Laura Palomo

Characterization of four novel BRCA2 large genomic rearrangements in Spanish breast/ovarian cancer families: review of the literature, and reevaluation of the genetic mechanisms involved in their origin

Large genomic rearrangements (LGRs) at the BRCA2 locus explain a non-negligible proportion of hereditary breast and ovarian cancer (HBOC) syndromes. The multiplex ligation and probe amplification (MLPA) assay has permitted in recent years to identify several families carrying LGRs at this locus, but very few such alterations have been fully characterized at the molecular level. Yet, molecular characterization is essential to identify recurrent alterations, to analyze the genetic mechanisms underlying such alterations, or to investigate potential genotype/phenotype relationships. We have used MLPA to identify BRCA2 LGRs in 7 out of 813 Spanish HBOC families previously tested negative for BRCA1 and BRCA2 small genomic alterations (substitutions and indels) and BRCA1 LGRs. We used a combination of long-range PCR, restriction mapping, and cDNA analysis to characterize the alterations at the molecular level. We found that Del Exon1-Exon2, Del Exon12-Exon16 and Del Exon22-Exon24 explain one family each, while Del Exon2 appears to be a Spanish founder mutation explaining four independent families. Finally, we have combined our data with a comprehensive review of the literature to reevaluate the genetic mechanisms underlying LGRs at the BRCA2 locus. Our study substantially increases the spectrum of BRCA2 LGRs fully characterized at the molecular level. Further on, we provide data to suggest that non-allelic homologous recombination has been overestimated as a mechanism underlying these alterations, while the opposite might be true for microhomology-mediated events.

Targeted deep sequencing improves outcome stratification in chronic myelomonocytic leukemia with low risk cytogenetic features

Clonal cytogenetic abnormalities are found in 20-30% of patients with chronic myelomonocytic leukemia (CMML), while gene mutations are present in >90% of cases. Patients with low risk cytogenetic features account for 80% of CMML cases and often fall into the low risk categories of CMML prognostic scoring systems, but the outcome differs considerably among them. We performed targeted deep sequencing of 83 myeloid-related genes in 56 CMML patients with low risk cytogenetic features or uninformative conventional cytogenetics (CC) at diagnosis, with the aim to identify the genetic characteristics of patients with a more aggressive disease. Targeted sequencing was also performed in a subset of these patients at time of acute myeloid leukemia (AML) transformation. Overall, 98% of patients harbored at least one mutation. Mutations in cell signaling genes were acquired at time of AML progression. Mutations in ASXL1, EZH2 and NRAS correlated with higher risk features and shorter overall survival (OS) and progression free survival (PFS). Patients with SRSF2 mutations associated with poorer OS, while absence of TET2 mutations (TET2wt) was predictive of shorter PFS. A decrease in OS and PFS was observed as the number of adverse risk gene mutations (ASXL1, EZH2, NRAS and SRSF2) increased. On multivariate analyses, CMML-specific scoring system (CPSS) and presence of adverse risk gene mutations remained significant for OS, while CPSS and TET2wt were predictive of PFS. These results confirm that mutation analysis can add prognostic value to patients with CMML and low risk cytogenetic features or uninformative CC.

Correlation of myelodysplastic syndromes with i(17)(q10) and TP53 and SETBP1 mutations.

Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal stem cell disorders that are highly prevalent in the elderly. Isochromosome 17(q10) [i(17q)] has a frequency of 0 4–0 8%, and cytogenetic risk stratification suggests that isolated i(17q) is of intermediate prognostic significance (Brunning et al, 2008; Greenberg et al, 2012; Schanz et al, 2012). It has been postulated that i(17q) may be associated with TP53 mutations, although a recent study found no association (Kanagal-Shamanna et al, 2012). SETBP1 is located at 18q21.1 and mutations of this gene have been reported in several haematological malignancies (Crist obal et al, 2010; Fernandez-Mercado et al, 2013; Piazza et al, 2013). We investigated the mutational status of TP53 and SETBP1 in 27 untreated MDS patients (who provided written informed consent) reported to the Spanish MDS group, as TP53 mutations and i(17q) have been shown to rarely co-exist in recent reports of concurrent i(17q) and SETBP1 mutations. Patients were classified according to the World Health Organization 2008 criteria (Brunning et al, 2008) and G-banding karyotypes were described according to the International System for Human Cytogenetic Nomenclature 2009 (Shaffer et al, 2009) (Table I). Genomic DNA was isolated from cells fixed in Carnoy’s fixative and from fresh bone marrow samples (QIAamp DNA mini-kit, Qiagen Inc, Valencia, CA, USA). TP53 exons 5–9 and SETBP1 exon 3 were amplified and sequenced as previously described (Meggendorfer et al, 2013). Patients were divided into three groups: no mutations, TP53 mutations and SETBP1 mutations. Statistical analyses (SPSS 22.0. SPSS Inc., Chicago, IL, USA) were performed for these three groups and for the overall patient population. Baseline characteristics and main clinical variables were compared by Kruskal–Wallis and Pearson or Fisher’s exact test for continuous or categorical variables, respectively. Overall survival (OS) was measured from haematological diagnosis to death from any cause or last follow-up. Survival curves were plotted according to the Kaplan–Meier method, and compared using the log-rank test. Univariate and multivariate analyses of OS were performed using the Cox proportional hazard ratio (HR) model. Two-sided P values <0 05 were considered statistically significant. TP53 mutations were analysed in all patients. Five of the 27 (18 5%) had non-synonymous point mutations (Table I). Two patients had mutations in exon 7, G245S and R175H [complex karyotype (CK) and isolated i(17q)]; two had mutations in exons 5 and 6 (H179P and Y220H, respectively, all with a CK); and one patient (with CK) had an intronic mutation (c.919 + 1G>A) at the splicing recognition site. SETBP1 was analysed in all patients. Eleven (41%) had SETBP1 non-synonymous point mutations (mainly located in residues 868–871). Five of 11 (45%) had heterozygous mutations in D868N. Four of these patients had isolated i (17q); the remaining one had i(17q) with one additional abnormality. SETBP1 heterozygous G870S mutations (27%) were found in 3/11 patients, all with isolated i(17q). The three remaining patients had single heterozygous mutations in D868Y, S869G and I871T [D868Y patient had a CK, while the others presented isolated i(17q)]. A statistically significant relationship was found between mutation group and karyotype classification (P = 0 009). Most TP53 mutated cases (80%) presented a CK, meanwhile 82% of SETBP1 mutations presented isolated i(17q). There were no statistically significant differences between the groups regarding haemoglobin, leucocytes, platelets, polymorphonuclear cells, absolute neutrophil count or blast percentage. The TP53-mutated group showed worse OS than non-mutated and SETBP1-mutated groups {median OS [95% confidence interval (CI)]: 2 9 (2 4, 3 5), 14 1 (0, 33 7) and 13 9 (7 9, 19 8), respectively, P = 0 001}. Patients with a CK showed worse OS than isolated i(17q) and i(17q) with one additional abnormality [median OS (95% CI): 3 8 (2 5, 5 1), 26 5 (7 2, 45 8) and 21 3 (8 3, 34 3), respectively, P < 0 001] (Figure S1). Univariate analysis of OS (Table II) revealed a statistically significant difference between non-mutated and TP53mutated patients [HR (95% CI): 0 07 (0 01, 0 3); P = 0 002], and between SETBP1-mutated and TP53mutated patients [HR (95% CI): 0 11 (0 02, 0 5); P = 0 006]. There were also significant differences between the cytogenetic groups: isolated i(17q) versus CK [HR (95% CI): 0 07 (0 02, 0 3); P < 0 001]and i(17q) with an additional abnormality versus CK [HR (95% CI): 0 07 (0 01, 0 3); P = 0 001]. In multivariate analysis (Table II), and because of the high correlation between karyotype and mutation type, only the karyotype remained as a prognostic factor, considering CK as the reference category: isolated i (17q) versus CK (P = 0 002), and i(17q) with an additional abnormality versus CK (P = 0 01). correspondence

Targeted deep sequencing of CD34+ cells from peripheral blood can reproduce bone marrow molecular profile in myelodysplastic syndromes

of related donors for bone marrow transplantation in sickle cell anemia. Am J Pediatr Hematol Oncol. 1994;16:27–29. [5] Sorror ML, Maris MB, Storb R, et al. Hematopoietic cell transplantation (HCT)-specific comorbidity index: a new tool for risk assessment before allogeneic HCT. Blood. 2005;106(8):2912–2919. [6] Walters MC, De Castro LM, Sullivan KM, et al. Indications and results of HLA-identical sibling hematopoietic cell transplantation for sickle cell disease. Biol. Blood Marrow Transplant. 2016;22:207–211.

