Genomic analysis in short- and long-term patients with malignant pleura mesothelioma treated with palliative chemotherapy.

BACKGROUND: Malignant pleural mesothelioma (MPM) is an aggressive tumour with poor prognosis. The aim of this study was to identify genetic mutations associated with poor or extended survival in patients who received palliative chemotherapy. METHODS: A total of 720 patients diagnosed with MPM between 2005 and 2015 were identified. Overall survival (OS) was longer than 30 months from diagnosis for 27 patients. Twelve of 27 (44%) of the pleural biopsies from long-term survivors were retrieved and matched with 12 biopsies from patients who survived less than 12 months; one biopsy was then excluded for poor DNA quality. RESULTS: A total of 11 patients had a mean OS of 5.5 months, whereas 12 patients lived more than 30 months (mean OS: 55.8 ± 25). Mutational analysis identified 428 alterations; of which, 148, classified as somatic and functional, were considered further. Among these, 85% were missense variants, 8% were variants causing a stop gain and 6% were splice variants. Loss-of-function mutations in UQCRC1 were significantly associated with reduced survival in patients with MPM (p = 0.027), while a higher frequency of mutations in MXRA5 and RAPGEF6 was registered in long-term survivors. CONCLUSION: This is the first study evaluating the relationship between the mutational profile and outcome in patients with MPM after palliative chemotherapy. UQCRC1 codes for cytochrome b-c1 complex subunit 1 which plays a fundamental role in normal mitochondrial functions and in cell metabolism. Recent studies described UQCRC1 deregulation in other cancers. Our results suggest a possible role for mitochondrial metabolism in the biology of mesothelioma.

DNA aneuploidy and tissue architecture in oral potentially malignant disorders with epithelial dysplasia assessed by a 10 locus FISH panel.

Subjectivity in oral dysplasia grading has prompted evaluation of molecular­based tests to predict malignant transformation. Aneuploidy detected by DNA image­based cytometry (ICM) is currently the best predictor but fails to detect certain high risk lesions. A novel multiplex fluorescence in situ hybridization (FISH) panel was used to explore possible explanations by detecting aneuploidy at the single cell level. FISH was compared to reference standard DNA ICM in 19 oral lesions with epithelial dysplasia and used to characterize the cellular architecture. Copy number variation at 3q28, 7p11.2, 8q24.3, 11q13.3 and 20q13.12 and matched chromosome specific loci were assessed by dual­color FISH to assess numerical and spatial patterns of copy number increase and gene amplification. FISH revealed wide variation in copy number at different loci. Only low level copy number gain was present and often in only a small proportion of cells, although usually with all or all but one locus (9/12). Four cases showed gene amplification, one at two loci. Some probes revealed an internal presumed clonal structure within lesions not apparent in routine histological examination. Both methods produced similar diagnostic results with concordance in detection of aneuploidy by both methods in 17 out of 19 samples (89%). We have shown that oral dysplastic lesions may contain very few aneuploid cells at a cellular level, high copy number gain is rare and changes appear to arise from large chromosomal fragment duplications. Single stem lines are relatively homogeneous for loci with copy number gain but there is a subclonal structure revealed by gene amplification in some lesions.

MET-EGFR dimerization in lung adenocarcinoma is dependent on EGFR mtations and altered by MET kinase inhibition.

Advanced lung cancer has poor survival with few therapies. EGFR tyrosine kinase inhibitors (TKIs) have high response rates in patients with activating EGFR mutations, but acquired resistance is inevitable. Acquisition of the EGFR T790M mutation causes over 50% of resistance; MET amplification is also common. Preclinical data suggest synergy between MET and EGFR inhibitors. We hypothesized that EGFR-MET dimerization determines response to MET inhibition, depending on EGFR mutation status, independently of MET copy number. We tested this hypothesis by generating isogenic cell lines from NCI-H1975 cells, which co-express L858R and T790M EGFR mutations, namely H1975L858R/T790M (EGFR TKI resistant); H1975L858R (sensitized) and H1975WT (wild-type). We assessed cell proliferation in vitro and tumor growth/stroma formation in derived xenograft models in response to a MET TKI (SGX523) and correlated with EGFR-MET dimerization assessed by Förster Resonance Energy Transfer (FRET). SGX523 significantly reduced H1975L858R/T790M cell proliferation, xenograft tumor growth and decreased ERK phosphorylation. The same was not seen in H1975L858R or H1975WT cells. SGX523 only reduced stroma formation in H1975L858R. SGX523 reduced EGFR-MET dimerization in H1975L858R/T790M but induced dimer formation in H1975L858R with no effect in H1975WT. Our data suggests that MET inhibition by SGX523 and EGFR-MET heterodimerisation are determined by EGFR genotype. As tumor behaviour is modulated by this interaction, this could determine treatment efficacy.

