gain of two copies of CKS1B at 1q21; tetrasomy for chromosome 3; trisomy and tetrasomy for chromosome 7; tetrasomy for chromosome 9; tetrasomy for chromosome 12; gain of one to twocopies of ATM at 11q22.3; deletion of chromosome 13 deletion positive; gain of one to two copies of TP53 at 17p13.1). Given her history of two cancers, you arrange for germline genetic testing using DNA from cultured skin fibroblasts, which demonstrates pathogenic variants in ATM [c.1898 + 2T > G] and CHEK2 [p.T367Metfs]. Her family history is significant for multiple cancers. (Fig.1)." Fig. 1 Representative pedigree from a patient with germline pathogenic ATM and CHEK2 variants who was affected by early onset breast cancer and chronic lymphocytic leukemia. Arrow indicates proband. Colors indicate cancer type/disease: purple, breast cancer; blue, lymphoma; brown, melanoma; yellow, colon cancer; and green, autoimmune disease.

Atypical immunophenotype of chronic lymphocytic leukemia

Assessment of the immunophenotype plays a crucial role in the diagnostic process of chronic lymphocytic leukemia (CLL). The expression of CD5, CD19 and CD23 antigens with a concomitant reduction or lack of surface immunoglobulin expression as well as CD22 and CD79b antigens is the basic part of CLL diagnosis. A significant diagnostic challenge is atypical CLL with cells devoid of CD5 or CD23 antigens. The assessment of additional antigens in flow cytometry, especially the CD200 glycoprotein, may facilitate the process of differential diagnosis of atypical CLL from other B-cell lymphoproliferative neoplasms. The results of current studies analyzing the influence of atypical CLL on prognosis are inconclusive. The analysis of a large group of patients with atypical CLL is difficult because of the rare occurrence of CD5(–) or CD23(–) CLL and the misdiagnosis of this disease as other B-cell lymphoproliferative neoplasms. The following paper aims to show how important it is to include atypical CLL in the diagnostic process of this disease and to re-standardize the commonly used immunophenotypic scales for its diagnosis.

An Unusual Hematologic Manifestation in a COVID-19 Patient Treated with COVID-19 Convalescent Plasma

Introduction/ObjectiveA subset of Chronic Lymphocytic Leukemia (CLL) patients with COVID-19 may manifest rapid elevations in lymphocyte counts and poor clinical outcomes. Here we report our observations regarding this unusual hematologic manifestation in a CLL patient after he experienced a COVID-19 convalescent plasma (CCP) transfusion reaction.Methods/Case Report56-year-old A Rh- male with stable, treatment-naive CLL (13q deletion positive) diagnosed three years prior was admitted for COVID-19 hypoxia. Before developing COVID-19, baseline white blood cell (WBC) count was stable (~ 64 K/ mm3). Due to worsening hypoxia he was treated with an ARh+ High Titer CCP unit, Remdesivir, tocilizumab, and dexamethasone. The Blood Bank was notified of a possible CCP reaction and performed its standard workup which was adjudicated to be a febrile TACO reaction. During this evaluation it was noted that the patient’s WBC count had initially decreased to 46 K/ mm3, but rose on HD 3, to 78.4 K/mm3 and by discharge on HD 10 had increased to 200 K/ mm3. Flow cytometry revealed a B cell CLL immunophenotype. Post discharge day (PDD) 6 he developed herpes zoster. On post-discharge day (PDD) 7 his WBC count was 124K/mm3. By PDD 77 his WBC count had returned to baseline (~50 K/mm3).Results (if a Case Study enter NA)NAConclusionThe pt’s initial drop in his WBC count followed by a gradual rise has been observed in patients with severe COVID-19 independent of CCP infusion. Lymphocytosis in a subset of CLL patients with COVID-19 has been reported (termed “COVID-19 Induced Lymphocytosis” (CIL)) and, in contrast to our patient with an improved outcome, has been associated with severe/fatal outcomes. Of note these other patients did not receive CCP. Mechanisms related to CIL are unknown. Such poor clinical outcomes heighten the need for further studies of CIL. The role of CCP in affecting this process, if any, also merits further clarification.

