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Chronic myeloid leukemia (CML) is a rare myeloproliferative disorder caused by the reciprocal translocation t(9;22)(q34;q11) in hematopoietic stem cells (HSCs). This chromosomal translocation results in the formation of an extra-short chromosome 22, called a Philadelphia chromosome (Ph), containing the BCR-ABL1 fusion gene responsible for the expression of a constitutively active tyrosine kinase that causes uncontrolled growth and replication of leukemic cells. Mechanisms behind the formation of this chromosomal rearrangement are not well known, even if, as observed in tumors, repetitive DNA may be involved as core elements in chromosomal rearrangements. We have participated in the explorative investigations of the PhilosoPhi34 study to evaluate residual Ph+ cells in patients with negative FISH analysis on CD34+/lin- cells with gDNA qPCR. Using targeted next-generation deep sequencing strategies, we analyzed the genomic region around the t(9;22) translocations of 82 CML patients and one CML cell line and assessed the relevance of interspersed repeat elements at breakpoints (BP). We found a statistically higher presence of LINE elements, in particular belonging to the subfamily L1M, in BP cluster regions of both chromosome 22 and 9 compared to the whole human genome. These data suggest that L1M elements could be potential drivers of t(9;22) translocation leading to the generation of the BCR-ABL1 chimeric gene and the expression of the active BCR-ABL1-controlled tyrosine kinase chimeric protein responsible for CML.

Chronic Myeloid Leukemia (CML) is a stem cell cancer that arises when t(9;22) translocation occurs in a hematopoietic stem cells. This event results in the expression of the BCR-ABL1 fusion gene, which codes for a constitutively active tyrosine kinase that is responsible for the transformation of a HSC into a CML stem cell, which then gives rise to a clonal myeloproliferative disease. The introduction of Tyrosine Kinase Inhibitors (TKIs) has revolutionized the management of the disease. However, these drugs do not seem to be able to eradicate the malignancy. Indeed, discontinuation trials (STIM; TWISER; DADI) for those patients who achieved a profound molecular response showed 50% relapsing within 12 months. We performed a comparative analysis on 15 CML patients and one B-ALL patient, between the standard quantitative reverse-transcriptase PCR (qRT-PCR) and our genomic DNA patient-specific quantitative PCR assay (gDNA qPCR). Here we demonstrate that gDNA qPCR is better than standard qRT-PCR in disease monitoring after an average follow-up period of 200 days. Specifically, we statistically demonstrated that DNA negativity is more reliable than RNA negativity in indicating when TKIs therapy can be safely stopped.

Background: Clonal chromosome changes are often found in the bone marrow (BM) of patients with Shwachman-Diamond syndrome (SDS). The most frequent ones include an isochromosome of the long arm of chromosome 7, i (7)(q10), and an interstitial deletion of the long arm of chromosome 20, del (20)(q). These two imbalances are mechanisms of somatic genetic rescue. The literature offers few expression studies on SDS. Results: We report the expression analysis of bone marrow (BM) cells of patients with SDS in relation to normal karyotype or to the presence of clonal chromosome anomalies: del (20)(q) (five cases), i (7)(q10) (one case), and other anomalies (two cases). The study was performed using the microarray technique considering the whole transcriptome (WT) and three gene subsets selected as relevant in BM functions. The expression patterns of nine healthy controls and SDS patients with or without chromosome anomalies in the bone marrow showed clear differences. Conclusions: There is a significant difference between gene expression in the BM of SDS patients and healthy subjects, both at the WT level and in the selected gene sets. The deletion del (20)(q), with the EIF6 gene consistently lost, even in patients with the smallest losses of material, changes the transcription pattern: a low proportion of abnormal cells led to a pattern similar to SDS patients without acquired anomalies, whereas a high proportion yields a pattern similar to healthy subjects. Hence, the benign prognostic value of del (20)(q). The case of i (7)(q10) showed a transcription pattern similar to healthy subjects, paralleling the positive prognostic role of this anomaly as well.

