Mutational analysis in BCR-ABL1 positive leukemia by deep sequencing based on nanopore MinION technology.

We report a third-generation sequencing assay on nanopore technology (MinION) for detecting BCR-ABL1 KD mutations and compare the results to a Sanger sequencing(SS)-based test in 24 Philadelphia-positive (Ph+) leukemia cases. Our data indicates that MinION is markedly superior to SS in terms of sensitivity, costs and timesaving, and has the added advantage of determining the clonal configuration of multiple mutations. We demonstrate that MinION is suitable for employment in the hematology laboratory for detecting BCR-ABL1 KD mutation in Ph+ leukemias.

Myelodysplastic syndrome with 5q deletion following IgM monoclonal gammopathy, showing gene mutation MYD88 L265P.

Patients affected by monoclonal gammopathy of undetermined significance (MGUS) very rarely develop a myelodysplastic syndrome (MDS). However, it was also demonstrated that MGUS patients had a significantly increased risk of developing MDS compared to the general population. We report a case of 5q-syndrome following a MGUS IgMk with mutation of MYD88 L256P. To our knowledge, this is the first case of del(5q) MDS following MGUS IgMk with the MYD88 L256P mutation in which there is coexistence of the markers of the two clonal diseases, but as an expression of distinct pathological features.

ADAMTS2 gene dysregulation in T/myeloid mixed phenotype acute leukemia.

BACKGROUND: Mixed phenotype acute leukemias (MPAL) include acute leukemias with blasts that express antigens of more than one lineage, with no clear evidence of myeloid or lymphoid lineage differentiation. T/myeloid (T/My) MPAL not otherwise specified (NOS) is a rare leukemia that expresses both T and myeloid antigens, accounting for less than 1% of all leukemias but 89% of T/My MPAL. From a molecular point of view, very limited data are available on T/My MPAL NOS. CASE PRESENTATION: In this report we describe a T/My MPAL NOS case with a complex rearrangement involving chromosomes 5 and 14, resulting in overexpression of the ADAM metallopeptidase with thrombospondin type 1 motif, 2 (ADAMTS2) gene due to its juxtaposition to the T cell receptor delta (TRD) gene segment. CONCLUSION: Detailed molecular cytogenetic characterization of the complex rearrangement in the reported T/My MPAL case allowed us to observe ADAMTS2 gene overexpression, identifying a molecular marker that may be useful for monitoring minimal residual disease. To our knowledge, this is the first evidence of gene dysregulation due to a chromosomal rearrangement in T/My MPAL NOS.

Immunophenotypic and molecular features of 'cuplike' acute myeloid leukemias.

Nuclear invaginations, also referred to as fishmouth or cuplike nuclei, have long been identified in microgranular APL, myelomonocytic and monocytic AMLs. More recently, this typical morphological feature has been associated with NPM1 and FLT3 mutations, as well as with the lack of CD34 and HLA-DR expression. In this study, we retrospectively analyzed the morphologic, immunophenotypic, cytogenetic, and molecular features of 68 patients with AML. A cuplike nuclear invagination was detected in more than 10% of blast cells in 15 (22%) cases. Our data show that a cuplike morphology is associated with FLT3-ITD positivity, as well as with the loss of CD34 and HLA-DR expression. The results were not significantly modified when a higher cutoff of cuplike cells was used. Our results are not sufficient to suggest that cuplike AML could represent a distinct subtype, but further investigations could yield a better characterization of this feature in patients with AML.

Gene expression profiling of chronic myeloid leukemia with variant t(9;22) reveals a different signature from cases with classic translocation.

