Summary Background and objective Genomic instability and reduced glutathione S-transferase (GST) activity have been identified as potential risk factors for malignant complications in celiac disease ...(CD). In this study, we assessed the possible influence of GST polymorphisms on genome instability phenotypes in a genetically characterised group of celiac patients from previous studies. Methods The deletion polymorphisms in GSTM1 and GSTT1 genes and the single-nucleotide polymorphism GSTP1 c.313A>G were genotyped using PCR in a set of 20 untreated adult patients with a known genomic instability phenotype and 69 age- and sex-matched healthy individuals. Results The frequencies of variant genotypes in patients were GSTM1 -null (30%), GSTT1 -null (5%), GSTP1 -AG (60%) and GSTP1 -GG (15%), and they showed no differences from controls. No significant differences were found in the genotype distribution based on telomere length. Cases with GSTM1 -null genotype (83%) and microsatellite stability were more frequent than those with genomic instability. Moreover, carriers of GSTP1 -variant genotype (73%) and stable phenotype were significantly increased compared to unstable patients (27%) ( P = 0.031). No differences were found according to the clinical-pathological characteristics of celiac cases. Conclusions No association between GST polymorphic variants and celiac-associated genomic instability was proven in our cohort. Future studies should explore the usefulness of other biomarkers to distinguish celiac patients who are susceptible to cancer development.
From the Instituto de Investigaciones Hematológicas Mariano R. Castex, Academia Nacional de Medicina de Buenos Aires, Argentina (LCR, CPR, MC, RPB, MdTP, IBL, CDDB); Division of Genomic Medicine, ...University of Sheffield Medical School, Sheffield, UK (AG)
Correspondence: Liliana C. Rossetti, Departamento de Genética, Academia Nacional de Medicina. Pacheco de Melo 3081, Cdad. de Buenos Aires (1425), Argentina. E-mail: rossetti{at}hematologia.anm.edu.ar
Hemophilia A (HA) is caused by heterogeneous mutations in the factor VIII gene ( F8 ). This paper reports 16 novel small F8-mutations and rearrangements in a series of 80 Argentinian families with severe-HA. Using an updated scheme for F8 -analysis, we found 37 F8 -inversions (46%), 10 large deletions (13%), 13 small ins/del (16%), 7 nonsense (9%) and 8 missense mutations (10%), including 4 new ones (p.T233K, p.W1942R, p.L2297P and p.L2301S). The potential changes leading to severe-HA of these latter mutations were suggested by bioinformatics. The F8 -mutation was characterised in 76 families (95%). They received genetic counselling and precise information about treatment design.
Key words: F8, HEMA, severe phenotype, mutation characterization.
BCR-ABL fusion gene is implicated in the pathogenesis of chronic myeloid leukemia (CML), encoding the oncoprotein p210
BCR-ABL with anti-apoptotic activity. The inability to undergo apoptosis is an ...important mechanism of drug resistance and neoplastic evolution in CML. The gene transcript expression of mitochondrial apoptotic related genes
BAX and
BCL-XL was evaluated by quantitative Real Time PCR (qPCR)
in vitro in K562 cells and
in vivo in peripheral blood of 66 CML patients in different stages of the disease: 13 cases at diagnosis, 34 in chronic phase (CP), 10 in accelerated phase (AP) and 9 in blast crisis (BC).
Our results in K562 cells showed that all treatments with different tyrosine kinase inhibitors (TKIs) induced a decreased expression of the antiapoptotic oncogene
BCL-XL, whereas the proapoptotic gene BAX remains constant with minor modifications.
A significantly lower
BAX/BCL-XL expression ratio (mean
±
SEM) than a group of healthy individuals (4.8
±
0.59) were observed in CML patients at diagnosis (1.28 ± 0.16), in AP (1.14
±
0.20), in BC (1.16
±
0.30) and in 18% of cases of patients in CP (2.71
±
0.40). Most CP cases (82%) showed a significantly increased ratio (10.03
±
1.30), indicating that the treatment with TKIs efficiently inhibited the expression of BCL-XL by blocking BCR-ABL oncoprotein.