The prognosis of MYC translocation positive diffuse large B-cell lymphoma depends on the second hit.

A proportion of MYC translocation positive diffuse large B-cell lymphomas (DLBCL) harbour a BCL2 and/or BCL6 translocation, known as double-hit DLBCL, and are clinically aggressive. It is unknown whether there are other genetic abnormalities that cooperate with MYC translocation and form double-hit DLBCL, and whether there is a difference in clinical outcome between the double-hit DLBCL and those with an isolated MYC translocation. We investigated TP53 gene mutations along with BCL2 and BCL6 translocations in a total of 234 cases of DLBCL, including 81 with MYC translocation. TP53 mutations were investigated by PCR and sequencing, while BCL2 and BCL6 translocation was studied by interphase fluorescence in situ hybridization. The majority of MYC translocation positive DLBCLs (60/81 = 74%) had at least one additional genetic hit. In MYC translocation positive DLBCL treated by R-CHOP (n = 67), TP53 mutation and BCL2, but not BCL6 translocation had an adverse effect on patient overall survival. In comparison with DLBCL with an isolated MYC translocation, cases with MYC/TP53 double-hits had the worst overall survival, followed by those with MYC/BCL2 double-hits. In MYC translocation negative DLBCL treated by R-CHOP (n = 101), TP53 mutation, BCL2 and BCL6 translocation had no impact on patient survival. The prognosis of MYC translocation positive DLBCL critically depends on the second hit, with TP53 mutations and BCL2 translocation contributing to an adverse prognosis. It is pivotal to investigate both TP53 mutations and BCL2 translocations in MYC translocation positive DLBCL, and to distinguish double-hit DLBCLs from those with an isolated MYC translocation.

Chromosomal anomalies in primary cutaneous follicle center cell lymphoma do not portend a poor prognosis.

BACKGROUND: The t(14;18)(q32;q21) chromosomal translocation is found in the majority of nodal follicular lymphomas but only rarely in primary cutaneous follicle center cell lymphomas (PCFCL). Recent studies have postulated that the translocation is more prevalent in PCFCL than previously described and that it might be a molecular prognostic marker. OBJECTIVES: The purpose of our study was to analyze cases of PCFCL for the presence of a t(14;18) translocation using fluorescence in situ hybridization to detect balanced translocations involving either the BCL2 or MALT1 loci and to correlate the results with growth pattern, immunophenotype, and clinical outcome. METHOD: In all, 57 patients with PCFCL were extracted from our cutaneous lymphoma database. Retrospective analysis of clinical parameters including lesion type, location, diagnostic stage, lactate dehydrogenase, initial treatment, relapse rate, and survival was performed. RESULTS: In all, 57 patients with PCFCL were included in this study. We detected 1 BCL2 chromosomal amplification, 4 translocations of BCL2, and 1 IGH/MALT1 translocation. LIMITATIONS: This was a case series retrospective study. CONCLUSIONS: PCFCL has an excellent 5-year overall survival (100% disease-specific survival). Chromosomal abnormalities of either BCL2 or MALT1 were detected in 10% of cases but do not correlate with a specific immune pathology or clinical outcome.

Fluorescence in situ hybridisation analysis of bone marrow trephine biopsy specimens; an additional tool in the diagnostic armoury.

Fluorescence in situ hybridisation (FISH) analysis is now widely employed in the diagnosis and risk stratification of a wide range of malignant diseases. While this technique is used successfully with formalin-fixed paraffin-embedded (FFPE) sections from numerous tissue types, FISH analysis of FFPE tissue sections from trephine biopsy specimens has been less widely reported, possibly due to technical limitations relating to the decalcification protocols employed. During the last 4 years FISH analysis has been carried out successfully in 42 out of 55 (76%) consecutive trephine biopsy specimens received as part of the standard diagnostic service at our institution. Samples decalcified using EDTA-based protocols were analysed successfully in 31/31 cases (100%), whereas only 11/24 samples (46%) decalcified using formic acid-based protocols were successful. In our experience, FISH analysis of trephine biopsy specimens is a highly reproducible technique and a very useful adjunctive tool in the diagnostic armoury; however, its use in a standard diagnostic setting relies on the use of EDTA-based decalcification protocols.