BACKGROUND: The t(9;22)(q34;q11) generating the BCR/ABL1 fusion gene represents the cytogenetic hallmark of chronic myeloid leukemia (CML). About 5-10% of CML cases show variant translocations with the involvement of other chromosomes in addition to chromosomes 9 and 22. The molecular bases of biological differences between CML patients with classic and variant t(9;22) have never been clarified. FINDINGS: In this study, we performed gene expression microarray analysis to compare CML patients bearing variant rearrangements and those with classic t(9;22)(q34;q11). We identified 59 differentially expressed genes significantly associated with the two analyzed groups. The role of specific candidate genes such as TRIB1 (tribbles homolog 1), PTK2B (protein tyrosine kinase 2 beta), and C5AR1 (complement component 5a receptor 1) is discussed. CONCLUSIONS: Our results reveal that in CML cases with variant t(9;22) there is an enhancement of the MAPK pathway deregulation and show that kinases are a common target of molecular alterations in hematological disorders.

Cytogenetic and Array-CGH Characterization of a Simple Case of Reciprocal t(3;10) Translocation Reveals a Hidden Deletion at 5q12.

Chromosome deletions, including band 5q12, have rarely been reported and have been associated with a wide range of clinical manifestations, such as postnatal growth retardation, intellectual disability, hyperactivity, nonspecific ocular defects, facial dysmorphism, and epilepsy. In this study, we describe for the first time a child with growth retardation in which we identified a balanced t(3;10) translocation by conventional cytogenetic analysis in addition to an 8.6 Mb 5q12 deletion through array-CGH. Our results show that the phenotypic abnormalities of a case that had been interpreted as "balanced" by conventional cytogenetics are mainly due to a cryptic deletion, highlighting the need for molecular investigation in subjects with an abnormal phenotype before assuming the cause is an apparently simple cytogenetic rearrangement. Finally, we identify PDE4D and PIK3R1 genes as the two major candidates responsible for the clinical features expressed in our patient.

Non random distribution of genomic features in breakpoint regions involved in chronic myeloid leukemia cases with variant t(9;22) or additional chromosomal rearrangements.

BACKGROUND: The t(9;22)(q34;q11), generating the Philadelphia (Ph) chromosome, is found in more than 90% of patients with chronic myeloid leukemia (CML). As a result of the translocation, the 3' portion of the ABL1 oncogene is transposed from 9q34 to the 5' portion of the BCR gene on chromosome 22 to form the BCR/ABL1 fusion gene. At diagnosis, in 5-10% of CML patients the Ph chromosome is derived from variant translocations other than the standard t(9;22). RESULTS: We report a molecular cytogenetic study of 452 consecutive CML patients at diagnosis, that revealed 50 cases identifying three main subgroups: i) cases with variant chromosomal rearrangements other than the classic t(9;22)(q34;q11) (9.5%); ii) cases with cryptic insertions of ABL1 into BCR, or vice versa (1.3%); iii) cases bearing additional chromosomal rearrangements concomitant to the t(9;22) (1.1%). For each cytogenetic group, the mechanism at the basis of the rearrangement is discussed.All breakpoints on other chromosomes involved in variant t(9;22) and in additional rearrangements have been characterized for the first time by Fluorescence In Situ Hybridization (FISH) experiments and bioinformatic analyses. This study revealed a high content of Alu repeats, genes density, GC frequency, and miRNAs in the great majority of the analyzed breakpoints. CONCLUSIONS: Taken together with literature data about CML with variant t(9;22), our findings identified several new cytogenetic breakpoints as hotspots for recombination, demonstrating that the involvement of chromosomes other than 9 and 22 is not a random event but could depend on specific genomic features. The presence of several genes and/or miRNAs at the identified breakpoints suggests their potential involvement in the CML pathogenesis.

Molecular evidence of the independent origin of multiple Wilms tumors in a case of WAGR syndrome.

