A rescue approach in refractory diffuse large B-cell lymphoma with obinutuzumab-redirected cytokine-induced killer cells: a first-in-human case report.

Not available.

Evans syndrome secondary to chronic lymphocytic leukaemia: presentation, treatment, and outcome.

Evans syndrome (ES) is defined by the combination (either simultaneous or sequential) of immune thrombocytopenia (ITP) and autoimmune haemolytic anaemia (AIHA). When related to secondary conditions, ES may arise in patients with chronic lymphocytic leukaemia (CLL), which is frequently associated to autoimmune cytopenias (AIC). We analysed 25 patients with ES secondary to CLL, which were identified from a large series of consecutive patients with CLL, diagnosed and followed up in two institutions. They represented 2.9 % of the whole series. Thirteen patients presented with concurrent ITP and AIHA (simultaneous ES), while others developed the two AIC sequentially. Occurrence of ES was associated with unfavourable biological prognostic factors like ZAP-70 expression, unmutated immunoglobulin heavy chain variable region gene status, 17-p13 deletion and TP53 gene mutations. Of note, the majority of patients with ES (66 %) had stereotyped B cell receptor configuration. Most patients had short-lasting remissions and required second-line treatments to control the autoimmune manifestations of ES. Patients with ES were associated with inferior survival compared to patients not developing AIC, especially when ES developed early in the course of CLL, although the reduced survival was not confirmed by multivariate analysis. In conclusion, ES secondary to CLL is a difficult-to-treat complication, characterised by adverse biological features and clinical outcome.

Epstein-Barr virus DNA load in chronic lymphocytic leukemia is an independent predictor of clinical course and survival.

The relation between Epstein-Barr virus (EBV) DNA load and clinical course of patients with chronic lymphocytic leukemia (CLL) is unknown. We assessed EBV DNA load by quantitative PCR at CLL presentation in mononuclear cells (MNC) of 220 prospective patients that were enrolled and followed-up in two major Institutions. In 20 patients EBV DNA load was also assessed on plasma samples. Forty-one age-matched healthy subjects were tested for EBV DNA load on MNC. Findings were validated in an independent retrospective cohort of 112 patients with CLL. EBV DNA load was detectable in 59%, and high (≥2000 copies/µg DNA) in 19% of patients, but it was negative in plasma samples. EBV DNA load was significantly higher in CLL patients than in healthy subjects (P < .0001). No relation was found between high EBV load and clinical stage or biological variables, except for 11q deletion (P = .004), CD38 expression (P = .003), and NOTCH1 mutations (P = .05). High EBV load led to a 3.14-fold increase in the hazard ratio of death and to a shorter overall survival (OS; P = .001). Poor OS was attributable, at least in part, to shorter time-to-first-treatment (P = .0008), with no higher risk of Richter's transformation or second cancer. Multivariate analysis selected high levels of EBV load as independent predictor of OS after controlling for confounding clinical and biological variables. EBV DNA load at presentation is an independent predictor of OS in patients with CLL.

Clonal populations of hematopoietic cells with paroxysmal nocturnal hemoglobinuria phenotype in patients with splanchnic vein thrombosis.

INTRODUCTION: Splanchnic vein thrombosis (SVT) is a serious complication in patients with paroxysmal nocturnal hemoglobinuria (PNH). Mutant PNH clones can be associated with an increased risk of SVT even in the absence of overt disease, but their prevalence in non-selected SVT patients remains unknown. MATERIALS AND METHODS: Patients with objective diagnosis of SVT and without known PNH were tested for the presence of PNH clone using high-sensitivity flow cytometric analysis. RESULTS: A total of 202 SVT patients were eligible, 58.4% were males, mean age was 54.6years (range 17-94), site of thrombosis was portal in 103 patients, mesenteric in 67, splenic in 37, and supra-hepatic in 10. SVT was associated with JAK2 V6167F in 28 of 126 (22.2%) screened patients, liver cirrhosis in 15.3% patients, recent surgery in 10.9%, and myeloproliferative neoplasm in 10.6%, whereas in 34.6% of patients neither permanent nor transient risk factors were detected. None of the patients had a clearly demonstrable PNH clone, but in two patients (0.99%, 95% CI 0.17-3.91) we observed very small PNH clones (size 0.014% and 0.16%) confirmed in two independent samples. One patient had portal vein thrombosis and no associated risk factors, the second had superior mesenteric vein thrombosis and inflammatory bowel disease. CONCLUSIONS: Very small PNH clones can be detected in patients with SVT and no clinical manifestations of disease. Future studies are needed to explore the potential role of this finding in the pathogenesis of SVT.