A Tissue Counterpart to Monoclonal B-Cell Lymphocytosis.

B-cell clones discovered in tissue biopsies, without overt lymphoma, may represent a tissue counterpart to peripheral blood monoclonal B-cell lymphocytosis (MBL), herein termed tMBL. To characterize the clinicopathologic features of tMBL. During a 10-year period, we retrospectively identified non-bone marrow/peripheral blood cases with monotypic B cells detected by tissue-based flow cytometry but without an identifiable lymphomatous infiltrate on routine histopathology. We excluded cases with prior diagnosis of chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma or MBL. Fifty-four cases were identified (35 lymph node, 3 splenic, and 16 soft tissue/viscera). Forty-six cases were CLL-type, 2 were atypical CLL, and 6 were non-CLL. tMBL was detectable by immunohistochemistry in 14 cases (26%, all CLL-type). Concurrent blood flow cytometry, available in 10 cases, showed 4 with low-count MBL (3 CLL-type, 1 with non-CLL-type), 5 with high-count MBL (all CLL-type), and 1 case negative for clonal population. With median follow-up of 51 months, 2 patients had progression of disease (CLL, 68.7 months; and diffuse large B-cell lymphoma, 5.9 months). Patients with immunohistochemistry-detectable tMBL had increased monoclonal B cells per total lymphocyte events (P = .01), morphologic evidence of bone marrow involvement (P = .04), higher white blood cell count (P = .02), and increased absolute lymphocyte count (P = .02). tMBL spans an immunophenotypic spectrum similar to MBL, is detectable by immunohistochemistry in a minority of cases (often CLL immunophenotype), and is likely systemic in most cases. Development of overt lymphoma is uncommon but may occur, warranting clinical follow-up.

The Case | Nephrotic syndrome in a patient with chronic lymphocytic leukemia

A 59-year-old man with chronic lymphocytic leukemia (CLL) was referred to the nephrology clinic for new-onset nephrotic syndrome. He had been diagnosed with CLL 1 year earlier after an incidental finding of lymphocytosis (lymphocyte count, 34 × 109/l) during an influenza A infection. Flow cytometry confirmed a typical immunophenotype of CLL. A watchful waiting approach to CLL management was adopted as the patient was asymptomatic. He was otherwise healthy with no significant medical history. He took no medications including over-the-counter medications.

Integration of automated morphological features resolves a distinct group of atypical chronic lymphocytic leukemias with chromosomal aberrations

Automated morphological assessment of peripheral blood slides has become an important modality facilitating characterization and quantification of cells in a uniform, fast and robust manner. In this study, we evaluated the morphological diversity in peripheral blood films of 94 chronic lymphocytic leukemia (CLL) patients using the DM1200 CellaVision automated microscopy system. Aberrant lymphocytes and smudge cells were enumerated and correlated with CLL immunophenotype, chromosomal aberrations and prognostic parameters. Herein, we show that the percentages of aberrant and smudge cells was highly variable between patients and did not correlate with each other. Increased aberrant lymphocytes and fewer smudge cells were associated with an atypical immunophenotype including low expression of CD23, higher levels of FMC7 and bright surface levels of CD20. High fraction of aberrant lymphocytes also was associated with trisomy 12. These cells were predominantly of small/medium size, sometimes with cleft nuclei. No correlation was noted between aberrant or smudge cells and clinical stage, CD38, ZA70 or time to first treatment. Taken together, automated morphological analysis of peripheral blood leukocytes emerged as a powerful and robust tool for the quantitative morphological stratification of CLL. Integration of the automated morphological features discriminates between different CLL phenotypes and distinct chromosomal aberrations.