BACKGROUND: This study investigated the genetic events leading to tumorigenesis in a patient affected with WAGR syndrome who developed multiple distinct Wilms tumors (WTs). PROCEDURE AND RESULTS: At 1 year of age, the child developed two synchronous bilateral WTs that were resected by partial nephrectomy. Histologically, these tumors were fetal rhabdomyomatous nephroblastomas. Immunohistochemical study revealed the absence of nuclear expression of WT1 protein, while beta-catenin protein was expressed at nuclear level by the large majority of tumor cells. Molecular investigations of WT1 gene and exon 3 of beta-catenin (CTNNB1) gene detected no mutations. At 4 years of age, 28 months after the chemotherapy completion, a third WT was diagnosed in the left kidney, and surgically removed before any further chemotherapy. Nine months after surgery, a metastasis was detected in the left lung. Both the third renal tumor and the lung metastasis showed a blastema-predominant morphology. Immunohistochemistry confirmed the lack of expression of WT1 protein, while beta-catenin protein was expressed at nuclear level by the large majority of tumor cells. Molecular analysis of the third renal tumor and the lung metastasis revealed a 4 bp deletion in exon 7 of WT1 gene, leading to a frameshift of the reading frame and to a premature stop of the translation (c.925_928delACTC, p.T309LfsX71); no mutations in the exon 3 of the beta-catenin gene were documented. CONCLUSIONS: These data demonstrate that multiple WTs can arise as a consequence of different genetic events in a patient with genetic predisposition, such as WAGR syndrome.

c-Kit/PDGFRA gene status alterations possibly related to primary imatinib resistance in gastrointestinal stromal tumors.

PURPOSE: To correlate morphologic changes with molecular, biochemical, and cytogenetic profiles in gastrointestinal stromal tumor (GIST) patients before and after imatinib treatment. EXPERIMENTAL DESIGN: We investigated 132 tumor samples obtained from 35 patients with advanced disease who underwent resective surgery after imatinib treatment according to the European Organization for Research and Treatment of Cancer-Soft Tissue and Bone Sarcoma Group protocol. On the basis of imaging findings, 27 patients were responders and 8 progressors, and retaining this radiological subdivision, we analyzed posttreatment morphologic changes correlating them with molecular, biochemical, and cytogenetic analyses. RESULTS: On the basis of morphology (residual viable cellularity/proliferation markers), three subgroups were identified showing high, moderate, or low response. All of the progressing cases clustered in the low-response subgroup, whereas the responding cases were distributed in all three subgroups. The correlation between morphology and the molecular findings showed that secondary mutations segregated with the low-response subgroup, whereas c-Kit primary resistance mutations were randomly distributed in the three subgroups. Fluorescence in situ hybridization analysis of c-Kit/PDGFRA genes showed that all of the progressing cases were disomic. Referring to morphology, among the responding cases, a disomic pattern was mainly restricted to the high responders, whereas the moderate and low responders were aneusomic. Comparison of post-imatinib genomic profiles with the 23 available primary tumors showed that 17 cases carried the same cytogenetic pattern. Overall, 12 of the 27 primary tumors presented a gain/loss of c-Kit/PDGFRA gene copy number. CONCLUSIONS: Our findings show that c-Kit/PDGFRA genomic alterations were present at disease onset in 1/3 of the examined cases. They therefore represent an early event possibly related to primary imatinib resistance in GISTs.

Cyclooxygenase-2 and platelet-derived growth factor receptors as potential targets in treating aggressive fibromatosis.

PURPOSE: To explore the molecular bases of potential new pharmacologic targets in aggressive fibromatosis (desmoid tumor). EXPERIMENTAL DESIGN: Tumor specimens from 14 patients surgically treated for aggressive fibromatosis (6 familial adenomatous polyposis and 8 sporadic cases), analyzed for adenomatous polyposis coli (APC) and CTNNB1 (beta-catenin) mutations, were further investigated for beta-catenin, cyclooxygenase-2 (COX-2), platelet-derived growth factor (PDGF) receptor alpha (PDGFRA)/PDGF receptor beta (PDGFRB), their cognate ligands (PDGFA and PDGFB), and KIT using a comprehensive immunohistochemical, biochemical, molecular, and cytogenetic approach. RESULTS: No CTNNB1 (beta-catenin) mutations were found in the familial adenomatous polyposis patients, but previously reported activating mutations were found in six of the eight sporadic patients. All of the cases carrying an altered WNT pathway showed nuclear and cytoplasmic immunoreactivity for beta-catenin, whereas beta-catenin expression was restricted to the cytoplasm in the sporadic patients lacking CTNNB1 mutations. COX-2 protein and mRNA overexpression was detected in all 14 cases, together with the expression and phosphorylation of PDGFRA and PDGFRB, which in turn paralleled the presence of their cognate ligands. No PDGFRB mutations were found. The results are consistent with PDGFRA and PDGFRB activation sustained by an autocrine/paracrine loop. CONCLUSIONS: Aggressive fibromatosis is characterized by WNT/oncogene pathway alterations triggering COX-2-mediated constitutive coactivation of PDGFRA and PDGFRB, and may therefore benefit from combined nonsteroidal anti-inflammatory drug + tyrosine kinase inhibitor treatment.

