Ilaria Giardini

World Health Organization classification in combination with cytogenetic markers improves the prognostic stratification of patients with de novo primary myelodysplastic syndromes

This study correlated chromosomal defects with French–American–British (FAB)/World Health Organization (WHO) classification subtypes, proposed a revised International Prognostic Scoring System (IPSS) cytogenetic grouping; and established which classification, when used with the IPSS cytogenetic categories, best predicted clinical outcome in the myelodysplastic syndromes (MDS). A higher prevalence of chromosomal defects and distinct defects were observed in patients with multi‐lineage dysplasia and a blast cell percentage >10%. Abnormalities of the long arm of chromosome 3, del(7)(q31q35), trisomy 8, del(11)(q14q23), del(12p) and 20q‐ could be segregated from their respective IPSS cytogenetic categories and used to develop new cytogenetic subgroups. Clinical parameters, FAB/WHO classification, IPSS score and standard or revised cytogenetic categories were statistically relevant for overall survival (OS) and progression‐free intervals (PFI) and were included within five distinct multivariate models compared by the Akaike Information Criterion. To predict OS, the best models included age, WHO classification and standard or revised IPSS cytogenetic categories; to predict PFI, the best model included the same variables and revised cytogenetic categories. In conclusion, (i) the WHO classification was associated with a more homogeneous cytogenetic pattern than the FAB classification, (ii) WHO classification and standard/revised IPSS cytogenetic categories were much more effective than IPSS for predicting MDS clinical outcome, (iii) revised cytogenetic subgroups predicted PFI more effectively than standard categories.

Clinical relevance of cytogenetics in myelodysplastic syndromes

Abstract:  Myelodysplastic syndromes (MDS) are a group of heterogeneous stem cell disorders with different clinical behaviors and outcomes. Conventional cytogenetics (CC) studies have demonstrated that the majority of MDS patients harbor clonal chromosome defects. The probability of discovering a chromosomal abnormality has been increased by fluorescence in situ hybridization (FISH), which has revealed that about 15% of patients with a normal chromosome pattern on CC may instead present cryptic defects. Cytogenetic abnormalities, except for the interstitial long‐arm deletion of chromosome 5 (5q−), are not specific for any French‐American‐British (FAB)/World Health Organization (WHO) MDS subtypes, demonstrate the clonality of the disease, and identify peculiar morphological entities, thus confirming clinical diagnosis. In addition, chromosome abnormalities are independent prognostic factors predicting overall survival and the likelihood of progression in acute myeloid leukemia.

Molecular Genetics of Acute Myeloid Leukemia

Abstract: Recurring chromosomal abnormalities are detected in most patients with acute myeloid leukemia (AML). They may be associated with a distinct AML FAB subtype or may identify distinct clinicobiological entities within the same FAB subtype. Therefore, cytogenetic investigation has a pivotal role in AML diagnosis. In addition, it is one of the most valuable prognostic determinants of the disease, as recently demonstrated. The development of new molecular techniques, such as reverse transcriptase polymerase chain reaction and fluorescence in situ hybridization, has allowed perfect definition of the chromosome regions containing genes with a crucial role in normal hemopoiesis and leukemia. Understanding the action of such genes provides new insights into AML pathogenesis and has led us to envisage new therapeutic options.

Long‐term follow up with conventional cytogenetics and band 13q14 interphase/metaphase in situ hybridization monitoring in monoclonal gammopathies of undetermined significance

