Genetic landscape of ultra-stable chronic lymphocytic leukemia patients.

Background: Chronic lymphocytic leukemia (CLL) has a heterogeneous clinical course. Beside patients requiring immediate treatment, others show an initial indolent phase followed by progression and others do not progress for decades. The latter two subgroups usually display mutated IGHV genes and a favorable FISH profile. Patients and methods: Patients with absence of disease progression for over 10 years (10-34) from diagnosis were defined as ultra-stable CLL (US-CLL). Forty US-CLL underwent extensive characterization including whole exome sequencing (WES), ultra-deep sequencing and copy number aberration (CNA) analysis to define their unexplored genetic landscape. Microarray analysis, comparing US-CLL with non-US-CLL with similar immunogenetic features (mutated IGHV/favorable FISH), was also carried out to recognize US-CLL at diagnosis. Results: WES was carried out in 20 US-CLL and 84 non-silent somatic mutations in 78 genes were found. When re-tested in a validation cohort of 20 further US-CLL, no recurrent lesion was identified. No clonal mutations of NOTCH1, BIRC3, SF3B1 and TP53 were found, including ATM and other potential progression driving mutations. CNA analysis identified 31 lesions, none with known poor prognostic impact. No novel recurrent lesion was identified: most cases showed no lesions (38%) or an isolated del(13q) (31%). The expression of 6 genes, selected from a gene expression profile analysis by microarray and quantified by droplet digital PCR on a cohort of 79 CLL (58 US-CLL and 21 non-US-CLL), allowed to build a decision-tree capable of recognizing at diagnosis US-CLL patients. Conclusions: The genetic landscape of US-CLL is characterized by the absence of known unfavorable driver mutations/CNA and of novel recurrent genetic lesions. Among CLL patients with favorable immunogenetics, a decision-tree based on the expression of 6 genes may identify at diagnosis patients who are likely to maintain an indolent disease for decades.

Increased serum levels of soluble CD44 standard, but not of variant isoforms v5 and v6, in B cell chronic lymphocytic leukemia.

The CD44 cell surface proteoglycan participates in a variety of functions including lymphohematopoiesis, lymphocyte homing and tumor metastasis. In addition to the standard form (CD44st), a large family of variant isoforms (CD44v) is generated by alternative splicing of a single gene. Certain CD44v (v5 and V6) are upregulated in the course of neoplastic progression and reflect the metastatic potential of tumor cells. CD44 v6 is expressed in high-grade non-Hodgkin's lymphoma cells and is released in the serum, thus providing a soluble marker that reflects tumor burden, disease progression and treatment response. Here we show that serum CD44st is elevated in approximately half of B-CLL patients. In contrast, CD44v5 and v6 are detected at normal levels in the large majority of the cases. CD44st serum levels correlate significantly with the number of circulating leukemic B cells and with the levels of another soluble B-CLL marker, beta2-microglobulin. Immunoprecipitation analyses of B-CLL sera allow detection of several high molecular weight bands and of a 78 kDa band that represents a soluble form of CD44st and is 4 kDa lower than a similar band (82 kDa) detected in B-CLL cell lysates. Elevated serum CD44st associates with a number of unfavorable prognostic factors such as high peripheral blood lymphocytosis, splenomegaly, advanced disease stage and therapy requirement. A follow-up study indicates that serum levels of CD44st are related to disease status, thus reinforcing our veiw that this molecule may represent a reliable tumor marker in B-CLL.

Evidence of biased immunoglobulin variable gene usage in highly stable B-cell chronic lymphocytic leukemia.

Recognition of biased immunoglobulin variable (IgV) gene usage in B-cell chronic lymphocytic leukemia (B-CLL) may yield insight into leukemogenesis and may help to refine prognostic categories. We explored Ig variable heavy (VH) and light (VL) chain gene usage in highly stable and indolent B-CLL (n=25) who never required treatment over 10 or more years. We observed an unexpectedly high usage of mutated VH3-72 (6/25; 24.0%), a gene that was otherwise rare in B-CLL (7/805; 0.87%; P<0.01), including mutated cases (6/432; 1.39%; P<0.01) and was exceptional among indolent (1/230, 0.435%; P<0.01), and aggressive B-cell lymphomas (0/105; P<0.01). Three of six VH3-72 B-CLL cases utilized the same VL Vkappa4-1 gene. Two V(H)3-72 B-CLL cases had highly homologous VH complementarity determining regions 3 (CDR3s), encoding Cys-XXXX-Cys domains, and utilized Vkappa4-1 genes with homologous IgVL CDR3s. An identical threonine to isoleucine change at codon 84 of V(H)3-72 framework region 3 (FR3) recurred in four cases of highly stable VH3-72 B-CLL. This mutation is expected to cause a conformational change of FR3 proximal to CDR3 that might critically affect high-affinity antigen binding. B-cell receptors encoded by VH3-72 may identify a specific B-CLL group and be implicated in leukemogenesis through an antigen-driven expansion of B cells.