Immunophenotypic, cytogenetic, and mutational characterization of cell lines derived from myelodysplastic syndrome patients after progression to acute myeloid leukemia

Leukemia cell lines have been widely used in the hematology field to unravel mechanistic insights and to test new therapeutic strategies. Myelodysplastic syndromes (MDS) comprise a heterogeneous group of diseases that are characterized by ineffective hematopoiesis and frequent progress to acute myeloid leukemia (AML). A few cell lines have been established from MDS patients after progression to AML but their characterization is incomplete. Here we provide a detailed description of the immunophenotypic profile of the MDS‐derived cell lines SKK‐1, SKM‐1, F‐36P; and MOLM‐13. Specifically, we analyzed a comprehensive panel of markers that are currently applied in the diagnostic routine for myeloid disorders. To provide high‐resolution genetic data comprising copy number alterations and losses of heterozygosity we performed whole genome single nucleotide polymorphism‐based arrays and included the cell line OHN‐GM that harbors the frequent chromosome arm 5q deletion. Furthermore, we assessed the mutational status of 83 disease‐relevant genes. Our results provide a resource to the MDS and AML field that allows researchers to choose the best‐matching cell line for their functional studies.

Serotonin receptor type 1B constitutes a therapeutic target for MDS and CMML

Myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML) are chronic myeloid clonal neoplasms. To date, the only potentially curative therapy for these disorders remains allogeneic hematopoietic progenitor cell transplantation (HCT), although patient eligibility is limited due to high morbimortality associated with this procedure coupled with advanced age of most patients. Dopamine receptors (DRs) and serotonin receptors type 1 (HTR1s) were identified as cancer stem cell therapeutic targets in acute myeloid leukemia. Given their close pathophysiologic relationship, expression of HTR1s and DRs was interrogated in MDS and CMML. Both receptors were differentially expressed in patient samples compared to healthy donors. Treatment with HTR1B antagonists reduced cell viability. HTR1 antagonists showed a synergistic cytotoxic effect with currently approved hypomethylating agents in AML cells. Our results suggest that HTR1B constitutes a novel therapeutic target for MDS and CMML. Due to its druggability, the clinical development of new regimens based on this target is promising.

Impact of mutational studies on the diagnosis and the outcome of high-risk myelodysplastic syndromes and secondary acute myeloid leukemia patients treated with 5-azacytidine

Myelodysplastic syndromes (MDS) are stem cell disorders caused by various gene abnormalities. We performed targeted deep sequencing in 39 patients with high-risk MDS and secondary acute myeloid leukemia (sAML) at diagnosis and follow-up (response and/or relapse), with the aim to define their mutational status, to establish if specific mutations are biomarkers of response to 5-azacytidine (AZA) and/or may have impact on survival. Overall, 95% of patients harbored at least one mutation. TP53, DNMT3A and SRSF2 were the most frequently altered genes. Mutations in TP53 correlated with higher risk features and shorter overall survival (OS) and progression free survival (PFS) in univariate analysis. Patients with SRSF2 mutations were associated with better OS and PFS. Response rate was 55%; but we could not correlate the presence of TET2 and TP53 mutations with AZA response. Patients with sAML presented more variations than patients with high-risk MDS, and usually at relapse the number of mutations increased, supporting the idea that in advanced stages of the disease there is a greater genomic complexity. These results confirm that mutation analysis can add prognostic value to high-risk MDS and sAML patients, not only at diagnosis but also at follow-up.

DNA methylation profile in chronic myelomonocytic leukemia associates with distinct clinical, biological and genetic features

ABSTRACT Chromosomal abnormalities are detected in 20–30% of patients with chronic myelomonocytic leukemia (CMML) and correlate with prognosis. On the mutation level, disruptive alterations are particularly frequent in chromatin regulatory genes. However, little is known about the consequential alterations in the epigenetic marking of the genome. Here, we report the analysis of genomic DNA methylation patterns of 64 CMML patients and 10 healthy controls, using a DNA methylation microarray focused on promoter regions. Differential methylation analysis between patients and controls allowed us to identify abnormalities in DNA methylation, including hypermethylation of specific genes and large genome regions with aberrant DNA methylation. Unsupervised hierarchical cluster analysis identified two main clusters that associated with the clinical, biological, and genetic features of patients. Group 1 was enriched in patients with adverse clinical and biological characteristics and poorer overall and progression-free survival. In addition, significant differences in DNA methylation were observed between patients with low risk and intermediate/high risk karyotypes and between TET2 mutant and wild type patients. Taken together, our results demonstrate that altered DNA methylation patterns reflect the CMML disease state and allow to identify patient groups with distinct clinical features.