Clinical Relevance of Immunophenotypic Characteristics in Chronic Lymphocytic Leukemia

Abstract 4604 Background:Chronic lymphocytic leukemia (CLL) has the highest incidence and prevalence of any leukemia in the United States. It has a characteristic immunophenotype, but variant forms (typically termed “atypical CLL”) are commonly encountered. It is not known if this immunophenotypic heterogeneity in CLL is clinically relevant. We hypothesized that the immunophenotype of CLL cells correlates with both biologic parameters and clinical outcomes. Methods:We performed a retrospective review of clinical, biologic, and flow cytometric data of patients undergoing an evaluation of an absolute lymphocytosis at the Duke University and Durham VA Medical Centers. Patients meeting immunophenotypic criteria for CLL based on cell surface expression of CD5, CD19, CD20, CD23, and surface immunoglobulin light chain (sIg) were included in this analysis. Atypical CLL was defined if any of the following were observed: (1) expression of CD5, CD20, CD23, or sIg that differed from a typical CLL immunophenotype (e.g. bright CD20 or low CD23; hereafter non-standard immunophenotype), (2) expression of T cell markers CD7 or CD8, or (3) expression of CD123. Through electronic chart review, clinical and biologic parameters were abstracted. These included age; gender; ethnicity; WBC, hemoglobin, platelet count, and Rai stage at diagnosis; time to first treatment; overall survival; CD38, ZAP70, IGHV mutation status, and interphase cytogenetics (FISH). A chi square test or Fisher's exact test for small sample sizes was used to test association of categorical data. The central tendencies of continuous measurement were compared using the Wilcoxon rank sum test. Overall survival and time to treatment were calculated using the Kaplan-Meier product limit method. Survival curves were compared using the log-rank test. This study was approved by IRBs at Duke University and Durham VA Medical Centers. Results:We reviewed 189 patients, including 119 (63%) with typical CLL immunophenotype and 70 (37%) with atypical. Among patients with atypical CLL, 93% (65 of 70) had a non-standard immunophenotype, 33% (23 of 70) had expression of T cell markers, and 33% (14 of 43) had expression of CD123. We observed expression of T cell markers in 12% (23 of 189) and CD123 in 12% (14 of 114) of the entire cohort, respectively. At the time of diagnosis, there was no association between typical or atypical CLL immunophenotype and age, gender, race/ethnicity, WBC, hemoglobin, platelet count, or Rai stage (p>0.05 for all comparisons). With regard to biologic parameters, there was no association of CLL immunophenotype with the proportion of patients with CD38 ≥30%; however, the central tendency of CD38 was higher in the atypical group when analyzed as a continuous variable (median value 38% vs. 10%, p=0.04). There was no difference in the proportion of ZAP70 positive cases between typical and atypical groups. Analysis of interphase cytogenetics (FISH) data showed that a higher percentage of typical than atypical immunophenotypes had del 13q as a sole abnormality and a lower percentage had trisomy 12 (p=0.02). Despite the increased frequency of del 13q as a sole abnormality, lower frequency of trisomy 12, and lower expression of CD38, there was no difference between groups in time to first treatment (median 5.3 years for typical vs. 2.1 years for atypical; p= 0.11) or in overall survival (median 13.6 yrs64 years for typical vs. 18.0 years for atypical; p=0.11) (Figure 1). However, when patients with non-standard CLL immunophenotype were compared to those with a standard immunophenotype, a significantly shorter time to first treatment was observed (median 6.0 years for standard immunophenotype vs. 2.2 years for non-standard, p=0.03). [Display omitted] Conclusion:Different immunophenotypic subtypes are commonly encountered in the care of patients with CLL. These groups have different biological characteristics, including differences in expression of CD38 and cytogenetic abnormalities. Exploratory analysis also showed differences in clinical outcomes based on immunophenotypic parameters. Validation studies are currently underway. Disclosures:No relevant conflicts of interest to declare.