Summary. One‐third of patients with monoclonal gammopathy of undetermined significance (MGUS) may progress to multiple myeloma (MM) and may develop a long arm deletion of chromosome 13 (13q–). As the incidence of 13q–, time of development and prognostic impact in MGUS patients is still under debate, we decided to perform serial sequential conventional cytogenetics (CC) and metaphase/interphase fluorescence in situ hybridization (FISH) analyses on bone marrow mononuclear cells obtained from 18 asymptomatic, untreated MGUS patients. Median follow up was 30 months (range 6–72). Interphase FISH identified a 13q14 deletion in five out of 18 patients (on clinical diagnosis in one patient and during the follow up in the remaining four patients). Subsequently, metaphase FISH and CC also identified the deletion in four out of five patients. All five of the patients progressed to MM 6–12 months after 13q– identification, without developing any FISH determined JH rearrangements. MM progression also occurred in two other karyotypically normal patients. We conclude that: (i) the extent of the 13q deletion does not vary during the clinical outcome; (ii)13q– plays a crucial role in MGUS/MM pathogenesis and confers a proliferative advantage to clonal plasma cells being initially demonstrated by interphase FISH and only afterwards by metaphase FISH and CC; and (iii) association of 13q– with t(4;14)(p16.3;q32) remains to be demonstrated. However, a transition from MGUS to MM may also occur in patients with normal karyotypes or other abnormalities, suggesting the possibility of distinct pathogenetic pathways.

Chronic lymphocytic leukemia with del13q14 as the sole abnormality: dynamic prognostic estimate by interphase-FISH

This study analyzed 140 patients with isolated del13q14 on interphase FISH (I‐FISH), to identify subsets with a different progression risk and to assess the acquisition of additional chromosomal abnormalities (clonal evolution) in treatment‐naïve del13q14 patients. A monoallelic deletion (del13qx1) was detected in 123 cases (88%), a biallelic deletion (del13qx2) in eight and a mosaic of monoallelic and biallelic deletions (del13qx1/del13qx2) in nine. In 33% of cases, deletion encompassed the Rb1 locus The median percentage of abnormal nuclei was 50% (15%–96%), and it was higher in patients with a biallelic/mosaic pattern in comparison with patients with monoallelic deletion. Sixty two patients (44%) have been treated; 5‐year treatment free survival rate was 56% and the median treatment free survival was 65 months. The baseline percentage of deleted nuclei, as a continuous variable, was related to progression (HR: 1.02; p = 0.001). According to deletion burden, three groups were identified: 64 cases (46%) had <50% deleted nuclei, 47 (33%) had 50–69% deleted nuclei, and 29 (21%) had ≥70% deleted nuclei. The 5‐year untreated rate was 70.5% , 52.6% and 28.7% (p < 0.0001), respectively. In multivariate analysis using IGHV mutational status, presence of a nullisomic clone, CD38 expression and percentage of deleted nuclei as covariates, only IGHV mutational status and the percentage of deleted nuclei were independent risk factors for treatment. In 103 patients serially monitored by I‐FISH before starting any treatment, we observed a significant increase in the proportion of del13q14 cells, and this increase affected the risk of subsequent treatment requirement (HR 2.54, p = 0.001). The appearance of a new clone was detected in 16 patients (15.5%) and chromosome 13 was involved in 14 of them. I‐FISH monitoring proves worthwhile for a dynamic risk stratification and for planning clinical surveillance. Copyright

Detection of TET2 abnormalities by fluorescence in situ hybridization in 41 patients with myelodysplastic syndrome

TET2 haplo-insufficiency occurs through different molecular mechanisms and is promptly revealed by array comparative genomic hybridization, single nucleotide polymorphism (SNP) array, and next-generation sequencing (NGS). Fluorescence in situ hybridization (FISH) can effectively demonstrate TET2 deletions and is often used to validate molecular results. In the present study 41 MDS patients with and without 4q abnormalities were analyzed with a series of bacterial artificial chromosome (BAC) probes spanning the 4q22.3-q25 region. On conventional cytogenetic (CC) studies, a structural defect of the long arm of chromosome 4 (4q) was observed in seven patients. In three, one each with a t(1;4)(p21;q24), an ins(5;4)(q23;q24qter), and a t(4;17)(q31;p13) as the sole chromosomal abnormality, FISH with the RP11-356L5 and RP11-16G16 probes, which cover the TET2 locus, produced one signal only. Unexpectedly, this same result was achieved in 3 of the remaining 34 patients. Thus, a TET2 deletion was observed in a total of six patients (14.6%). TET2 deletion was not correlated with any particular clinical findings or outcome. These findings demonstrate that 1) FISH is an effective and economical method to reveal cryptic abnormalities of band 4q22-q24 resulting in TET2 deletions; 2) in these patients, TET2 deletion is the unifying genetic event; and 3) the different breakpoints within the 4q22-q25 region suggest that deletions are not mediated by repetitive sequences.

