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Background An appreciable proportion of patients in need of salvage high-dose chemotherapy (HDC) and autologous peripheral blood stem cell (PBSC) transplantation (PBSCT) fail to mobilize adequate numbers of hematopoietic progenitors, and plerixafor is applied for that purpose. Limited data exist on remobilization of PBSCs in patients who have relapsed after prior HDC + PBSCT. Herein, we report on consecutive patients that had undergone successful prior single or tandem HDC for a variety of malignant neoplasms in our institution, and later required re-mobilization of PBSCs in order to support further HDC cycles. Patients and Methods Plerixafor was administered in combination with granulocyte-colony stimulating factor alone, or after mobilizing chemotherapy. Five patients, 2 B-cell non-Hodgkin lymphomas, 1 multiple myeloma, 1 germ-cell tumor, and 1 Ewing sarcoma, having relapsed after prior HDC + PBSCT, were deemed candidates for further cycle(s) of PBSC-supported HDC. Plerixafor was applied in a “just-in-time” strategy after low CD34+ numbers were measured on the first day of anticipated hematopoietic stem cell collection (non-Hodgkin lymphoma, germ-cell tumor, and Ewing sarcoma), or pre-emptively in multiple myeloma. Results Successful collection of adequate PBSCs was achieved in all patients, from 1.8 to 3.8 × 106/kg after a median of 2 (range, 1-3) leukaphereses; 4 of 5 patients underwent subsequent HDC + PBSCT and engrafted after a median of 11 days (range, 9-55 days) and 25 days (range, 17-76 days) for neutrophils and platelets, respectively. Conclusion Plerixafor proved effective to mobilize adequate numbers of PBSCs in individual patients with relapsed malignancies after prior single or tandem HDC + PBSCT. These PBSCs could establish sustained multi-lineage hematopoietic engraftment without any sequelae.

Chronic lymphocytic leukemia (CLL) is the most common type of leukemia in adults and is characterized by the presence of specific cytogenetic abnormalities. CLL research has been focused on epigenetic processes like gene promoter methylation of CpG islands. In the present study, the methylation status of the RAD21 gene is studied and associated with cytogenetic findings in CLL patients in order to investigate its possible implication in CLL pathogenesis and the formation of CLL chromosomal abnormalities.

This study aimed to investigate whether occupational and environmental exposures, lifestyle, family, and medical history are associated with chronic lymphocytic leukemia (CLL) risk and its chromosomal abnormalities. The study included 138 CLL cases and 141 age- and sex-matched controls. Information data were collected through in-person interviews from cases and controls. Cytogenetic analysis was performed on CLL bone marrow cells. Positive associations were found between CLL and cancer family history, smoking, pneumonia, and exposure to petroleum, metals, pesticides/chemical fertilizers, detergents, and medical radiation. Chromosome deletions of 11q and 13q were more frequent in patients exposed to pesticides and rubber, respectively. This study investigated for the first time specific risk factors in relation to CLL aberrations and presented positive correlations. Moreover, it indicates the possible involvement of specific occupational and lifestyle risk factors in the onset of CLL.

The dual role of caffeine as a chromosomal damage inducer and G2/M-checkpoint abrogator is well known but it is observed mainly at relatively high concentrations. At low concentrations, caffeine enhances the cytogenetic effects of several carcinogens and its intake during pregnancy has been recently reported to cause adverse birth outcomes. Interestingly, a threshold below which this association is not apparent was not identified. Since chromosomal abnormalities and aneuploidy are the major genetic etiologies of spontaneous abortions and adverse birth outcomes, we re-evaluate here the effects of caffeine at the cytogenetic level and propose a model for the mechanisms involved. Our hypothesis is that low caffeine concentrations affect DNA replication and cause chromosomal aberrations and asymmetric cell divisions not easily detected at metaphase since damaged cells are delayed during their G2/M-phase transition and the low caffeine concentrations cannot abrogate the G2-checkpoint. To test this hypothesis, caffeine-induced chromatid breaks and micronuclei in peripheral blood lymphocytes (PBLs) were evaluated in vitro after low caffeine concentration exposures, followed by a short treatment with 4mM of caffeine to abrogate the G2-checkpoint. The results show a statistically significant increase in chromatid breaks at caffeine concentrations ≥1mM. When caffeine was applied for G2/M-checkpoint abrogation, a statistically significant increase in chromatid breaks, compared to an active checkpoint, was only observed at 4mM of caffeine. The potential of low concentrations to induce asymmetric cell divisions was tested by applying a methodology combining the cytochalasin-B mediated cytokinesis-block micronucleus assay (CBMN) with interphase FISH (iFISH), using selected centromeric probes. Interestingly, low caffeine concentrations induce a dose dependent aneuploidy through asymmetric cell divisions, which are caused by misalignment of chromosomes through a mechanism unrelated to the formation of chromatid breaks. The cytogenetic approach used, combining CBMN with iFISH, is proposed as a valuable tool to test chemically induced asymmetric cell divisions.