The detection of minimal residual disease status in chronic lymphocytic leukemia

In this issue of Clinical Cytometry, Durrieu et al. on behalf of GEIL-GOELAMS present evidence that circulating levels of CD19+ CD5+ chronic lymphocytic leukemia (CLL)-like cells can be used to define a threshold for the detection of minimal residual disease (MRD) in CLL (this issue: page 346). This study was performed in 16 different hematology laboratories or services, and the data is analyzed centrally. The panel consisted of five tubes of four reagents each with CD19 and CD5 being present in each tube. They determined that the level of two CLL-like immunophenotypes CD19+/CD5+/CD43int/CD79blo and CD19+/ CD5+/CD81lo/CD22lo were less than 4 × 10−4 per WBC in unwashed, lyzed normal whole blood. Levels greater than this found in post treatment CLL samples were taken to signify the presence of MRD. The author further state that they are unable to determine monoclonality using light chain restriction due to their polyclonal nature in normal blood donors and the presence of cytophilic antibodies being present in the unwashed, lyzed method. And that bright CD5+ B cells are often lambda positive, and polyclonal. I have several preferences (biases) in this matter, and they include the use of CD20 rather than CD19 or preferably both CD20 and CD19 in combination to define B cells and the clone, demonstration of monoclonality or monotypia, and that reports be based on the absolute cell-count (WBC or absolute lymphocyte count ALC) and not just the percentage < or > 10−4. If the WBC is used, it should be used in conjunction with the ALC and even a B cell absolute count. I sympathize with the problem of cytophilic antibodies but in monoclonal B cell lymphocytosis studies, values of 5–50 light chain restricted B cells per microliter can be routinely detected (1). I was also surprised to see that the lower range of 1–3% CD5+ B cells seems to be the norm, whereas values of 10–25% seem to be more the rule. The European Research Initiative on CLL (ERIC) has published a standardized four-color approach to MRD detection in CLL (2). Both the authors of that study and this study find a WBC threshold of 10−4 below which is considered MRD negativity. The ERIC study allows for the primary role of kappa lambda tube for historical purposes but favors the combination of CD20/CD38/CD19/CD5 over the combinations of CD43/CD79b and CD81/CD22. They further point out the need for the evaluation of gate contamination, number of total events collected (500,000), and operator training. Certain precautions are required for differentiating between blood and marrow involvement in the setting of antibody therapies. However, due to the correlation of MRD between blood and marrow, blood should be the first site tested for MRD. The complete absence of B cells (B cell aplasia) might also be taken as a clinical measure of MRD. If ascertainment of marrow involvement in this setting were considered important, a bone marrow study would be indicated. Conversely, the timing of the blood MRD analysis could be postponed. The German CLL Study Group have also standardized three four tube panels of four-color reagents using a larger volume of blood, and where appropriate 2 million leucocytes were collected (3). This paper also shows that the overall ability of MRD flow is to detect and quantify residual CLL cells and is not affected by the addition of rituximab to FC chemotherapy (3). In a 2005 editorial, Montserrat (4) called for standardization and revision of 1996 NCI response guidelines to include MRD negative status as a goal. In 2008, Hallek et al. (5) revised the NCI guidelines to include not only a definition of MRD but also encouraged that the MRD detection become part of future CLL clinical trials because MRD negative status has prognostic impact. The GCLLSC, ERIC, and GEIL studies have all demonstrated that multicolor flow cytometry can meet the needs of a robust MRD assay. All three groups used a four-color, five tube panel and a sequential gating strategy. Newer clinical flow cytometers now allow for the use of 6–8 colors. The Salamanca group has developed an automated method for doing MRD detection by merging files on the same patient with the addition normal donor virtual 1:1 dilution as a control (6). Knowledge of the original CLL immunophenotype would be useful. One could envision an integration of the GCLLSC, ERIC, and GEIL methods combining CD19/CD20/CD5/CD38/CD79b/ CD22/CD41/CD81 and CD19/CD20/CD5/CD45/CD3/CD14/kappa/lambda. Del Poeta et al. (7) concluded that the addition of rituximab consolidation and maintenance therapy prolonged response duration significantly in patients with MRD + CLL. MRD detection is indicated in the investigation, care, and treatment of the CLL patient. Clinical trials will be required to answer the question of overall survival in CLL. Regardless, CLL remains a model disease for the appropriate treatment of the elderly with progressive disease.