The
BAX/BCL-XL ratio showed a significant inverse correlation (Spearman
P
<
0.0001) with
BCR-ABL/ABL relative expression indicating that low BAX/BCL-XL was associated with disease progression. Accordingly, the follow up of a cohort of eight cases during 6
months from diagnosis showed that while the
BAX/BCL-XL ratio rapidly increased after treatment in seven cases with good evolution, it decreased in the single case that showed rapid evolution and short survival.
Our data suggest that
BAX/BCL-XL expression ratio may be a sensitive monitor of disease progression and an early predictor of TKI therapy responsiveness in CML patients.
BACKGROUND AND AIMSMalignant complications of celiac disease (CD) include carcinomas and lymphomas. The genetic basis behind cancer development in CD is not known, but acquisition of genetic ...abnormalities and genomic instability has been involved. The aim of this study was to explore molecular characteristics of genomic instability in CD patients by analyzing microsatellite instability (MSI) and loss of heterozygosis (LOH) with carefully selected microsatellites.
METHODSWe genotyped small bowel biopsies and peripheral blood samples from 20 untreated CD patients using five microsatellites related to MMR genes (panel A), and five repeats associated with tumor suppressor genes, chromosome instability, inflammation, and cancer (panel B).
RESULTSGenomic instability was found in seven out of 20 (35%) cases atD5S107, D18S58, GSTP, TP53 or DCC, being TP53 the most frequently affected (five out of seven cases; 71%). Microsatellite alterations were significantly found using panel B markers (P=0.04). No cases with high frequency of MSI and replication error phenotype were detected. Only one case displayed MSI-L alone. Three patients exhibited LOH and three other cases showed LOH with low level of MSI, being classified as having chromosome instability phenotype.
CONCLUSIONTwo novel observations were found in this studyfirst, the finding that non-neoplastic cells from a group of untreated CD patients present genomic instability at nucleotide level; and second, the advantage to use carefully selected microsatellites to identify celiac patients with molecular instability. Our data support the existence of chromosome instability phenotype in CD, suggesting that stable and unstable patients are genomically distinct subtypes that may follow a different evolution.
Philadelphia (Ph) chromosome in acute lymphoblastic leukemia (ALL) is found nearly in 20-30% adults and 5% children. Ph chromosome results from the reciprocal translocation t(9;22)(q34;q11) joining ...the tyrosine kinase c-ABL1 proto-oncogene on chromosome 9 to BCR gene on chromosome 22. The majority of Ph breakpoint in ALL occurs in the minor breakpoint cluster region at first intron of the BCR gene, and results in the fusion of BCR exon 1 to ABL1 exon 2 (e1a2) generating the oncoprotein p190BCR-ABL1. Ph+ ALL patients present an aggressive disease with a poor prognosis. Current treatment includes potent chemotherapy and tyrosine kinase inhibitors, such as imatinib. The aim of this study was to quantify BCR-ABL1 transcripts in ALL Ph+ patients at different stages of treatment in order to evaluate the kinetics of the tumor clone in response to treatment.
We reported a preliminary analysis of the clinical trial GATLA LLA-Ph+2011 (Grupo Argentino de Tratamiento de la Leucemia Aguda LLA-Ph+) that includes patients from 1 to 21 years old newly diagnosed Ph+ ALL. Written informed consent was obtained from all subjects. Diagnosis of Ph+ALL was confirmed by the presence of Ph chromosome and/or positive molecular analysis for detection of BCR-ABL1 fusion transcripts. All patients received induction treatment consisting of vincristine, daunorubicin, prednisolone, asparaginase, and metrotexate. Imatinib was administered 300 mg/m2/day from day 15 of induction until the initiation of conditioning for alloHSCT or to the end of maintenance therapy. The clinical protocol consist in an induction IA (33 days) and IB (28 days), consolidation RA1, RA2 and RA3, re-induction (Protocol II A and B, 36 days each) and maintenance to complete 2 years of treatment. Eligible patients are transplanted after the third block.
Minimal residual disease (MRD) monitoring was performed by one-step quantitative RT-PCR assays in bone marrow and/or peripheral blood, using RotorGene® Qiagen; at the end of induction AI, previous RA1, RA2, RA3 and protocol II Fase IIA, IIB and during maintenance or post-alloHSCT. BCR-ABL1 quantification was expressed relative to the amount of ABL1 mRNA. We studied a control group of pediatrics positive P190 isoform bone marrow samples at diagnosis in order to establish our basal reference to estimate the logarithmic reduction of BCR-ABL1 transcripts for our cohort. The obtained mean value of BCR-ABL1(e1a2)/ABL1 in the control group was 55.5% (range 29% - 92%). The molecular response was defined as optimal (OMR, OMR-undetectable) if the BCR-ABL1/ABL1 ratio was lower than 0.055%.