Genomic aberrations are important indicators of prognosis, clinical course and treatment of chronic lymphocytic leukemia (CLL). Two cytogenetic methods, karyotype, and FISH, with still ongoing improvements, are used for CLL investigation, but the panel of chromosomal abnormalities, their prognostic significance and contribution in CLL pathogenesis have not been elucidated yet.Our study deals with the cytogenetic investigation of 237 CLL patients trying to answer ambiguous issues of the disease in the light of new CLL stimulation methodology. More specifically, we compared the detection rate and type of chromosomal aberrations between cultures stimulated with and without the new mitogens and we combined them with the data obtained from interphase (iFISH) and metaphase FISH (mFISH).Approximately 70% of the abnormal karyotypes and all the subclonal abnormalities were detected exclusively in DSP-30/IL-2 cultures. DSP-30/IL-2 exhibited ∼10-fold greater ability to detect abnormalities compared to TPA and unstimulated cultures, revealing60 different chromosomal aberrations. Moreover, the comparison between DSP-30/IL-2 cultures and unstimulated cultures indicated that loss of chromosome Y is rather an age-related phenomenon and not a specific aberration of CLL. Clonal evolution was also detected in 50% of patients with available follow-up karyotypic data and changed the prognosis in 86.4% of them. Finally, it was shown that mFISH must be performed in DSP-30/IL-2 cultures in addition to iFISH to uncover submicroscopic translocations or insertions undetectable by iFISH.All the above argue in favor of the parallel application of karyotype, iFISH and mFISH after DSP-30/IL-2 stimulation for CLL clinical practice and research.

Chronic lymphocytic leukemia (CLL) is associated with recurrent cytogenetic abnormalities that are important prognostic indicators and may influence treatment choices. Nonetheless, several chromosome abnormalities have not yet been completely determined, because of the low mitotic in vitro activity of B-CLL cells. Now, novel technologies such as stimulation of CLL cells with new B-cell mitogens have helped to overcome this problem. We present here a novel conventional and molecular cytogenetic study of a CLL patient with t(6;13)(p21;q14.1), a rare chromosomal aberration. The findings contribute to the identification of rare recurrent aberrations and of any prognostic effect in CLL that could be used for prognostic and therapeutic purposes. The present study demonstrates that t(6;13)(p21;q14.1) as a secondary event to the interstitial deletion in 13q14 region, resulting in the loss of RB1, is a rare but nonrandom abnormality in CLL, resistant to the current treatment CLL protocols with a rather favorable or intermediate prognosis but definitely not an adverse prognosis. Further studies in more CLL patients are required to delineate the prognostic value of t(6;13)(p21;q14.1) and to identify any candidate genes with potential role in the pathogenesis of the disease.

Chronic lymphocytic leukemia (CLL) has been recently attributed to a combination of genetic predisposition and exposure to environmental factors. UDP-glucuronosyltransferase (UGT)1A1*28 is an inborn polymorphism that results in significant downregulation of uridine diphosphate glucuronyltransferase 1-1 (UGT1A1) activity, one of the most critical metabolizing enzymes involved in the detoxification of toxic substances, some of which contribute to CLL pathogenesis. Here, for the first time, we investigated the putative impact of UGT1A1*28 on CLL incidence and on the formation of the most common chromosomal abnormalities of CLL. UGT1A1*28 was investigated in 109 CLL patients and 108 healthy controls, and was associated with karyotypic and fluorescence in situ hybridization (FISH) results. A significant high frequency of the mutant genotype was observed in patients carrying abnormal FISH patterns, especially del(11q) and +12, which are CLL-specific abnormalities. We also observed a significant association between UGT1A1*28 and the intermediate to unfavorable cytogenetic CLL risk groups. No difference, though, was observed in genotypes between patients and controls. Therefore, we could suggest that UGT-deficient individuals may be at a greater risk for developing CLL-specific abnormalities. Our study might serve as a starting point to consider UGT1A1*28 polymorphism as one of the possible predisposing factors of CLL pathogenesis.

Gilbert’s syndrome is characterized by a benign indirect hyperbilirubinemia. It has often been underestimated and undiagnosed because of its mild symptoms; although it is not as rare as was once believed when its frequency was estimated using data originating from biochemical tests. Based on molecular techniques, the occurrence of Gilbert’s syndrome has changed, increasing to 10% in the Caucasian population. This molecular defect was described, by Bosma et al, in 1995, and affects the promoter region of the UGT 1A1 gene. In this case report, our aim is to present a new combination of two molecular defects in a Greek patient with Gilbert’s syndrome. A 13-year-old Greek girl was examined for Gilbert’s syndrome using molecular techniques, and an uncommon genotype was revealed comprising the rare mutation G71R in trans with A(TA)7TAA motif. The G71R mutation according to the literature, as well as our epidemiological data, is rare in Caucasians, while it is common in Asian populations. This is the first case study in the Greek population to report a new genotype for Gilbert’s syndrome manifestation in the Caucasian population.