Reassessment of small lymphocytic lymphoma in the era of monoclonal B-cell lymphocytosis

In the 2008 World Health Organization classification, small lymphocytic lymphoma is defined as a neoplasm with the tissue morphology and immunophenotype of chronic lymphocytic leukemia, but with absence of leukemia. Minimal criteria of tissue involvement to separate small lymphocytic lymphoma from monoclonal B-cell lymphocytosis have not been defined. We reviewed the clinicopathological features of 36 patients with extramedullary tissue biopsies containing chronic lymphocytic leukemia-type cells and less than 5×10(9)/L peripheral blood monoclonal B cells. Pathological features (extent and patterns of involvement, architectural preservation, presence of proliferation centers) as well as cytogenetic and radiological findings were examined in relation to clinical outcome. The biopsies were performed to evaluate lymphadenopathy in 20 patients and for other reasons (most frequently staging of a non-hematologic neoplasm) in 16 patients. At latest follow-up (median 23 months), 21 untreated patients had no or stable lymphadenopathy, 3 had regressed lymphadenopathy, and 12 had developed progressive lymphadenopathy and/or received therapy for chronic lymphocytic leukemia/small lymphocytic lymphoma. Features associated with progression/treatment included lymph nodes 1.5 cm or greater on imaging studies (P=0.01) and presence of proliferation centers in the biopsied tissue (P=0.004). Neither the size nor extent of involvement of the excised lymph node correlated with progression/treatment. Our findings suggest that biopsies containing chronic lymphocytic leukemia-type cells, but lacking proliferation centers and with non-enlarged or only slightly enlarged lymph nodes on imaging, represent a very indolent disease that may best be considered a tissue equivalent of monoclonal B-cell lymphocytosis rather than overt small lymphocytic lymphoma. We propose that such cases be designated as tissue involvement by chronic lymphocytic leukemia/small lymphocytic lymphoma-like cells of uncertain significance.

Minimal Residual Disease following Allogeneic Hematopoietic Stem Cell Transplantation

Minimal residual disease (MRD), both before and after transplantation, is a clinically important yet relatively poorly defined aspect of allogeneic hematopoietic stem cell transplantation (alloHSCT). The clinical relevance of MRD in the context of alloHSCT has been demonstrated by its association with the development of clinical relapse. However, with the possible exception of chronic myeloid leukemia (CML), the specific techniques, timing, frequency, and clinical utility, relative to improvement in patient outcomes, for monitoring MRD in the setting of alloHSCT has yet to be clearly defined. A concise overview of monitoring techniques for detecting MRD, as well as treatment strategies and biological and clinical research initiatives for MRD suggested by the National Cancer Institute First International Workshop on the Biology, Prevention, and Treatment of Relapse after Allogeneic Hematopoietic Stem Cell Transplantation, is covered in this article.

Detection of a t(4;14)(p16;q32) in two cases of lymphoma showing both the immunophenotype of chronic lymphocytic leukemia

Reciprocal IGH/14q32 translocations are detectable in 55–70% of patients with plasma cell myeloma; e.g., the adverse t(4;14)(p16;q32) fusing the IGH and FGFR3 genes (immunoglobulin heavy chain/fibroblast growth factor receptor 3). In a minority of patients with B-lineage chronic lymphocytic leukemia (CLL), reciprocal IGH/14q32 translocations have been reported as well. We describe the occurrence of a t(4;14)(p16;q32) in two lymphoma patients showing the immunophenotype of B-CLL, which, to our knowledge, is the first report on such an association. The first patient, a 72-year-old female, showed mature lymphocyte infiltration of the bone marrow and marked splenomegaly. Immunophenotyping revealed aberrant CD5 expression and light-chain lambda restriction on mature B-lymphocytes corresponding to a B-CLL. Interphase fluorescence in situ hybridization (FISH) plus chromosome banding revealed a t(4;14)(p16;q32) in addition to an unbalanced der(16)t(8;16)(q23;p13) and a del(8)(p11). The second patient, a male of 65 years, showed marked peripheral leukocytosis. Immunophenotyping revealed the phenotype of CLL/PLL (chronic lymphocytic leukemia/prolymphocytic leukemia). Again, FISH together with karyotyping revealed a t(4;14)(p16;q32). These two cases with an t(4;14), but the immunophenotype of B-CLL, demonstrate the genetic variability of B-cell lymphomas and the potential of specific metaphase cultivation techniques using oligonucleotides to increase our insights in the genetic pathways of these heterogeneous disorders.