Transcriptomic rationale for synthetic lethality-targeting ERCC1 and CDKN1A in chronic myelomonocytic leukaemia

Despite the absence of mutations in the DNA repair machinery in myeloid malignancies, the advent of high‐throughput sequencing and discovery of splicing and epigenetics defects in chronic myelomonocytic leukaemia (CMML) prompted us to revisit a pathogenic role for genes involved in DNA damage response. We screened for misregulated DNA repair genes by enhanced RNA‐sequencing on bone marrow from a discovery cohort of 27 CMML patients and 9 controls. We validated 4 differentially expressed candidates in CMML CD34+ bone marrow selected cells and in an independent cohort of 74 CMML patients, mutationally contextualized by targeted sequencing, and assessed their transcriptional behavior in 70 myelodysplastic syndrome, 66 acute myeloid leukaemia and 25 chronic myeloid leukaemia cases. We found BAP1 and PARP1 down‐regulation to be specific to CMML compared with other related disorders. Chromatin‐regulator mutated cases showed decreased BAP1 dosage. We validated a significant over‐expression of the double strand break‐fidelity genes CDKN1A and ERCC1, independent of promoter methylation and associated with chemorefractoriness. In addition, patients bearing mutations in the splicing component SRSF2 displayed numerous aberrant splicing events in DNA repair genes, with a quantitative predominance in the single strand break pathway. Our results highlight potential targets in this disease, which currently has few therapeutic options.

Calreticulin mutations are not present in patients with myeloproliferative chronic myelomonocytic leukemia

Dear Editor, Chronic myelomonocytic leukemia (CMML) has been classified into a new category of myelodysplastic/myeloproliferative diseases (MDS/MPN) according to the last WHO classification of myeloid malignancies [1]. However, some patients with CMML show proliferative features (MP-CMML) such as leukocytosis and organomegaly, making them clinically and biologically similar to myeloproliferative neoplasms (MPN). At biological level, JAK2V617F mutations in MP-CMML have been described at a lower frequency than in MPN [2]. Recently, CALR gene mutations have been described in 67–88 % JAK2V617F and MPL-negative MPN; these cases also have a good prognosis [3–7]. The role of CALR mutations in CMML is not well known, and the few studies carried out so far have not focused on MP subtype [3, 4, 8]. The objective of this study was to characterize the frequency, type, and prognostic implications of CALR mutations in MP-CMML. CALR exon 9 mutations were studied in 30 patients (22 males, median age 66 years) diagnosed of CMML according toWHO classification [1], and all of them belonged to MP-CMML according to the French-American-British Co-operative Leukaemia Group (Table 1). All patients provided informed consent for sample collection. Biological analyses were carried out in accordance with the Declaration of Helsinki. Mutation analysis was performed as previously described [3]. An informative result was obtained in 29/30 patients, and no CALR exon 9 mutation was found. Although a small percentage of mutated MP-CMML patients was expected [4], no mutations were detected in the samples studied. These results are in concordance with those of Klampfl T et al. [3] and Hou et al. [8], who found no CALR mutations in 64 and 48 CMML patients, respectively (subtype not specified), but they do differ from the results of Nangalia et al., who found 1/33 CMML mutated patient (subtype not specified) [4]. There is also another study [9] of other MDS/ MPN groups (atypical CML and unclassifiable MDS/MPN) where CALR mutations were not seen in either entity (n=30). Therefore, outside essential thrombocythemia and primary myelofibrosis, CALR mutations are only found in (1) a few patients with MDS/MPN, most of them defined as having refractory anemia with ring sideroblasts associated with marked thrombocytosis, even though the percentage described is very variable depending on the series (0 to 12.5 %) [3, 4, 10] and (2) a few MDS patients, mainly with refractory anemia with ring sideroblasts, refractory anemia, and refractory anemia with excess blasts [3, 4, 8, 10]. This observation could indicate a relationship between CALR mutations and the thrombocytosis phenotype within myeloid neoplasms, suggesting that calreticulin mutations primarily affect the biology of megakaryocytes [11]. L. Zamora (*) :M. Cabezon : S. Marce : L. Palomo : F. Milla : E. Feliu : B. Xicoy ICO Badalona, Hospital Germans Trias i Pujol, Institut de Recerca contra la Leucemia Josep Carreras, Badalona, Spain e-mail: lzamora@iconcologia.net