BCL3 translocation in CLL with typical phenotype: assessment of frequency, association with cytogenetic subgroups, and prognostic significance

BCL3 translocation, occurring as a consequence of t(14;19) (q32.3;q13.2), is a recurrent genetic lesion shared by several B-cell malignancies, including chronic lymphocytic leukaemia (CLL), chronic B-cell lymphoproliferative disorders, aggressive lymphomas and Hodgkin lymphoma (Michaux et al, 1996, 1997; McKeithan et al, 1997; Au et al, 2002; Martı́n-Subero et al, 2006, 2007; Huh et al, 2007, Chapiro et al, 2008; Kelly et al, 2008). Few series of CLL harbouring BCL3 translocation have been so far reported. All these reports agreed that CLL harbouring BCL3 translocation is preferentially associated with atypical morphology and phenotype, and may be best described as: (i) small B-cell leukaemias with atypical morphology (7/7 cases) and a phenotypic score ranging from 1 to 3 (7/7 cases) (Huh et al, 2007); (ii) CLL with atypical morphology (20/30 cases) and low phenotypic score (14/18 cases) (Chapiro et al, 2008); and (iii) CLL with atypical morphology/phenotype (12/27 cases), or lacking CD23 expression (5/8 cases) (Martı́n-Subero et al, 2007); (iv) CLL lacking CD23 expression (3/4 cases) (Kelly et al, 2008). From a biological point of view, BCL3 translocations in atypical CLL have been associated with presence of +12, unmutated IGHV genes, and biased usage of the IGHV4-39/IGHD6-13/IGHJ5 rearrangement (Martı́nSubero et al, 2007; Chapiro et al, 2008). From a clinical point of view, BCL3 translocations in atypical CLL might mark an aggressive course and an increased risk of transformation to aggressive lymphoma (Huh et al, 2007; Martı́n-Subero et al, 2007; Chapiro et al, 2008; Kelly et al, 2008). Based on these observations, it has been suggested that B-cell lymphoproliferative disorders carrying the t(14;19)(q32.3;q13.2) translocation might represent an entity distinct from CLL, provisionally termed as ‘t(14;19)-positive small B-CLL leukaemia’ (Huh et al, 2007). Though rarely, however, BCL3 translocation may also be detected in true CLL with a typical phenotype (Martı́n-Subero et al, 2007; Chapiro et al, 2008). A comprehensive characterization of the frequency, distribution and prognostic relevance of BCL3 translocation in CLL showing a typical phenotype is still lacking, and represents the aim of this study. The study was based on 225 CLL harbouring a typical phenotype (phenotypic score > 3). Patients provided informed consent in accordance with local Institutional Review Board requirements and the Declaration of Helsinki. BCL3 translocation was analysed by fluorescent in situ hybridization (FISH; split signal probe, Dako, Glostrup, Denmark) on fresh/ cryopreserved peripheral blood mononuclear cells collected at diagnosis. At least 500 interphase cells with well-delineated fluorescent spots were examined. Median age at diagnosis was 70 years. Male:female ratio was 126:99. Binet stage was A in 177/225 (78Æ7%) cases, B in 27/225 (12Æ0%), and C in 21/225 (9Æ3%). Median absolute lymphocyte count was 11Æ0 · 10/l. CD38 ‡ 30% was observed in 61/225 (27Æ1%) cases, ZAP70 was ‡20% in 60/202 (29Æ7%), IGHV homology was ‡98% in 74/221 (33Æ5%), del13q14 was found in 120/225 (53Æ3%), +12 in 47/225 (20Æ9%), del11q22-q23 in 15/225 (6Æ7%), and del17p13 in 24/225 (10Æ7%). BCL3 translocation was observed in 4/225 (1Æ8%) CLL (Tables I and II). The partner chromosome was 14q32 (IGH)

Alternative splicing of hTERT: a further mechanism for the control of active hTERT in acute myeloid leukemia