A total of 25 subjects (male: female = 16: 9) were enrolled into the study from November 2011 to July 2014. The median age was 7 (range 2 - 19) years old. Type of BCR breakpoint was minor (e1a2) in 96% of patients, and only one present a rearrangement corresponding to major BCR (b3a2). During the follow up (median: 15 months), five patients (20%) died: 3 by sepsis, 1 by post alloHCT complications, and 1 due to disease progression. Two patients had to rotate to dasatinib because of disease relapse: one in central nervous system and the other post alloHCT. Our preliminary results showed that the overall survival was 79%±10% and the survival free of event (death, molecular and disease relapse) was 67%±10% at 12 months.
We serially measured BCR-ABL1 at different trial stage (AI, RA1, RA2, RA3, Protocol II) and OMR was 13.3%, 41.2%, 33.3%, 45.5% and 76.9%, respectively. Among 17 evaluable patients on maintenance and post alloHCT, 88% (15/17) obtained OMR values, and among them, 87% (13/15) presented OMR-undetectable values.
We evaluated the kinetics of the tumor clone under treatment regarding prognostic factors. Patients older than 10 years showed a higher rate of OMR at RA1 (83% vs 27%, p=0.05). However, all evaluated younger patients reached an OMR at protocol II stage (100% vs 33%, p=0.045), showing a sustained and deeper molecular response. Other evaluated parameters did not show a relationship with OMR at different stages.
Our preliminary data showed that BCR-ABL1 quantification may be a good marker to evaluate response to treatment in Ph+ positive ALL patients. BCR–ABL1 transcripts dropped to undetectable levels in 76% (13/17) of evaluable cases at the later stage of treatment. Also, 77% of patients achieved an OMR at Protocol II not only might provide a better chance for receiving alloHSCT but also might contribute to durable remissions.
No relevant conflicts of interest to declare.
Allogeneic hematopoietic stem cell transplantation (HSCT) using sibling donors is a life-saving intervention for patients with hematological malignancies. It is recognized that numerous genetic ...factors in both patient and donor play a role in outcome. TGF beta 1 is a member of a highly pleiotrophic family of growth factors involved in the regulation of numerous immunomodulatory processes. Several functional polymorphisms have been identified in TGF-B1 gene, such as a single nucleotide polymorphism (SNP) at codon 10 of exon 1. We have previously published significant impact of this SNP on unrelated donor (UD) transplant outcome, predominantly on Non Relapse Mortality (NRM) and acute Graft-vs.-Host-Disease (aGVHD). To date there are no published data in large sibling donor HSCT cohorts that analyze survival outcomes. We have hypothesized TGF-B1 SNP may influence the outcome of sibling donor HSCT similarly to UD. One hundred and sixty six patient/donor pairs who underwent a sibling donor HSCT in our centers were genotyped for the presence of a SNP at codon 10 (rs1982073) by an allele-specific PCR. The transplant took place between January 2000 and March 2014 and the median follow up time was 4.4 years. Median age was 33 years and 59% were male. The prevalent diagnoses were acute myeloid leukemia 54 (32%), acute lymphoid leukemia 33 (20%), lymphoproliferative disorders 24 (14%), myelodysplastic syndrome 21 (13%), myeloproliferative neoplasm 9 (5%), and 42% were in early stage. Myeloablative conditioning regimens were used in 58% of transplants; the source was PBSC in 93.3% of the patients. The patients’ observed SNP frequencies were TT 22%, TC 56% and CC 22%, and for the donors were 25%, 57% and 18% respectively. No significant impacts were observed in the full cohort analysis. When we analyzed the myeloablative cohort significant differences were founded. Wild-type donors (TT) experienced a significant increase in relapse incidence compared to other genotypes (1-3 years TT 37-41% vs. TC 15-23% vs. CC 0-0%, Gray’s test p=0.01), with a significant decrease in disease free survival (DFS) (1-3 years TT 53-39% vs. TC/CC 73-65%, log-rank p=0.04) and overall survival (OS) (1-3 years TT 64-46% vs. TC/CC 77-67%, log-rank p=0.04) (figures). No differences in terms of NRM were founded. We conclude that in sibling donor-HSCT TGF-B1 wild-type donors might be associated with an increase in relapse and subsequent decrease in DFS and OS. These results should be confirmed in larger series. Identification of these SNPs pre-transplant will allow for transplant conditioning and immunosuppression regimens to be tailored to the individual patient, as well as assisting in the most appropriate choice of donor.