Genomic BCR-ABL1 breakpoint characterization by a multi-strategy approach for "personalized monitoring" of residual disease in chronic myeloid leukemia patients.

For monitoring minimal residual disease (MRD) in chronic myeloid leukemia (CML) the most recommended method is quantitative RT-PCR (RT-qPCR) for measuring BCR-ABL1 transcripts. Several studies reported that a DNA-based assay enhances the sensitivity of detection of the BCR-ABL1 genomic rearrangement, even if its characterization results difficult. We developed a DNA-based method for detecting and quantifying residual BCR-ABL1 positive leukemic stem cells in CML patients. We propose two alternative approaches: the first one is a fluorescence in situ hybridization (FISH)-based step followed by Sanger sequencing; the second one employs MinION, a single molecule sequencer based on nanopore technology. Finally, after defining the BCR-ABL1 genomic junction, we performed the target CML patient-specific quantification, using droplet digital PCR (ddPCR). FISH and MinION steps, respectively, together with ddPCR analysis, greatly reduce the complexity that has impeded the use of "personalized monitoring" of CML in clinical practice. Our report suggests a feasible pipeline, in terms of costs and reproducibility, aimed at characterizing and quantifying the genomic BCR-ABL1 rearrangement during MRD monitoring in CML patients.

RARA and RARG gene downregulation associated with EZH2 mutation in acute promyelocytic-like morphology leukemia.

Most acute promyelocytic leukemia (APL) patients express PML-RARA fusion; in rare cases, RARA is rearranged with partner genes other than PML. To date, only 2 patients presenting features similar to APL showing the RARG gene rearrangement have been described. We report an acute myeloid leukemia patient with morphology resembling APL without involvement of the RARA gene. Molecular and fluorescent in situ hybridization analyses excluded PML-RARA fusion and variant rearrangements involving RARA and RARG loci. Targeted next-generation sequencing showed EZH2- D185H mutation. As this mutation involved the region of interaction with DNA methyltransferases, we speculate an epigenetic alteration of genes involved in the APL-like phenotype. Expression analysis by droplet digital polymerase chain reaction revealed downregulation of the RARA and RARG genes. We hypothesize a novel mechanism of EZH2 function alteration, which may be responsible for an acute myeloid leukemia with APL-like phenotype featuring dysregulation of the RARA and RARG genes.

Design and MinION testing of a nanopore targeted gene sequencing panel for chronic lymphocytic leukemia.

We report a customized gene panel assay based on multiplex long-PCR followed by third generation sequencing on nanopore technology (MinION), designed to analyze five frequently mutated genes in chronic lymphocytic leukemia (CLL): TP53, NOTCH1, BIRC3, SF3B1 and MYD88. For this purpose, 12 patients were selected according to specific cytogenetic and molecular features significantly associated with their mutational status. In addition, simultaneous analysis of the targets genes was performed by molecular assays or Sanger Sequencing. Data analysis included mapping to the GRCh37 human reference genome, variant calling and annotation, and average sequencing depth/error rate analysis. The sequencing depth resulted on average higher for smaller amplicons, and the final breadth of coverage of the panel was 94.1%. The error rate was about 6% and 2% for insertions/deletions and single nucleotide variants, respectively. Our gene panel allows analysis of the prognostically relevant genes in CLL, with two PCRs per patient. This strategy offers an easy and affordable workflow, although further advances are required to improve the accuracy of the technology and its use in the clinical field. Nevertheless, the rapid and constant development of nanopore technology, in terms of chemistry advances, more accurate basecallers and analysis software, offers promise for a wide use of MinION in the future.