Clinical significance of LAIR1 (CD305) as assessed by flow cytometry in a prospective series of patients with chronic lymphocytic leukemia.

Most patients affected by chronic lymphocytic leukemia are diagnosed by flow cytometry. Several immunophenotypic markers have been identified as significant and independent prognostic variables, especially from retrospective cohorts. However, while attractive because their detection is inexpensive and feasible in most laboratories, only few have been validated by independent series. The expression of leukocyte-associated immunoglobulin-like receptor-1 (also known as LAIR1, LAIR-1 or CD305), an inhibitor of B-cell receptor-mediated signaling, has been reported to be lacking in high-risk chronic lymphocytic leukemia. However, its correlation with biological variables and its prognostic significance remain unknown. We investigated 311 consecutive patients, prospectively enrolled since 2007. Methods for studying patients were standardized and included clinical assessment, immunophenotype, fluorescence in situ hybridization, and status of immunoglobulin heavy chain variable region genes. Overall, 22.1% of patients had Binet stage B or C disease, 38.5% had unmutated immunoglobulin genes, 15.1% had high-risk cytogenetic abnormalities, 23.4% were CD38(+), 37.8% CD49d(+), and 59.8% LAIR1(+). Expression of LAIR1 was inversely related to that of CD38 (P=0.0005), but was not associated with CD49d expression (P=0.96). A significantly lower expression of LAIR1 was observed in patients with Binet stage B or C disease (P=0.023), and in the presence of high-risk cytogenetic abnormalities (P=0.048) or unmutated immunoglobulin heavy chain variable region genes (P<0.0001). At univariate analysis LAIR1(+) was significantly associated with longer time to first treatment (P=0.0002). This favorable effect of LAIR1(+) was confirmed by multivariate analysis (hazard ratio=2.1, P=0.03 for LAIR1). Our results indicate that LAIR1 expression is a reliable and inexpensive marker capable of independently predicting time to first treatment in newly diagnosed unselected patients with chronic lymphocytic leukemia.

B-cell receptor configuration and mutational analysis of patients with chronic lymphocytic leukaemia and trisomy 12 reveal recurrent molecular abnormalities.

Trisomy 12 (+12) is the third most frequent cytogenetic aberration in chronic lymphocytic leukaemia (CLL) retrievable both as the sole chromosomal abnormality or in association with additional alterations. NOTCH1 mutations are known to be more prevalent among +12 patients, whereas mutations of FBXW7, a gene involved in NOTCH1 degradation, that lead to the constitutional activation of NOTCH1 have not been investigated in this setting. We analyzed a unicentric cohort of 44 +12 patients with CLL for mutations of TP53, NOTCH1 and FBXW7 genes, and we correlated them with B-cell receptor (BCR) configurations. FBXW7, TP53 and NOTCH1 mutations were identified in 4.5%, 6.8% and 18.2% of patients, respectively. FBXW7 and NOTCH1 mutations appeared in a mutually exclusive fashion, suggesting that both aberrations might affect the same biological pathway. We found that 44.1% of +12 CLL patients had stereotyped B-cell receptors, which is significantly higher than that observed in patients with CLL and no +12 (27%, p = 0.01). Subsets #1, #8, #10, #28 and #59 were the most represented stereotyped patterns, and IGHV4-39*01 was the gene configuration most commonly used. There was a significantly higher risk for Richter's syndrome (RS) transformation in patients with NOTCH1 or FBXW7 mutations, with four of the seven (57%) patients developing RS and characterized at least by one of the two abnormalities. These observations suggest that, similarly to the aberrations of NOTCH1, FBXW7 gene mutations may also result in cell proliferation and evasion from apoptosis in patients with +12 CLL. Together with the extremely high frequency of stereotyped BCRs and RS transformation, these abnormalities appear to cluster in these CLL patients with additional chromosome 12, suggesting a connection with the prognosis of the disease.

Double productive immunoglobulin sequence rearrangements in patients with chronic lymphocytic leukemia.