Malignant Pleural Mesothelioma (MPM) is a heterogeneous disease. Morphologically, three different phenotypes are distinguishable: epithelioid (e-), sarcomatoid (s-) and biphasic (biph-) MPM, the latest, being a mixture of e-and s-MPM cells. Being an intermediate entity, management of biph-MPM, remains debatable and controversial, with different guidelines recommending distinct approaches. Identification of biph-MPM associated genetic alterations, through deep sequencing analysis, may provide useful tools to understand these lesions. A retrospective cohort of 69 surgically resected MPMs, 39 biph-MPMs (56.5%) and 30 e-MPMs (43.5%) was selected. A separate set of 16 biph-MPM was used as validation set. Deep sequencing analysis on an MPM-specific custom panel (MPM_geneset) comprising 1041 amplicons spanning 34 genes was performed. A total of 588 variants and 5309 mutational events were detected. In total, 91.3% of MPMs showed at least one mutation and 76.8% showed co-occurrence of more than one alteration. Mutations in MXRA5 (p = 0.05) and NOD2 (p = 0.018) were significantly associated with biph-MPM both in the training and validation cohort and correlated with the extent of the sarcomatoid component. Mutations in NOD2 and XRCC6 correlated with patients’ survival. We demonstrated that biph-MPM are associated with a specific mutation set, and that genetic analysis at diagnosis may improve patients’ risk stratification.

In Shwachman-Diamond syndrome (SDS), deletion of the long arm of chromosome 20, del(20)(q), often acquired in bone marrow (BM), may imply a lower risk of developing myelodysplastic syndrome/acute myeloid leukaemia (MDS/AML), due to the loss of the EIF6 gene. The genes L3MBTL1 and SGK2, also on chromosome 20, are in a cluster of imprinted genes, and their loss implies dysregulation of BM function. We report here the results of array comparative genomic hybridization (a-CGH) performed on BM DNA of six patients which confirmed the consistent loss of EIF6 gene. Interestingly, array single nucleotide polymorphisms (SNPs) showed copy neutral loss of heterozygosity for EIF6 region in cases without del(20)(q). No preferential parental origin of the deleted chromosome 20 was detected by microsatellite analysis in six SDS patients. Our patients showed a very mild haematological condition, and none evolved into BM aplasia or MDS/AML. We extend the benign prognostic significance of del(20)(q) and loss of EIF6 to the haematological features of these patients, consistently characterized by mild hypoplastic BM, no or mild neutropenia, anaemia and thrombocytopenia. Some odd results obtained in microsatellite and SNP-array analysis demonstrate a peculiar genomic instability, in an attempt to improve BM function through the acquisition of the del(20)(q).

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Background: The majority of the cases of bone marrow failure syndromes/aplastic anaemias (BMFS/AA) are non-hereditary and considered idiopathic (80-85%). The peripheral blood picture is variable, with anaemia, neutropenia and/or thrombocytopenia, and the patients with idiopathic BMFS/AA may have a risk of transformation into a myelodysplastic syndrome (MDS) and/or an acute myeloid leukaemia (AML), as ascertained for all inherited BMFS. We already reported four patients with different forms of BMFS/AA with chromosome anomalies as primary etiologic event: the chromosome changes exerted an effect on specific genes, namely RUNX1, MPL, and FLI1, leading to the disease. Results: We report two further patients with non-hereditary BM failure, with diagnosis of severe aplastic anaemia and pancytopenia caused by two different constitutional structural anomalies involving chromosome 8, and possibly leading to the disorder due to effects on the RUNX1T1 gene, which was hypo-expressed and hyper-expressed, respectively, in the two patients. The chromosome change was unbalanced in one patient, and balanced in the other one. Conclusions: We analyzed the sequence of events in the pathogenesis of the disease in the two patients, including a number of non-haematological signs present in the one with the unbalanced anomaly. We demonstrated that in these two patients the primary event causing BMFS/AA was the constitutional chromosome anomaly. If we take into account the cohort of 219 patients with a similar diagnosis in whom we made cytogenetic studies in the years 2003-2017, we conclude that cytogenetic investigations were instrumental to reach a diagnosis in 52 of them. We postulate that a chromosome change is the primary cause of BMFS/AA in a not negligible proportion of cases, as it was ascertained in 6 of these patients.