Complex Immunophenotypic Changes as Prognosis Panel in Chronic Lymphocytic Leukemia

Brief Abstract The present study proposes to asses the prognosis with consecrated markers CD38 and the new ZAP-70 related to the various patterns of CLL complex immunophenotype: cyclin D1, CD38, CD20, FMC7, CD23. This particular combination could be used as a prognosis panel in CLL. Brief Abstract Background Chronic lymphocytic leukemia (CLL) has many other prognostic markers established in the past years related to disease and to the patient status, and the most important seem to now be immunophenotypical, genetic, and molecular. CD38 and ZAP-70 are now used in a strong prognosis panel of markers for CLL. Aims The present study proposes to assess the prognosis with consecrated markers CD38 and the new ZAP-70 related to the various patterns of CLL immunophenotype. Patients and Methods We have analyzed 187 patients diagnosed with CLL in order to find correlations between clinical stage, immunophenotype, and outcome. Results We found correlations between expression of CD38 related to clinical outcome, (dr: 0.541; P P = .018). The expression of BCL-2 was correlated to outcome (dr: 0.533; P P P = .014) and BCL-2 expression ( P = .034). Lower expression CD23 was associated with poor outcome and expression of CD38 and ZAP-70 ( P value + (28% vs. 3%), FMC7 + (33% vs. 8%), and lower CD23 (30%-60% vs. > 60%). Also, CD38 was positive in 44% vs. 10% and ZAP-70 in 66% versus 5%. This association defines a lymphoma-like immunophenotype for cyclin D1—positive cases. Treatment was chlorambucil, fludarabine/cyclophosphamide (FC), or FC + rituximab (FCR). Conclusion Flow cytometry is the most practical method used in CLL for diagnosis and prognosis evaluation. The immunophenotypical markers surrogate for IgVH mutation status, as CD38 and ZAP-70 have a strong correlation with outcome in CLL, and our results found that analysis by flow cytometry of both CD38 and ZAP-70 could be used in the evaluation of patients with CLL as strong prognosis markers. The complex immunophenotype in CLL could be used to define 2 main prognosis patterns: (1) cyclin D1—positive, CD38 positive, CD20 high, FMC7 positive, CD23 weak with poor prognosis; and (2) cyclin D1—negative, CD38 negative, CD20 low, FMC7 negative, CD23 high with good prognosis. These patterns have strong association with expression of ZAP-70 and could be used as prognosis assessment of patients with CLL.

Clinical progression and outcome of patients with monoclonal B-cell lymphocytosis

Monoclonal B-cell lymphocytosis (MBL) is a clonal lymphoproliferation with the immunophenotype of chronic lymphocytic leukemia (CLL) but a B-lymphocyte count of less than 5 × 109/l and no lymphadenopathy, organomegaly, cytopenias or symptoms. We performed a retrospective analysis of patients with MBL (n = 46), Rai stage 0 CLL (n = 112) and Rai stage ≥1 CLL (n = 54). Median follow-up and range was 30 (0.1 – 120) months for MBL, 60 (0.1 – 309) months for stage 0 CLL and 54 (0.1 – 309) months for stage ≥1 CLL. None of the MBL patients required treatment compared with 24 of 112 (21%) stage 0 CLL and 28 of 54 (52%) stage ≥1 CLL patients (p < 0.0003). No MBL underwent aggressive transformation compared with 1 of 112 (0.8%) stage 0 CLL and 6 of 54 (11%) stage ≥1 CLL patients (p < 0.0003). Progression-free survival (PFS) appeared improved in MBL compared to stage 0 CLL, although this did not reach statistical significant (p = 0.07) due to the relatively short follow-up in the MBL group; two year PFS was 97.2% for MBL, 93.1% for stage 0 CLL, and 68% for stage ≥1 CLL patients (p < 0.0001 for stage ≥1 CLL compared with MBL and stage 0 CLL). This is the first study of outcome in MBL which demonstrates that patients have an improved disease course compared to stage 0 CLL patients. Over a median 2.5 years of follow-up, no MBL patients required treatment or died of CLL-related causes.

Clinical Progression and Outcome of Patients with Monoclonal Lymphocytosis of Undetermined Significance.