Linkage analysis for ATM in familial B cell chronic lymphocytic leukaemia.

B cell chronic lymphocytic leukaemia (CLL) shows evidence of familial aggregation, but the inherited basis is poorly understood. Mutations in the ATM gene have been demonstrated in CLL. This, coupled with a possibly increased risk of leukaemia in relatives of patients with Ataxia Telangiectasia, led us to question whether the ATM gene is involved in familial cases of CLL. To examine this proposition we typed five markers on chromosome 11q in 24 CLL families. No evidence for linkage between CLL and ATM in the 24 families studied and the best estimates of the proportion of sibling pairs that share no, one or both haplotypes at ATM were not different from their null expectations. This would imply that ATM is unlikely to make a significant contribution to the three-fold increase in risk of CLL seen in relatives of patients.

Diagnostic and prognostic role of PET/CT in patients with chronic lymphocytic leukemia and progressive disease.

In order to evaluate the predictive value of positron emission tomography-computed tomography (PET/CT) in discriminating the presence of a Richter's syndrome (RS) or a second malignancy (SM), as well as to evaluate its prognostic value in patients with chronic lymphocytic leukemia (CLL), we retrospectively analyzed the data of 90 patients who, in the suspicion of a RS or a SM, underwent PET/CT followed by the biopsy of the involved tissue. The median maximum Standardized Uptake Value (SUV max) in the presence of a CLL/small lymphocytic lymphoma, a diffuse large B-cell lymphoma (DLBCL), a Hodgkin lymphoma (HL), a SM were 3.5, 14.6, 7.0 and 6.3, respectively (P ⩽ 0.0001). A SUV max cutoff value ⩾ 5 showed a sensitivity, specificity, positive and negative predictive values of 88.2, 71.2, 51.3 and 94%, respectively, for the presence of a more aggressive disease (DLBCL, HL and SM). A SUV max ⩾ 5 identified also a subset of treatment naive patients with an inferior progression-free survival (P = 0.011) and overall survival (P = 0.067). These findings suggest that PET/CT may helpfully integrate the biologically-based prognostic stratification of CLL. Prospective clinical trials including larger cohorts of patients are needed to conclusively define the role and prognostic impact of PET/CT in the routine management of CLL patients.

Standard conditioning regimen and T-depleted donor bone marrow for transplantation in chronic myeloid leukemia.

Between January 1984 to June 1985, 18 Ph1 positive chronic myeloid leukemia (CML) patients in chronic phase (CP) underwent allogeneic bone marrow transplantation (BMT) from HLA identical and MLC negative siblings. The median age was 32.5 yr and median disease duration of CML at time of BMT was 19.3 months. The pretransplant conditioning regimen consisted of cyclophosphamide (CTX) (120 mg/kg) and 10.20 Gy total body irradiation (TBI) at 6 doses of 1.7 Gy each, administered in 3 daily fractions over 2 days at a dose rate of 15-20 cGy/min. To prevent graft-vs-host disease (GvHD) we used methotrexate (MTX) in one patient and cyclosporin-A (CYA) in the other 17 patients. In addition to CYA, given until day +365, 10 patients received donor marrow depleted of T cells with CAMPATH-1. The residual marrow lymphocytes were always less than 1%. The rate of engraftment was significantly correlated with the number of nucleated cells infused. Neither GvHD nor graft failure were observed among CAMPATH-1 patients. In this group one cytogenetic and one hematologic relapse occurred. The overall actuarial survival at 24 months is 78%. Of the 10 patients treated with donor marrow depleted of T cells, 9 are alive after a median follow-up of 9 months (range 5-18), with an actuarial survival of 90%. Of the other 8 patients transplanted with untreated marrow, 5 are alive after a median follow-up of 19.3 months (range 3.7-24) and the actuarial survival is 63.8%. This pilot study seems to demonstrate that T-cell depletion of donor bone marrow with CAMPATH-1 is effective to prevent GvHD, while the risk of graft failure can be avoided using a "standard" conditioning regimen including a fractionated TBI with a fast dose rate and a prolonged administration of CYA at the maximum tolerable dosage. While the high frequency of relapses suggests the employ of more aggressive anti-leukemic conditioning regimens in CAMPATH-1 treated marrow recipients.

Interferon therapy for Ph1 positive CML patients relapsing after T cell-depleted allogeneic bone marrow transplantation.