The immunoglobulin heavy chain variable (IGHV) gene mutational status represents a major prognostic marker in chronic lymphocytic leukemia (CLL). Usually, the prognostic implications of IGHV gene analysis can be reliably ascertained but, occasionally, double productive rearrangements have been detected. Clinical presentation and biological features of such cases are unknown. Sixty patients with morphologically and phenotypically monoclonal CLL but double productive IGHV rearrangements were retrospectively identified by mRNA analysis from three Hematology Institutions. Clinical and biological features and survival of these 60 patients were compared with a control group of patients with CLL and single IGHV rearrangement. A prospective registry was used to assess the epidemiology of double productive IGHV among incidental patients with CLL. Using standard criteria to define IGHV-mutated (M) or unmutated (U) cases, 39 of the 60 patients (65%) with double productive IGHV rearrangement had concordant status (23 MM, 16 UU), while 21 (35%) had discordant IGHV status. As compared with M patients, the MM ones had lower CD38 expression, more favorable cytogenetics and more indolent clinical behavior. Cases with UU had similar characteristics of U patients. Discordant cases presented with adverse prognostic features and had an aggressive clinical behavior requiring early treatment, similar to U patients. The prevalence of double IGHV was 3.1%. Patients with CLL with double concordant mutational status (MM or UU) have a clinical course similar to that of the corresponding single IGHV status, while those exhibiting discordant status represent a high risk population. This may help correct stratification within clinical trials.

Immune thrombocytopenia in patients with chronic lymphocytic leukemia is associated with stereotyped B-cell receptors.

PURPOSE: To assess biologic features related to the development of immune thrombocytopenia (ITP) in patients with chronic lymphocytic leukemia (CLL). EXPERIMENTAL DESIGN: We retrospectively analyzed 463 patients with CLL with available immunoglobulin heavy-chain variable (IGHV) gene status and B-cell receptor (BCR) configuration [heavy-chain complementary-determining region 3 (HCDR3)], of whom thirty-six developed ITP, according to previously defined criteria. Most of them had available cytogenetic analysis. RESULTS: We observed a significant association between ITP occurrence and IGHV unmutated gene status (P < 0.0001), unfavorable cytogenetic lesions (P = 0.005), and stereotyped HCDR3 (P = 0.006). The more frequent stereotyped HCDR3 subsets were #1 (IGHV1-5-7/IGHD6-19/IGHJ4; 16 of 16 unmutated) and #7 (IGHV1-69 or IGHV3-30/IGHD3-3/IGHJ6; 13 of 13 unmutated), both being significantly more represented among patients developing ITP (P = 0.003 and P = 0.001, respectively). Moreover, restricting the analysis to unmutated patients, subset #7 confirmed its independent significant association with the occurrence of ITP (P = 0.013). Both unmutated IGHV mutational status, del(11)(q23) and stereotyped BCR were significantly associated with shorter time to ITP development (P < 0.0001, P = 0.02, and P = 0.005, respectively) than other patients. CONCLUSION: Our data suggest that patients with CLL and peculiar BCR conformations are at higher risk of developing secondary ITP and that stereotyped BCR may be involved in the pathogenesis of this complication.

Telomere length and telomerase levels delineate subgroups of B-cell chronic lymphocytic leukemia with different biological characteristics and clinical outcomes.

BACKGROUND: B-cell chronic lymphocytic leukemia is a clinically heterogeneous disease; some patients rapidly progress and die within a few years of diagnosis, whereas others have a long life expectancy with minimal or no treatment. Telomere length and telomerase levels have been proposed as prognostic factors; however, very few cases have been characterized for both parameters and no study has analyzed the prognostic value of the telomere/telomerase profile. DESIGN AND METHODS: One hundred and seventy-three cases of chronic lymphocytic leukemia were characterized for telomere lengths and telomerase levels by real-time polymerase chain reaction. Data were correlated with established prognostic markers, IGVH mutational status and chromosomal aberrations, and clinical outcome. RESULTS: Telomere lengths were inversely correlated with telomerase levels (r(s) = -0.213; P = 0.012), and most of the cases of chronic lymphocytic leukemia with high levels (above median) of telomerase had short (below median) telomeres (P = 0.0001). Telomerase levels were higher and telomeres were shorter in unmutated IGVH cases than in mutated IGVH ones (P<0.0001). Chronic lymphocytic leukemias with 11q, 17p deletion or 12 trisomy had significantly higher levels of telomerase and shorter telomeres than those with no chromosomal aberration or the sole 13q deletion (P < 0.001). Telomere length/telomerase level profiles identified subgroups of patients with different clinical outcomes (P < 0.0001), even within the subsets of chronic lymphocytic leukemia defined by IGVH mutational status or chromosomal aberrations. Short telomere/high telomerase profile was independently associated with more rapid disease progression. CONCLUSIONS: Comprehensive analyses of telomeres, telomerase, chromosomal aberrations, and IGVH mutational status delineate groups of chronic lymphocytic leukemias with distinct biological characteristics and clinical outcomes. The telomere/telomerase profile may be particularly useful in refining the prognosis of chronic lymphocytic leukemia patients with mutated IGVH and no high-risk chromosomal aberrations.