Monoclonal Lymphocytosis of Undetermined Significance (MLUS) is a clonal lymphoproliferation, usually of B-cells. MLUS has the immunophenotype of Chronic Lymphocytic Leukemia (CLL) but an absolute lymphocyte count of 10 X 109/L or less and no lymphadenopathy, organomegaly, cytopenias, or symptoms attributable to the lymphoproliferation. There is little published information as to whether the clinical course of MLUS differs significantly from that of CLL. We performed a retrospective analysis of 335 consecutive patients with clonal lymphocytosis seen at St. Paul's Hospital diagnosed between January 1969 and July 2005. Patients were identified by a search of the practice database and clinical and pathological data were abstracted by chart review. MLUS was present in 106 patients and CLL in 229. All MLUS were of B-cell phenotype and 227 patients had B-CLL. Median age at diagnosis was 64.5 (range 33–86) y for MLUS and 65 (30–94) y for CLL (p<0.83). 45% of MLUS pts were male vs. 58% of CLL pts (p<0.036). ECOG Performance Status was 0 in 307 pts and was not analyzed further. Lymphocyte count at diagnosis was 7 (range 3–10) x 109/L for MLUS and 14 (range 3–394) x 109/L for CLL (p<0.0001). Lymphocyte doubling time (LDT) ≤12 mo (to an absolute value >50 X 109/L) occurred in 14% of MLUS and 11% of CLL (p<0.84) pts. Rai CLL stage 0, 1, 2, 3 and 4 were 111 pts, 48, 47, 3 and 6 respectively. Immunophenotyping was available in 202 pts; 173 were CD5+ CD19+, 2 were CD5− and 29 were CD19−. Analysis for CD38 was available in 50 pts. At a median follow-up of 49 (0–229) mo for MLUS and 64 (0–369) mo for CLL, 15 (15%) of MLUS pts required treatment, as did 96 (42%) of pts with CLL (p<0.0001). The median time to treatment (TTT) for the whole group was 35 (0–243) mo. Median TTT was 65 (0–202) mo for MLUS and 26 (0–243) mo for CLL; there was no significant difference in progression to active disease as measured by TTT between MLUS and CLL either for the whole groups (p<0.29) or according to age (p<0.54), gender (p<0.91), LDT (p<0.29) or CD38 status (p<0.26). Median OS for all patients was 191 (0–369) mo, for MLUS 218 (0–225) mo, and for CLL 165 (0–369) mo (p<0.04). There was a significant difference in OS favoring female MLUS pts over CLL, but not for males; median OS for females not reached at 225 mo for MLUS and median 153 (0–259) mo for CLL (p<0.02, see Table). There was no significant difference in OS between MLUS and CLL according to age, gender, LDT or CD38 status. One pt with MLUS underwent transformation to aggressive disease (Richter's transformation) as compared to 15 pts with CLL (8 Richter's and 7 prolymphocytic, p<0.025).CharacteristicOS at 10 years (%)P (log-rank)MLUSCLLAge≤6568810.07>6559450.59GenderM38680.47F100760.02LDT≤12 mo47740.10>12 mo80470.93CD38negative90730.60(positive ≥ 30% of cells)3 of 30 of 20.32Transformation eventsn=1n=150.025In conclusion, in this series of 335 patients with clonal lymphoproliferation, MLUS pts had significantly better OS than did pts with CLL, a difference which was seen in female MLUS pts in particular. Significantly fewer MLUS pts required treatment over their course than did pts with CLL. In CLL pts, there appeared to be an increased rate of transformation to aggressive non-Hodgkin's lymphoma or PLL.

Interphase Fluorescence In Situ Hybridization (FISH) with an IgH Probe Is Important in the Management of Patients with a Clinical Diagnosis of Chronic Lymphocytic Leukemia (CLL).