Eighteen chronic myeloid leukemia patients with hematological (four patients) or only cytogenetic (14 patients) relapse occurring after T cell-depleted allogeneic bone marrow transplantation (BMT) have been treated with alpha 2b interferon (IFN) at a starting dose of 5 x 10(6) i.u./m2 subcutaneously three times a week. All four patients with hematological relapse achieved long-lasting hematological remission without reduction of bone marrow Ph1 positive cells. When IFN was started the median percentage of bone marrow Ph1-positive metaphases was 50% (range 9-100) for the 14 patients with cytogenetic relapse. Twelve (85.7%) of these patients are alive with a median follow-up of 25 months (range 20-37 months) from cytogenetic relapse and 33 months (range 27-49 months) from BMT. Six (43%) of the 14 patients progressed to hematological relapse and eight patients (57%) are still in hematological remission with two patients achieving complete cytogenetic remission confirmed at molecular level by disappearance of the M-BCR rearranged band. IFN therapy may be a good alternative to conventional chemotherapy for transplanted CML patients with hematological relapse and the treatment of choice for patients with a persistent cytogenetic relapse occurring after T cell-depleted BMT.

Hodgkin/Reed-Sternberg cells and Hodgkin's disease in patients with B-cell chronic lymphocytic leukaemia: an immunohistological, molecular and clinical study of four cases suggesting a heterogeneous pathogenetic background.

We report the immunohistological, molecular and clinical findings in four patients affected by B-cell chronic lymphocytic leukaemia (CLL) who developed "Richter's syndrome with Hodgkin's disease (HD) features" or "CLL with Hodgkin's transformation", all characterised by the presence of typical Hodgkin/Reed-Sternberg (H/RS) cells in lymph node biopsies. In three cases the nodal involvement by CLL was demonstrated both by the presence of a predominant background of CD5/CD19/CD23+ small lymphocytes and an IgH monoclonal rearrangement revealed by PCR analysis. Conversely, in the remaining case there was neither immunohistological nor molecular evidence of lymph node involvement by CLL. In all four cases H/RS cells were Epstein-Barr virus (EBV) latent membrane protein (LMP-1) positive. These findings suggest that the presence of H/RS cells in the first three patients, who had CLL/HD nodal involvement, might be related to transformation or clonal evolution of CLL cells in H/RS cells, which is in keeping with use of the term "CLL with Hodgkin's transformation". In the fourth case a de novo HD may be postulated, representing a second malignancy presumably not clonally related to CLL. In all cases a key pathogenetic role of EBV is suggested by the expression of LMP-1 in H/RS cells. Our findings indicate that the presence of typical H/RS cells in lymph node biopsies in CLL patients may reflect a heterogeneous pathogenetic background. The different clinico-pathologic settings should be taken into consideration because of their possible implications for patients' treatment and prognosis.

Combination of Cytosine-Arabinoside (ARA-C), Cyclophosphamide and Prednisone in the Treatment of B-Chronic Lymphocytic Leukemia in Advanced Stages and Progressive Disease.

Recently the major advances in B-chronic lymphocytic leukemia (B-CLL) have been in defining biological characteristics and prognostic criteria. However it remains to be established which is the best therapeutic approach following the first line treatment, particularly when the patients are completely unresponsive to the standard treatment using Chlorambucil (CHL) and Prednisone (PDN) and the disease is progressive. We report the results of a combination regimen using Cytosine-Arabinoside (ARA-C), Cyclophosphamide (CTX) and PDN in 19 B-CLL patients with advanced disease, resistant to CHL + PDN. The treatment schedules were as follows: Schedule A) ARA-C 60mg/sqm from day 1 to 4 s.c., CTX 75 mg/sqm from day 1 to 4 i.v., PDN 40 mg/sqm from day 1 to 4 p.o.: courses were repeated every 4 weeks for 6 months; Schedule B) ARA-C 100 mg/sqm from day 1 to 7 s.c., CTX 100 mg/sqm from day 8 to 14 i.v., PDN 40 mg/sqm from day 1 to 21 p.o.: courses were repeated every 4 weeks for 6 months. Fourteen pts were treated with schedule A and 5 pts with schedule B. We observed 9 partial remissions (PR), 5 cases with no response and 5 patients with progression. The median duration of PR is 20 months and two patients remain in PR after 18 and 60 months, respectively. The combination of ARA-C, CTX plus PDN is highly effective in advanced stages and in pretreated B-CLL but we need larger randomized studies to draw more definitive conclusions.

Adhesion molecule expression on B-cells from acute and chronic lymphoid leukemias.