Abstract hTERT component is the key regulator of telomerase. Alternatively spliced variants of hTERT generate different telomerase activity. The goal of the study was to determine the role of different hTERT isoforms in the regulation of telomerase expression in AML patients. Among the 97 studied patients, 45 had a complex karyotype and 52 a normal karyotype. hTERT isoforms expression was determined in bone marrow samples by q-RT-PCR, using SYBR Green I. hTERT expression was lower in AML patients than controls (median 2.5 vs. 10.1, p = .003), though no difference was observed between the complex and normal karyotype (median 3.2 vs. 2.3, p = .37). High trans-dominant negative isoform expression increased the response rate by two. High expression of inactive product (–α – β) was shown to increase the risk of relapse by about three times. In conclusion, our data suggest an intriguing link between the control of hTERT isoforms expression and AML outcome.

IGHV Unmutated Status Influences Outcome More Than IGHV1-69 Gene Usage Per Se in Patients With Chronic Lymphocytic Leukemia

In this study, IGHV1-69 gene usage was detected in 46 out of 379 cases (12%) of chronic lymphocytic leukemia (CLL). In comparison with patients using alternative immunoglobulin heavy-chain variable (IGHV) genes, patients with IgHV1-69 CLLs more often presented at advanced stage, lacked somatic hypermutation (unmutated cases, 87% vs. 35%; P = .00001), and expressed unfavorable biologic characteristics. In 12 patients (26%), common amino acid motifs within the heavy-chain third complementarity-determining region were identified, allowing assignment to previously reported stereotyped subsets. In our study, treatment-free survival of patients with unmutated IGVH1-69 did not differ significantly from that of patients expressing unmutated alternative IGHV genes. As such, IGHV1-69 gene usage per se did not seem to be predictive of progressive disease, progression being primarily related to the unmutated IGHV profile.

Development of a Richter syndrome with a monoclonal component from a true B-cell chronic lymphocytic leukemia (B-CLL) treated with fludarabine

Dear Editor, B-cell chronic lymphocytic leukemia (B-CLL) patients with unmutated IgV genes frequently show trisomy 12 and a poor clinical outcome, while those with mutated IgV genes often show 13q14 deletions (13q−) and a favorable prognosis [1]. A small B-CLL proportion may present with a serum monoclonal component and lympho-plasmacytoid elements, distinguishable from those CD5− and CD23− cells of lympho-plasmacytoid lymphoma [2]. Fludarabine is a very effective drug for the treatment of B-CLL [3] and indolent lymphomas [4]. However, it is associated with decreased immuno-surveillance determining a high incidence of opportunistic infections and secondary cancers [5]. Moreover, the occurrence of a large B-cell lymphoma, referred to as Richter’s syndrome (RS), is one possible natural evolution even in untreated B-CLL patients [6]. Herein, we report on a 58-year-old woman in apparent good health who came to our clinic for evaluation because of the incidental discovery of hyperleucocytosis. Her clinical parameters and biological findings at diagnosis and during the follow-up are summarized in Table 1. She never showed any monoclonal component in either serum or urine samples; however, 1 month after the fourth course of fludarabine dual monoclonal components (12.0 g/dl), both typified as IgG kappa were detected in the serum (Fig. 1c and d). On admission to our ward, a lymphnode biopsy demonstrated the complete disruption of the normal architecture by a lymphomatous B-cell population, which presented a parafollicular and nodular pattern of infiltration. This cell population was formed by small mature B-cells, lympho-plasmacytoid elements and plasma cells, prolymphocytes and paraimmunoblasts. These last cells were grouped in nodular aggregates, that constituted 24% of the entire cell population. In conclusion, the lymphnode histology pointed to a B-CLL in RS evolution. On clinical diagnosis and during the follow-up, flourescent in situ hybridization (FISH) was always performed with the CLL probe panel, including the following five multicolor probes: LSI p53/LSI ATM/LSI D13S319/LSI 13q34/CEP 12 (Vysis, Downers Grove, IL, USA). At diagnosis, loss of one D13S319 locus was revealed in 48% of marrow cells (Fig. 1e), whereas upon progression, the same locus was Ann Hematol (2007) 86:619–622 DOI 10.1007/s00277-007-0281-y