Display omitted
No relevant conflicts of interest to declare.
There are previous reported data describing differences between European and Asian patients with Myelodysplastic Syndromes (MDS), and little is known about South-American (SA) patients. Our aim was ...to describe clinical characteristic of SA MDS population, to compare our series with diverse ethnicity, to evaluate scoring systems and prognostic factors.
We retrospectively analysed a series of 1080 patients with de novo MDS (1981-2014) from Argentine (Ar-635), Brazil (Br-345), and Chile (Ch-100). Patients were classified following FAB and WHO criteria, excluding patients with bone marrow (BM) blasts >30% and CMML with white blood cells count >12x109/L. Patients undergoing hematopoietic stem cell transplantation (5%) or hypomethylating therapy (12%) were censored at treatment initiation.
Chilean patients were younger (mean age: 59 vs 66-Ar, p<.001 and vs 65 years old-Br, p=.003), with a female preponderance (ratio M/F: 0.8-Ch, 1.3-Ar, 1.3-Br, p=.061). Since Ch did not include patients with CMML, distributions among FAB and WHO categories were different (p<.001). Br series showed a higher predominance of RARS (17% vs 9%-Ar vs 1%-Ch), and a lower percentage of RCMD (38% vs 43%-Ar vs 49%-Ch), while the frequency of the other subtypes were comparable. BM blast categories according to IPSS and IPSS-R were similar (p=.626 and =.508). Hemoglobin (Hb) level was significantly higher in Ar series (9.6g/dL vs 8.6g/dL-Ch, p<.001, and vs 8.7g/dL-Br, p=.002), with no differences in ANC and platelets (p=.842 and .763). There were no differences in the distribution of cytogenetic groups of risk (CGR) according to IPSS (p=.157). With respect to IPSS-R CGR distribution, Ar series showed a higher frequency of Very Good and Intermediate findings (4% and 19%, vs 2% and 15%–Br, 0% and 9%-Ch, respectively, p=.037).
The distribution among IPSS categories was similar with a higher predominance of the high risk group in Ch (17%, 9%-Ar, 9%-Br, p=.056). IPSS-R distribution confirmed a higher frequency of very high risk in Ch series (20%, 13%-Br, 10%-Ar) and a lower predominance of very low risk (5%, 15%-Br and 22%-Ar) (p<.001).
We also evaluated different prognostic factors in each MDS population and in the whole population (table). Overall survival (OS) was lower in Ch patients, consistent with the higher prevalence of higher risk group categories (36 months vs 56m-Ar; 63m-Br, p=.026). Hb, BM blast, CGR, IPSS and IPSS-R were useful to predict survival in the three series and in the overall SA MDS population. Age was not useful for Br patients and showed a borderline influence for the whole SA series. The ANC was neither a predictive variable for Br nor for Ch patients, and platelets count was not useful in Ch series.
Epidemiological and clinical characteristics, distribution among prognostic subgroups and OS were similar for Ar and Br compared to Ch MDS series. This will need further analysis in a larger group of patients. Nevertheless, the IPSS-R system and its variables showed a good reproducibility to predict clinical outcome for the whole SA population. Abstract 4651. Table 1ArgentineBrazilChileWhole SASurvival (50%, m)pSurvivalpSurvivalpSurvivalpAll patients56633655.026Gender M/F42/66<.00140/87=.00117/49ns41/70<.001Age ≤/>60 years77/50=.01944/68ns74/31=.00264/49=.052Hb ≥10/8-<10/<8g/dL98/50/31<.001135/44/36<.001NR/15/13=.002121/43/30<.001ANC ≥/<800/µL58/37=.03668/36=.06535/NRns57/37=.046Platelets ≥100/50-<100/<50 x103/µL71/44/40<.00170/47/40=.02941/36/17ns63/44/29<.001BM Blast ≤2/>2-<5/5-10/>10%80/63/21/17<.00175/70/36/11<.00148/36/15/7<.00177/60/25/13<.001CGR VG-G/I/P/VP66/35/20/12<.00170/20/9/16<.00174/13/4/7<.00168/32/16/12<.001IPSS121/44/19/14<.001116/55/13/9<.00150/49/6/7<.001116/49/18/10<.001IPSS-R136/64/34/18/14<.001135/70/32/17/11<.001NR/50/NR/13/6<.001135/70/41/18/11<.001
No relevant conflicts of interest to declare.