Genetic analysis has an increasing role in the diagnosis, classification, and management of patients with CLL. Although translocations involving the IgH locus are well characterized in B-cell malignancies such as follicular cell lymphoma, their frequency and significance in CLL are less well defined. We studied a series of patients with a clinical diagnosis of CLL using a FISH probe set including IgH.Methods: FISH was performed on blood using a DNA probe set designed to detect common chromosome anomalies associated with CLL and translocations involving IgH. Patients with an IgH abnormality were further studied using FISH probes sets for IgH and cyclin D1, BCL-2, BCL-3, BCL-6, BCL-11a, c-MYC, MALT-1, c-MAF, FGFR-3 or PAX-5. The clinical presentation, pathology, and flow cytometric immunophenotype was reviewed in patients with various translocations involving IgH.Results: Between 1/1/03 and 6/15/04, samples from 1032 patients with a clinical diagnosis of CLL were analyzed by FISH. Of these, 76 patients (7%) had translocations involving the IgH locus. Sixty one patients had a single translocation partner gene (cyclin D1 45%, BCL-2 24%, BCL-3 8%, c-MYC 2.5%, or BCL-11a 1.3%). Two patients had two independent clones (cyclin D1 and BCL-2; BCL-3 and BCL-11a). In 13 pts (17%) we could not identify the translocation partner. Thirty five (46%) of the 76 patients with IgH translocations had been seen at Mayo Clinic Rochester and had clinical and laboratory data for detailed review. Among these 35 patients, 16 (45%) had fusion of IgH and cyclin D1 and were diagnosed with mantle cell lymphoma (MCL). The immunophenotype of the monoclonal B cells was typical of MCL (CD5+, CD23-) in 6 patients and typical of CLL (CD5+, CD23+) in 4 patients. Six MCL patients had non-diagnostic immunophenotypes: CD5-, CD 23+ (n = 3) and CD5-, CD23- (n = 3). Twelve (34%) of the 35 patients had the IGH/BCL-2 fusion gene. Of these, 3 patients had leukemic phase follicular lymphoma, one had both CLL and MCL, and 8 had the diagnostic immunophenotype of CLL. The remaining 7 patients (19%) had a variety of translocation partners: BCL-3 (n = 1), BCL-3 and BCL-11a (n = 1), c-MYC (n = 2), and unknown (n = 3). The patients with a translocation involving BCL-3 had a typical CLL immunophenotype.Discussion: The IgH FISH probe detected a molecular defect in 7% of patients with a circulating monoclonal B cell population. In patients with a CLL immunophenotype, use of the IgH probe identified the IgH/cyclin D1 translocation characteristic of MCL in 4 patients, a finding of major clinical significance. Interestingly, after exclusion of MCL patients, more than 4% patients with CLL had other translocations involving IgH, a proportion comparable with other better defined chromosomal abnormalities in CLL. Although IgH/BCL-2 translocation is usually associated with follicular lymphoma, we identified 8 patients with CLL and this translocation. Translocations involving BCL-3 had been described before in CLL with an “atypical” immunophenotype. In contrast both of our patients with BCL-3 had typical phenotypes. Translocations involving IgH and BCL-11a have not been previously described. We conclude that FISH studies using the IgH probe improve the precision of diagnosis in CLL and could also have prognostic value.

Chronic lymphocytic leukemia patients with highly stable and indolent disease show distinctive phenotypic and genotypic features

AbstractDifferent biologic features have been associated with a more or less aggressive clinical course in chronic lymphocytic leukemia (CLL). In the present study, 20 patients with highly stable CLL observed at a single institution over a period of 10 to 23 years and who never required treatment were extensively characterized. The aim was to identify a distinct and reproducible biologic profile associated with disease stability that may be used to recognize at presentation CLL patients who are likely to have a very benign clinical course and for whom treatment is not indicated. The results obtained indicate that numerous parameters are closely associated with disease stability: a typical CLL morphology and immunophenotype, the lack of expression of the CD38 antigen, the mutated immunoglobulin (Ig) heavy (H) chain variable (V) pattern, the absence of p53 mutations, a CD4/CD8 ratio more than 1, the lack of 17p and 11q deletions and of complex karyotypic aberrations, and the occurrence of the 13q14 deletion. No case displayed the VH3-21 gene, linked in mutated CLL with a poor outcome. In addition, the VH1-69 gene associated with unmutated CLL cases was never detected. These biologic features were coupled with an indolent clinical course characterized by an unmodified clinical stage over time, and by lack of autoimmune phenomena and of major infections requiring parental antibiotics. At a time when aggressive therapeutic strategies are always more frequently used in the management of CLL, the distinctive features of patients with long-lived stable disease should be prospectively identified at presentation.