Identical IGHV-D-J gene rearrangement may precede the clinical onset of chronic lymphocytic leukemia by several years.

The pathogenesis of chronic lymphocytic leukemia (CLL) has not been fully elucidated. Moreover, the time required for the initial B lymphocyte IGH gene rearranged clone to manifest as a clinical entity remains unknown. We searched for previous IGH gene rearranged B lymphocyte clones in healthy people who developed CLL and estimated the time for the clone to become clinically detectable. To this aim, we identified all incident cases of CLL diagnosed in a cohort of 15,055 healthy subjects aged 18-65 years enrolled in a prospective survey on thrombophilia. Seven CLL cases were identified at a median follow-up of 54 months (range, 18-89). The estimated incidence was 0.46 cases/10,000 person-years (CI: 0.17-1.00). A PCR was performed to detect IGH gene rearrangement at enrollment and at CLL diagnosis. Comparison was possible in six subjects. In five, the same IGH gene rearrangement and gene sequence were already present 39-89 months before CLL diagnosis. A mutated status was identified in four of five cases. The median age at diagnosis was 66.2 years, and all subjects were asymptomatic. Two patients expressing the IGHV1-69 gene with an unmutated status required treatment 16 and 40 months after diagnosis. The IGHV4 family genes were rearranged in the remaining cases, all showing a mutated status. No additional rearrangements or mutations in the rearranged gene were found during follow-up. An identical clonal IGH gene rearrangement may precede CLL diagnosis by several years.

ZAP-70 expression is associated with increased risk of autoimmune cytopenias in CLL patients.

Autoimmune cytopenias (AIC) are frequent in chronic lymphocytic leukemia (CLL) patients, but risk factors and prognostic relevance of these events are controversial. Data about the influence on AIC of biological prognostic markers, as ZAP-70, are scanty. We retrospectively evaluated AIC in 290 CLL patients tested for ZAP-70 expression by immunohistochemistry on bone marrow biopsy at presentation. They were 185 men, median age 63 years, 77.9% Binet stage A, 17.6% B and 4.5% C. AIC occurred in 46 patients (16%): 31 autoimmune hemolytic anemias, 10 autoimmune thrombocytopenias, four Evans syndromes, and one pure red cell aplasia. Of the 46 cases of AIC, 37 (80%) occurred in ZAP-70 positive patients and nine (20%) in ZAP-70 negatives. ZAP-70 expression [Hazard Ratio (HR) = 7.42; 95% confidence interval (CI): 2.49-22.05] and age >65 years (HR = 5.41; 95% CI: 1.67-17.49) resulted independent risk factors for AIC. Among the 136 patients evaluated both for ZAP-70 expression and IGHV status, the occurrence of AIC was higher in ZAP-70 positive/IGHV unmutated cases (35%) than in patients ZAP-70 negative/IGHV mutated (6%) or discordant for the two parameters (4%; P < 0.0001). In ZAP-70 positive patients, occurrence of AIC negatively influenced survival (HR = 1.75; 95% CI: 1.06-2.86). The high risk of developing AIC in ZAP-70 positive CLL, particularly when IGHV unmutated, should be considered in the clinical management.

Impact of immune thrombocytopenia on the clinical course of chronic lymphocytic leukemia.