Recently, the IPSS-R has proposed five cytogenetic groups of risk (CGR) based on Schanz et al, 2011, proposal. Nevertheless, nearly 70% of patients belong to lower CGR. Our aim was to characterize ...the cytogenetic profile of South American (SA) MDS population trying to find differences among Argentine (A) and Brazil (B), both with diverse ethnicity, to evaluate CGR, and to define the impact of more frequent aberrations and of monosomal karyotype (MK) in our population.
This is a multicenter retrospective study of 943 SA (634 from A and 309 from B) de novo MDS patients (pts) evaluated from 1981 to 2014. Pts were classified following FAB and WHO criteria. The median age was 69 (15-99) years old with a male/female ratio of (533/410) 1.3. During the follow-up, censored up to receiving a disease modifying therapy (median: 21 months), 159 (17%) evolve to AML and 351 (37%) died. Regarding 130 pts (14%) who received hypomethylating therapy (HMT), 46 (35%) evolved to AML, and 61 (47%) died.
Although A population was larger than B series, no differences were observed concerning to: CGR distribution according to the IPSS (p=0.565) and to the IPSS-R (p=0.343), percentage of abnormal karyotypes (42%-A vs 40%-B; p=0.499), and presence of deletions and/or monosomies (77%-A vs 80%-B, p=0.700). B showed a higher proportion of karyotypes involving, at least, one chromosome Y, 5, 7, 8 and/or 20 (83% vs 73%-A, p=0.039), mostly due to a higher proportion of 5q- (39% vs 22%-A, p<0.001).
Karyotypes were classified as Very Good/Good (72%), Intermediate-Int (17%), Poor (5%), and Very Poor CGR (6%), with median survival of 68, 32, 18 and 12 months (p<0.001), and time to AML progression (25%) of 72, 26, 10, and 4 months, respectively (p<0.001). In order to find the prognostic value of certain cytogenetic findings, we evaluated the presence of 5q-, chr 7 alterations and +8 as isolated or accompanied by one (noncomplex- non CK) or more alterations (complex-CK). Our results were consistent with previous data. Interestingly +8 non CK showed better outcome than +8 isolated (p<0.001), in agreement with Haase et al, 2007. We also evaluated the presence of MK in the context of CK or non CK. MK non CK, that included 3 Good, 6 Int and 4 Poor karyotypes according to the IPSS-R, showed similar survival than CK, MK CK and other Poor risk findings (p=0.629). Therefore, MK non CK might be considered, at least, as a Poor risk finding, as proposed for primary Myelofibrosis by Teffery et al, 2011. In order to identify karyotypes with diverse outcome within the Int CGR, we applied different grouping criteria. The presence of a trisomy or of a deletion accompanied by other alteration (both excluding patients with +8), were associated with short survival (p=0.019 and p=0.092, respectively).
We also evaluated the impact of CGR on the outcome of patients under HMT. Patients with Int CGR (23%) showed similar median survival to the Very Good/Good (57%) group and a better outcome than the Poor/Very Poor (20%) (25 m vs 24 m vs 10 m, p=0.001), and a longer time to evolve to AML (22 m vs 12 m vs 6 m, p=0.005).
Cytogenetic findings had a clear impact in our population, and except for a higher incidence of 5q- in B, both series showed a high concordance of cytogenetic findings. Our results are in agreement with previously reported data from European and North American series. Also, our data suggest that MK are indicators of poor prognosis, not different from CK, and that the heterogeneous Int CGR might be further clarified taking into account the nature of different cytogenetic alterations. However, the wide spectrum of low frequency aberrations and the highly variant combinatory of aberrations stress the importance of large study groups to stablish a consensus in the way of grouping Int CGR findings.
No relevant conflicts of interest to declare.
PDF