Droplet digital PCR for the quantification of Alu methylation status in hematological malignancies.

BACKGROUND: Alu repeats, belonging to the Short Interspersed Repetitive Elements (SINEs) class, contain about 25% of CpG sites in the human genome. Alu sequences lie in gene-rich regions, so their methylation is an important transcriptional regulation mechanism. Aberrant Alu methylation has been associated with tumor aggressiveness, and also previously discussed in hematological malignancies, by applying different approaches. Moreover, today different techniques designed to measure global DNA methylation are focused on the methylation level of specific repeat elements. In this work we propose a new method of investigating Alu differential methylation, based on droplet digital PCR (ddPCR) technology. METHODS: Forty-six patients with hematological neoplasms were included in the study: 30 patients affected by chronic lymphocytic leukemia, 7 patients with myelodysplastic syndromes at intermediate/high risk, according with the International Prognostic Scoring System, and 9 patients with myelomonocytic leukemia. Ten healthy donors were included as controls. Acute promyelocytic leukemia-derived NB4 cell line, either untreated or treated with decitabine (DEC) hypomethylating agent, was also analyzed. DNA samples were investigated for Alu methylation level by digestion of genomic DNA with isoschizomers with differential sensitivity to DNA methylation, followed by ddPCR. RESULTS: Using ddPCR, a significant decrease of the global Alu methylation level in DNA extracted from NB4 cells treated with DEC, as compared to untreated cells, was observed. Moreover, comparing the global Alu methylation levels at diagnosis and after azacytidine (AZA) treatment in MDS patients, a statistically significant decrease of Alu sequences methylation after therapy as compared to diagnosis was evident. We also observed a significant decrease of the Alu methylation level in CLL patients compared to HD, and, finally, for CMML patients, a decrease of Alu sequences methylation was observed in patients harboring the SRSF2 hotspot gene mutation c.284C>D. CONCLUSIONS: In our work, we propose a method to investigate Alu differential methylation based on ddPCR technology. This assay introduces ddPCR as a more sensitive and immediate technique for Alu methylation analysis. To date, this is the first application of ddPCR to study DNA repetitive elements. This approach may be useful to profile patients affected by hematologic malignancies for diagnostic/prognostic purpose.

Droplet Digital PCR Is a Robust Tool for Monitoring Minimal Residual Disease in Adult Philadelphia-Positive Acute Lymphoblastic Leukemia.

The breakpoint cluster region-abelson 1 p190 fusion transcript is the most frequent variant observed in Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL). Qualitative-PCR and real-time quantitative PCR are the currently used methods to monitor minimal residual disease (MRD) in Ph+ ALL patients; for the latter, full standardization and an international quality validation are lacking. Here, we developed a droplet digital PCR (ddPCR) assay for MRD monitoring in p190+ ALL cases. The analytical performance was assessed by the limit-of-detection determination, showing a reliability, sensitivity, and precision of the assay of up to 0.001%. Comparison of results obtained with qualitative PCR and ddPCR in 117 follow-up samples from 16 of 26 Ph+ ALL patients showed discordant results in 27% of cases (32 of 117). Real-time quantitative PCR analysis of 19 ddPCR-positive samples with a low tumor burden failed to provide quantitative results in 63% of cases (12 of 19). These results highlight that in p190+ ALL the ddPCR method has a sufficient analytical performance for very low MRD monitoring and for predicting molecular relapse several months before hematologic relapse. In conclusion, MRD monitoring by ddPCR may better stratify Ph+ ALL patients at risk of disease progression.

Heterochromatin protein 1 interacts with 5'UTR of transposable element ZAM in a sequence-specific fashion.

