Employment of Oligodeoxynucleotide plus Interleukin-2 Improves Cytogenetic Analysis in Splenic Marginal Zone Lymphoma

To compare the efficiency of novel mitogenic agents and traditional mitosis inductors, 18 patients with splenic marginal zone lymphoma (SMZL) were studied. Three cultures using oligodeoxynucleotide (ODN) plus interleukin-2 (IL-2), or TPA, or LPS were setup in each patient. Seventeen/18 cases with ODN + IL2 had moderate/good proliferation (94, 4%) as compared with 10/18 cases with TPA and LPS (55%) (P = .015); 14/18 (77, 7%) cases with ODN + IL2 had sufficient good quality of banding as compared with 8/18 cases (44, 4%) with TPA and LPS. The karyotype could be defined from ODN + IL2-stimulated cultures in all 18 patients, 14 of whom (77, 7%) had a cytogenetic aberration, whereas clonal aberrations could be documented in 9 and in 3 cases by stimulation with LPS and TPA, respectively. Recurrent chromosome aberrations in our series were represented by aberrations of chromosome 14q in 5 patients, by trisomy 12 and 7q deletion in 4 cases each, and by abnormalities involving 11q and 13q in two cases each. These findings show that stimulation with ODN + IL2 offers more mitotic figures of better quality and results in an increased rate of clonal aberrations in SMZL, making this method ideal for prospective studies aiming at the definition of the prognostic impact of cytogenetic aberrations in this disorder

Identifying High-Risk Chronic Lymphocytic Leukemia: A Reappraisal of the Impact of Prognostic and Predictive Factors

Identifying High-Risk Chronic Lymphocytic Leukemia: A Reappraisal of the Impact of Prognostic and Predictive Factor

LATE APPEARANCE of 14q32/IGH TRANSLOCATION In CHRONIC LYMPHOCYTIC LEUKEMIA.

Up to 80% of Chronic Lymphocytic Leukemia (CLL) harbour clonal chromosome aberrations having important clinical implications (i.e. 13q deletion, +12, 11q/ATM and 17p/TP53 deletions). 14q32/IGH rearrangements were recently found in 6-19% of CLL patients and were associated with therapy-demanding disease and inferior outcome. Whereas evidence was provided that some of the classical aberrations, such as 11q-, 17p-, may appear late in CLL clinical history, no information is presently available concerning 14q32/IGH translocations. The aim of this study was i) to analyze the incidence of 14q32/IGH translocations occurring at clonal evolution in CLL, ii) to analyze the clinicobiologic significance of late-appearing 14q32/IGH translocations. One hundred-five CLL cases seen at our institution in a 10-year period were submitted to FISH analysis at diagnosis or before 1st line treatment as part of routine diagnostic workup. In 47 patients with indolent disease (untreated or treated with 1 line without relapse, group 1) FISH analysis was repeated after 48-96 months (median 72). In 58 relapsed patients who started 2nd line treatment (group 2), FISH was performed sequentially before administration of the 2nd line and before each subsequent line of therapy. These 105 patients fulfilled the following criteria: a) diagnosis of bona fide CLL based on morphology and immunophenotyping (CD5/CD19+, CD23+ as minimal requirement), b) clinical records available for review, c) successful FISH analysis at diagnosis and during follow-up. Those cases with t(11;14)(q13;q32)/CCND1-IGH or other 14q32/IGH translocations present at diagnosis were excluded from this study. Sequential FISH studies were performed in all patients on peripheral blood (PB) samples using commercially available probes for the identification of deletions at 13q14, 11q22/ATM, 17p13/TP53, of trisomy 12 and of 14q32/IGH translocations. In 10 patients bone marrow (BM) aspiration and/or lymph node (LN) biopsy were studied by FISH as well. The patients were treated at disease progression as defined by NCI criteria. Refractory disease was defined by stable disease or progressive disease during treatment or disease progression within 6 months of from antileukemic treatment using fludarabine alone or in combination with other agents. Time to chemorefractoriness was measured from date of first line treatment to date of refractoriness to fludarabine containing regimen or date of last follow-up. Overall survival was measured from diagnosis to date of last follow-up or death and from initiation of first line treatment to the date of death or last follow-up. At diagnosis 39% of the cases had 13q-, 14% had +12, 7% had 11q- and 3% had 17p-. A late-appearing 14q32/IGH rearrangement was not detected among 47 patients in group 1, whereas 7/58 cases (12,1%) in group 2 showed a 14q32/IGH break in 16-25% of the cells. These 7 patients had the following aberrations at diagnosis: 13q- and 11q- in 1 case, 13q- in 2 cases; 11q- in 1 case, +12 in 2 cases, no aberrations 1 case. The 14q32 translocation appeared after a median time of 64 months (range 51-91). It was associated with the appearance of 17p- in 3/7 cases with one of these presenting also biallelic del13q. In two cases paired BM or LN sample and PB samples were available for FISH studies and the appearance of IgH translocation in the BM or in the LN sample preceded its appearance in PB by 13-58 months. All 7 cases with late appearing 14q32/IGH translocation developed chemorefractoriness to fludarabine regimen with a median TTC of 27 months (range 12-40 months), as compared with a TTC of 67 months (range 1-143 months) in 51 treated patients who did not develop the 14q32 translocation (p=0.0002). Overall survival did not differ significantly either when measured from diagnosis or from 1st line treatment in 7 patients with 14q32 translocation as compared with the appropriate control. We arrived at the following conclusions: i) a late-appearing 14q32/IGH translocation occurred at a relatively high incidence (12,1%) in patients with relapsing disease and not in patients with stable disease, ii) this aberration involved a minority of cells and, in approximately half of the cases, it was associated with other aberrations, reflecting complex clonal evolution, iii) in 2 assessable cases it first appeared first in the BM or LN; iv) the appearance of 14q32/IGH translocation was associated with shorter TTC