Deletions of the long arm of chromosome 5 [del(5q)] are frequent chromosome aberrations with known prognosis in myelodysplastic syndromes and acute myeloid leukemia (AML). However, in chronic lymphocytic leukemia (CLL), they are rare and have been reported only as karyotypic results without known prognosis. In the present study, we report a novel conventional and molecular cytogenetic study of two CLL patients carrying interstitial del(5q) in order to contribute to the identification of rare recurrent aberrations and their prognostic impact in CLL. Karyotypic and fluorescence in situ hybridization analysis that used probes for the most common aberrations of CLL demonstrated that del(5q) was the sole chromosome abnormality in both patients at the time of diagnosis. None of these patients had a history of exposure to chemotherapy or radiotherapy. Both patients had disease that was still staged as Binet A at 28 and 18 months after diagnosis, respectively, without receiving any therapy because of their good clinical condition. Therefore, it could be suggested that del(5q) may not be associated with an adverse prognosis in CLL and is not related with therapy-induced chromosome changes. Further studies are required to elucidate the prognostic value of these deletions in more CLL patients, which could be advisable for prognostic and therapeutic purposes, as well as to identify candidate genes that may potentially play a role in the pathogenesis of CLL.

Isochromosome of the long arm of the derivative chromosome 17, originating from the translocation t(15;17) [ider(17)(q10)t(15;17) or ider(17q)] in acute promyelocytic leukemia (APL), is a rare chromosome aberration which has been associated with a poor prognosis. In the present study, we report on 4 male APL patients with ider(17q) and review the clinical, cytogenetic and molecular characteristics of all previously reported APL patients with ider(17q) in order to clarify the clinical features and outcome of these patients. The data presented in this study demonstrated that ider(17q), which resulted in an extra RARA-PML fusion gene, was more frequent in males than females (male/female ratio of 2.12/1), was associated with a rather low initial white blood cell count and did not confer an adverse prognosis in APL patients treated with all-trans-retinoic acid and chemotherapy. The most frequent additional chromosome change to ider(17q) was trisomy 8. Ider(17q) was observed in all subtypes of the PML-RARA fusion gene, but the frequency of the bcr1 subtype was increased. Cases of overrepresentation of the RARA-PML fusion gene and ider(17q) cases may help in elucidating the role of RARA-PML in leukemogenesis.

Abstract 2513 Acute myeloid leukemia (AML) is a heterogeneous disease with well-known clinical and pathological aspects, characterized by the acquisition of somatic mutations in haematopoietic progenitors leading to disruption of differentiation. However, the genetic etiology of AML, which include gene mutations and chromosomal aberrations, is largely unknown. Altered forms of genes that differ by a single nucleotide polymorphisms (SNPs) have been shown to predispose individuals to AML development. Recently it has been reported that interindividual differences based on detoxification genes polymorphisms may contribute to the AML susceptibility. Human cytochrome P450 (CYP) enzymes play a key role as phase I enzymes in the metabolism of drugs and environmental chemicals. Several CYP enzymes metabolically activate procarcinogens to genotoxic intermediates. CYP2B6 blocks the transformation of precarcinogens to their biologically active forms that provoke chromosomal instability and leukemia. CYP2B6 G516T SNP change the aminoacid sequence (Gln172His), resulting in enzymatic inactivation. Thus, individuals homozygous for the mutant allele (T/T) or heterozygotes (G/T) present decreased enzymatic activity. We performed a case-control study in a large series of AML patients to investigate the potential relation between genotype of CYP2B6 G516T SNP and the risk of de novo AML. We also compared the genotypic frequencies in AML patients in respect to chromosome abnormalities, FAB classification and clinical characteristics. The CYP2B6 G516T genotyping was performed on 195 de novo AML patients at diagnosis and 215 sex and age matched healthy controls using a PCR-RFLP assay and LightSNP assay. Unstimulated bone marrow cells were used for karyotypic analysis and karyotypes were described according to ISCN. Statistical analysis was performed using Chi-square test and P Karyotypic analysis was successfully performed in 97.8% of AML patients at diagnosis. Among them, 136 (69.7%) showed clonal karyotypic abnormalities. The genotypic distribution in patients and healthy groups was statistically significant and showed: homozygous wild type G/G 54,5% vs 67.2%, heterozygotes G/T 40.4% vs 27.5% and homozygous mutant T/T 6.7% vs 5.5% respectively (p The increased frequency of mutant allele leading to reduced enzyme activity suggests that the CYP2B6 gene may be a predisposing factor for the development of AML. Furthermore, the CYP2B6 G516T polymorphism seems to be associated with the presence of lesions on chromosomes 5 and / or 7 which are poor prognostic lesions in AML. Therefore, the high frequency of mutant genotypes (G/T and T/T) of CYP2B6 G516T SNP in these cytogenetic groups may be involved in the development of these specific chromosomal abnormalities. Disclosures: No relevant conflicts of interest to declare.