The realization of cross talks between transposable elements of class I and their host genome involves non-histonic chromatin proteins. These interactions have been widely analyzed through the characterization of the gypsy retrotransposon leader region, which holds a particularly strong insulator element, and the proteins required for its function, Su(Hw), Mod(mdg4), and Cp190. Here we provide evidence that a similar interaction should occur between ZAM, a gypsy-like element, and HP1, one of the most extensively studied chromatin proteins. We first assayed the existence of this binding using the yeast cells one-hybrid system and then we verified it in vivo by ChIP assay. In order to characterize the interaction between HP1 and the ZAM 5' untranslated region we performed a series of gel shift analyses. Our observations confirm an HP1 co-operative DNA-binding and display for the first time the HP1 DNA target motif that, we hypothesize, should be one of its nucleation sites.

Molecular and cytogenetic subgroups of oropharyngeal squamous cell carcinoma.

PURPOSE: The aim of this study was to acquire further insights into the pathogenetic pathways of head and neck squamous cell carcinomas (HNSCC) that may be useful for identifying new biomarkers instrumental in developing more specific treatment approaches. EXPERIMENTAL DESIGN: Cell cycle regulators and epidermal growth factor receptor (EGFR) and BRAF genes were analyzed in a series of 90 oropharyngeal SCCs of a cohort of surgically treated patients from a single institution, and the results were matched with the presence of high-risk human papillomavirus (HR-HPV) DNA and the TP53 status. RESULTS: At least four distinct groups of tumors were identified sharing a common histology but displaying different molecular/cytogenetic patterns: (a) 19% were HPV-positive SCCs whose lack of alterations of the investigated genes could explain their particular natural history, which requires less aggressive treatment; (b) 37% were HPV-negative SCCs carrying TP53 mutations, which may be more effectively treated by drugs acting through p53-independent apoptosis; (c) 34% were HPV-negative SCCs carrying wild-type TP53 and loss of 9p21 (p16INK4a and p15INK4b) and/or cyclin D1 overexpression that justify treatment with DNA-damaging drugs followed by cell cycle inhibitors; and (d) 10% were HPV-negative lacking tumor suppressor genes and cell cycle alterations. The second, third, and fourth groups also showed an increased copy number of EGFR and chromosome 7 (43%) that might justify the additional or alternative use of EGFR inhibitors. CONCLUSIONS: Our findings suggest that assessing HPV, TP53, 9p21, and EGFR status may be crucial to finding more tailored and beneficial treatments for oropharyngeal SCCs.

FLAG-Ida Regimen as Bridge Therapy to Allotransplantation in Refractory/Relapsed Acute Myeloid Leukemia Patients.

BACKGROUND: Patients with primary refractory or first relapse acute myeloid leukemia (AML) are considered to have worse clinical outcomes after treatment. For these patients, the achievement of complete remission appears crucial for them to be able to undergo allotransplantation, which might be the only possible treatment. PATIENTS AND METHODS: We used the FLAG-Ida (fludarabine, cytarabine [cytosine arabinoside], granulocyte colony-stimulating factor, idarubicin) regimen in patients with primary refractory/first relapse AML as a bridge to transplantation. We studied its efficacy in terms of overall response and overall survival to assess which variables (age, lactate dehydrogenase, bone marrow blast count, peripheral blood blast count, platelet count, white blood cell count, de novo or secondary AML, molecular-cytogenetic risk, duration of response, and relapsed or refractory disease) might have an effect on outcome. RESULTS: We analyzed the data from 108 consecutive adult patients (52 males, 66 females; median age, 49 years; range, 17-72 years) with newly diagnosed AML refractory to standard induction regimens or relapse after first complete remission, who had received the FLAG-Ida protocol as salvage therapy from January 2005 to December 2015. An overall response was achieved in 48 patients (44%). On multivariate analysis, the variables with a positive effect on the response rate were molecular-cytogenetic risk (P = .009), duration of first response in relapsed AML (P = .003), AML status (relapsed or refractory; P = .047), and peripheral blood blast count (P = .016). On multivariate analysis, overall survival was significantly associated with FLAG-Ida response (hazard ratio, 0.343; P = .001) and receipt of allotransplantation (hazard ratio, 0.277; P < .001). CONCLUSION: Our data seem to confirm the value of FLAG-Ida in this setting and might suggest its best usage as bridge therapy for patients awaiting allotransplantation.