Identifying High-Risk Chronic Lymphocytic Leukemia: A Pathogenesis-Oriented Appraisal of Prognostic and Predictive Factors in Patients Treated with Chemotherapy with or without Immunotherapy

Chronic lymphocytic leukemia (CLL) displays an extremely variable clinical behaviour. Accurate prognostication and prediction of response to treatment are important in an era of effective first-line regimens and novel molecules for high risk patients. Because a plethora of prognostic biomarkers were identified, but few of them were validated by multivariable analysis in comprehensive prospective studies, we applied in this survey stringent criteria to select papers from the literature in order to identify the most reproducible prognostic/predictive markers. Each biomarker was analysed in terms of reproducibility across the different studies with respect to its impact on time to first treatment (TTFT), progression free survival (PFS), overall survival (OS) and response to treatment. We were able to identify the following biomarkers as the most reliable in guiding risk stratification in the daily clinical practice: 17p-/TP53 mutations, IGHV unmutated configuration, short telomeres and 11q-. However, the method for measuring telomere length was not validated yet and 11q- was predictive of inferior OS only in those patients who did not receive FCR-like combinations. Stage and lymphocytosis were predictive of shorter TTFT and age, high serum thymidine kinase levels and poor performance status were predictive of shorter OS. Using our criteria no parameter was found to independently predict for inferior response to treatment

Diagnostic work-up for clinical and prognostic assessment of acute leukaemia

Acute leukaemia is a neoplastic disorder of haematopoietic precursors characterized by a rapid clinical course and a relatively high early death rate in spite of recent therapeutic progress. Prompt and reliable diagnostic and prognostic assessment has a favourable impact on patient outcome. Diagnostic suspicion relies on signs and symptoms of bone marrow failure or quali-/quantitative abnormalities of blood cells, which are accurately detected by modern automated counters. Cytomorphological examination of a stained blood and bone marrow film plays a key role in the diagnostic process, with relevant additional information coming from immunophenotyping of blast cells. Cytogenetic and molecular genetic data are the basis of prognostic stratification. In this context, a coordinated intervention of specialized laboratories combining solid expertise in each field involved in the diagnostic work-up is essential; at the same time, the availability of all the required technologies at the same hospital structure would probably lack efficiency due to the low number of tests performed. The organization of laboratory networks, either at regional or national level, especially in the context of clinical trials, may offer a great opportunity for centralization of more sophisticated technologies in reference laboratories, each highly specialized in a particular field of investigation and all interconnected. The challenge for laboratory haematology is the reorganization of clinical and scientific activity according to this model, without loosing educational potential in favour of new generations of medical doctors, haematologists, biologists and laboratory technicians

Chromosome aberrations detected by conventional karyotyping using novel mitogens in chronic lymphocytic leukemia: Clinical and biologic correlations

CHROMOSOME ABERRATIONS DETECTED BY CONVENTIONAL KARYOTYPING USING NOVEL MITOGENS IN CHRONIC LYMPHOCYTIC LEUKEMIA: CLINICAL AND BIOLOGIC CORRELATION

Chromosome aberrations detected by conventional karyotyping using novel mitogens in chronic lymphocytic leukemia: Clinical and biologic correlations.

To clarify whether karyotype aberrations (KA) involving regions not covered by the standard fluorescence in situ hybridization (FISH) panel have independent prognostic relevance, we evaluated KA by conventional cytogenetics in a learning cohort (LC; n = 166) and a validation cohort (VC; n = 250) of untreated chronic lymphocytic leukemia (CLL) patients. In the VC, novel mitogens were used to improve metaphase generation and TP53, NOTCH1, and SF3B1 mutations were assessed. KA undetected by FISH were found in 35 and 35% of the cases in the LC and VC, respectively. In addition to FISH, KA allowed reclassification of 23 and 26% of cases in the LC and VC, respectively, into a higher cytogenetic risk group. By multivariate analysis, both in the LC and VC, KA other than isolated 13q deletion correlated with a shorter time to first treatment (TFT; P < 0.001 and 0.003, respectively), while a complex karyotype predicted a worse overall survival (OS, P = 0.015 and 0.010, respectively). In the VC, where a comprehensive biologic assessment was performed, a shorter TFT was also predicted by stage (P < 0.001), IGHV mutational status (P = 0.05), and del(17p)/TP53 mutations (P = 0.033) while stage (P = 0.023) and del(17p)/TP53 mutations (P = 0.024) independently predicted a shorter OS. FISH results did not independently impact on TFT and OS, in the LC and VC cohorts; this was also the case for NOTCH1 and SF3B1 mutations in the VC. We suggest that in CLL, conventional karyotyping with novel mitogens could be more effective than FISH for the detection of KA allowing for a more precise refinement of prognosi