Adhesion molecule expression on acute and chronic lymphoid leukemia cells of B lineage (B-ALL and B-CLL) may subserve several functions. Adhesion of leukemic cells to endothelial cells and to extracellular matrix components is relevant to homing, trafficking and spread of the malignant cells, and thus to clinical presentation, course and disease prognosis. Adhesive interactions between malignant cells and accessory cells, particularly stromal cells in the bone marrow environment, may support growth of the malignant cells via cytokine-delivered messages. They may also deliver signals that prevent or trigger programmed cell death of tumor cells. Here we review data on the adhesive phenotype of leukemic blasts from pro-B (CALLA +) ALL and of cells from B-CLL cases. We show that expression of certain adhesion molecules may help define disease subsets with distinctive clinical and prognostic features. One adhesion molecule, the lymphocyte homing receptor CD44, allows definition of two groups of B-CLL patients with significantly different survival.

Bendamustine in combination with ofatumumab in relapsed or refractory chronic lymphocytic leukemia: a GIMEMA Multicenter Phase II Trial.

We conducted a phase II, noncomparative, open-label, multicenter GIMEMA (Gruppo Italiano Malattie EMatologiche dell'Adulto) study (CLL0809) to assess the efficacy and safety of bendamustine in combination with ofatumumab (BendOfa) in relapsed/refractory chronic lymphocytic leukemia (CLL). Forty-seven patients from 14 centers were evaluated. Therapy consisted of bendamustine (70 mg/m(2)) for 2 consecutive days every 28 days, and ofatumumab 300 mg on day 1 and 1000 mg on day 8 during the first cycle, and 1000 mg on day 1 subsequently. Treatment was administered up to six cycles. The overall response rate (ORR), as per intention-to-treat analysis, was 72.3% (95% confidence of interval (CI), 57-84%), with 17% complete responses. After a median follow-up of 24.2 months, the overall survival was 83.6% (95% CI, 73.0-95.7%) and the progression-free survival (PFS) was 49.6% (95% CI, 35.9-68.6%). The median PFS was 23.6 months. Univariate and multivariate analyses were used to identify clinical and biological characteristics associated with ORR and PFS. Myelosuppression was the most common toxicity; grade ≥3 neutropenia was observed in 61.7% of patients; however, grade ≥3 infections occurred in 6% of patients. BendOfa is feasible and effective in relapsed/refractory CLL patients, including patients with high-risk clinical and biological features.

Late listeriosis after fludarabine plus prednisone treatment.

Fludarabine plus prednisome treatment in patients with chronic lymphocytic leukaemia has recently been correlated with an increased incidence of Listeria monocytogenes infections. We observed a patient with chronic lymphocytic leukaemia who received fludarabine plus prednisone and developed a fatal listeriosis 7 months later. A further case of listeriosis has recently been described in a man who developed the infection 2 years after successful fludarabine treatment while he had a normal CD4 lymphocyte count. These cases suggest that the risk period for the development of such an unusual infection could be unexpectedly and unpredictably long. This data could be important in the evaluation of a possible antibacterial prophylaxis schedule and in the wariness about the potential danger of food known to contain L. monocytogenes.

Chronic lymphocytic leukemia: from palliative therapy to curative intent.

High dose therapy and stem cell transplantation is increasingly being used for treatment of CLL. The present article summarizes available results reported in literature on the use of high dose therapy followed by allogeneic or autologous hemopoietic precursor infusion. Transplant procedures seem a feasible approach, especially autografts, while allogeneic transplant has been associated with a higher mortality rate. Interesting clinical/biological results have been reported for both allogeneic and autologous transplants but prospective large clinical trials are needed to establish their real value. We consider important issues of stem cell transplantation in CLL patients, such as the kind of transplant (allogeneic vs autologous), the optimum timing, the selection of patients, the value and type of purging and, above all, impact on survival.

Fludarabine and prednisone in pretreated and refractory B-chronic lymphocytic leukemia (B-CLL) in advanced stages.

BACKGROUND: The use of fludarabine, an analog of vidarabine with significant lymphocytolytic activity, has provided encouraging results in the treatment of patients with non Hodgkin's lymphoma (NHL) and sometimes in phase II studies of solid tumors and acute leukemias. Fludarabine has also been shown to be effective in B-CLL. Some studies referred results using fludarabine in high-risk untreated or pretreated CLL patients. METHODS: We report treatment results with fludarabine and prednisone in 22 pretreated B-CLL patients in progressive disease, refractory to CHL and other regimens. RESULTS: One patient obtained a complete response (CR), 8 obtained a partial response (PR). Thirteen failed to respond to treatment. Among the partial responders, 1 patient had fatal pneumonia while neutropenic, another died of disease progression. Among the non responders, 7 died of progressive disease, and 3 evolved into high-grade NHL. Three are still alive in disease progression. CONCLUSIONS: We confirm the effectiveness of fludarabine in pretreated and resistant B-CLL, but in the light of the considerable toxicity observed in this series, we believe that the drug would be better employed as a first-line approach to "younger" untreated B-CLL patients.