The prevalence, clinical characteristics, and prognostic significance of immune thrombocytopenia (IT) in patients with chronic lymphocytic leukemia (CLL) have not been clearly determined. To clarify this, we retrospectively analyzed 1278 consecutive newly diagnosed patients with CLL. Criteria for IT diagnosis included the following: rapid (< 2 weeks) and severe fall (half of the initial level and below 100 x 10(9)/L) in platelet count; normal or augmented megakaryocytes in bone marrow; no or limited (not palpable) splenomegaly; no cytotoxic treatment in the preceding month. Sixty-four patients (5%) were diagnosed with IT. The median time to IT from CLL diagnosis was 13 months (range, 0-81 months), and median platelet count at IT diagnosis was 14 x 10(9)/L (range, 1-71 x 10(9)/L). Fifty-six of the 64 patients (87%) received treatment for IT. The probability of responding to treatment for IT was significantly higher for patients receiving chemotherapy with or without steroids than for patients treated with intravenous immunoglobulins with or without steroids (P = .01). The development of IT was significantly associated with unmutated IgVh, a positive direct antiglobulin test, and the occurrence of autoimmune hemolytic anemia. Patients with CLL and IT had poorer survival than other patients with CLL (5-year overall survival 64% vs 82%, P < .001), and this effect was independent from common clinical prognostic variables.

Gene therapy of thyroid cancer via retrovirally-driven combined expression of human interleukin-2 and herpes simplex virus thymidine kinase.

OBJECTIVE AND DESIGN: Based on our clinical experience with combined gene therapy of glioblastoma, we developed a retroviral vector expressing two therapeutic genes (i.e. thymidine kinase of herpes simplex virus, HSV-TK, and interleukin-2, IL-2) and evaluated its efficiency in vitro and in vivo. METHODS: Expression of therapeutic genes in transduced thyroid carcinoma cells was analyzed by real-time RT-PCR. Ganciclovir sensitivity of infected cells was assessed in vitro in thyroid carcinoma cell lines and in vivo in nude mice bearing xenografted thyroid cancers. The combined effect of IL-2/HSV-TK was compared with the effect of IL-2 alone. RESULTS: Expression of therapeutic genes was higher in differentiated than in anaplastic thyroid carcinoma cells. Ganciclovir treatment led to dose- and time-dependent killing of transduced cells in vitro. A bystander effect was demonstrated by using mixtures of infected and non-infected cells. In vivo studies showed a significant reduction of growth and the presence of an inflammatory infiltrate in transduced thyroid tumors expressing IL-2 alone, as compared with non-infected tumors. By using the retroviral vector expressing IL-2/HSV-TK, treatment with ganciclovir led to complete eradication of anaplastic tumors and a >80% reduction of the size of differentiated thyroid carcinomas. Histological analysis of tumor specimens showed extensive necrosis and inflammatory cell infiltrates. The combination of IL-2/HSV-TK plus ganciclovir was significantly more efficient than IL-2 alone in eradicating tumor masses. The bystander effect was also obtained in vivo. CONCLUSIONS: These findings demonstrate the feasibility and efficiency of a combined immunomodulating and suicide gene therapy approach for thyroid carcinomas.

Fluorescent polymerase chain reaction and capillary electrophoresis for IgH rearrangement and minimal residual disease evaluation in multiple myeloma.

BACKGROUND AND OBJECTIVES: Several polymerase chain reaction (PCR)-based techniques for tracking minimal residual disease (MRD) in B-lymphoproliferative disorders have been recently proposed. These procedures show significant variation in sensitivity and specificity. We describe an alternative assay based on fluorescent PCR combined with capillary electrophoresis and GeneScan analysis, to identify the monoclonal immunoglobulin heavy chain (IgH) rearrangement in multiple myeloma (MM) and to provide a semi-quantitative evaluation of MRD by limiting dilutions. DESIGN AND METHODS: Different sets of family specific primers derived from the leader region and from the framework-1 of IgH were used, with a unique reverse fluorescent primer JH. The malignant clone was identified by GeneScan and sequenced. Two tumor primers, mapping in the complementarity determining regions CDRII and CDRIII, were designed for each patient. A comparison between the nested-PCR approach and direct fluorescent PCR was performed for three patients in complete clinical remission after autologous or allogeneic bone marrow transplantation. RESULTS: Thirty-six consecutive patients with MM were screened and monoclonality was identified in about 70% of the cases. Molecular MRD evaluation was performed in 18 patients using tumor primers. This method allowed identification of 1 neoplastic cell among 10(4)-10(6) normal cells. In three cases, negative by nested-PCR and agarose gel electrophoresis, gene scanning showed persistence of the neoplastic clone, despite the negativity of the immunofixation. INTERPRETATION AND CONCLUSIONS: Capillary electrophoresis of fluorescent fragments with gene scanning provides a simple, rapid and reproducible method to detect IgH rearrangement and to evaluate MRD. Furthermore, the sensitivity reached is up to 1 log higher than that of the conventional approach with nested-PCR, even though two steps of specificity are maintained.