Fluorescence in situ hybridisation analysis of formalin-fixed paraffin-embedded tissue sections in the diagnostic work-up of non-Burkitt high grade B-cell non-Hodgkin's lymphoma: a single centre's experience.

AIMS: In recent years the genetic aberrations associated with diffuse large B-cell lymphoma and the new subtype described in the 2008 revision of the WHO classification, 'B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma' have been increasingly well defined. Recurrent genetic abnormalities include rearrangements involving MYC (8q24), BCL2 (18q21) and BCL6 (3q27); as the prognostic and therapeutic implications associated with these abnormalities are clarified their accurate identification at diagnosis is becoming increasingly critical. We describe our experience of using a panel of fluorescence in situ hybridisation (FISH) probes on formalin-fixed paraffin-embedded tissue sections in the diagnostic work-up of 162 patients with non-Burkitt high grade B-cell non-Hodgkin's lymphomas (HG-BNHL). METHODS: BCL6, IGH-BCL2 and MYC status were determined prospectively in sequential patients presenting with HG-BNHL, with respect to the presence of rearrangements and copy number changes. Small numbers of samples were analysed retrospectively or were studied at relapse in previously untested patients. RESULTS: FISH analysis was successful in 160/162 (99%) cases, with abnormalities detected in 118/160 (74%). CONCLUSIONS: FISH analysis of formalin-fixed paraffin-embedded tissue sections is a highly reproducible technique with an excellent success rate for the detection of genetic abnormalities which will play an increasingly important role in improving risk stratification of patients with HG-BNHL.

Molecular diagnostics-an emerging frontier in dermatopathology.

Advances made within the field of genomics and proteomics have facilitated the emergence of a new era of molecular diagnostics. However, ongoing rapid developments in molecular methodology ensure that this remains a complex field, accessible primarily to scientists who routinely utilize these techniques. For this reason, in this article we provide a concise overview of established and emerging molecular methods and discuss their role as diagnostic adjuncts in Dermatopathology. Important nonmolecular techniques that are used in conjunction with molecular methods are also highlighted.

Detection of cryptic and variant IGH-MYC rearrangements in high-grade non-Hodgkin's lymphoma by fluorescence in situ hybridization: implications for cytogenetic testing.

In recent years it has become increasingly evident that MYC rearrangements are not confined to classical Burkitt lymphoma (BL), but also occur in diffuse large B-cell lymphoma (DLBCL) and in the new subtype, "B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and BL" (BCLU), which was recently described in the 2008 revision of the World Health Organization classification. The accurate identification of MYC rearrangements in these three subtypes of high-grade lymphoma is becoming increasingly critical both in terms of diagnosis of classical BL and in light of the prognostic implications in cases of DLBCL and BCLU. We describe three cases of high-grade lymphoma in which cryptic insertion events, resulting in clinically significant IGH-MYC rearrangements, were detectable using an IGH/MYC three-color, dual-fusion fluorescence in situ hybridization (FISH) probe set, but were not detected using break-apart MYC FISH probes, thus highlighting the limitations of using break-apart probes as a stand-alone test, particularly with the increased use of interphase FISH analysis of formalin-fixed, paraffin-embedded tissue sections in the diagnostic work-up of these patients.

Minimal residual disease detection in childhood acute lymphoblastic leukaemia patients at multiple time-points reveals high levels of concordance between molecular and immunophenotypic approaches.

In this single centre study of childhood acute lymphoblastic leukaemia (ALL) patients treated on the Medical Research Council UKALL 97/99 protocols, it was determined that minimal residual disease (MRD) detected by real time quantitative polymerase chain reaction (RQ-PCR) and 3-colour flow cytometry (FC) displayed high levels of qualitative concordance when evaluated at multiple time-points during treatment (93.38%), and a combined use of both approaches allowed a multi time-point evaluation of MRD kinetics for 90% (53/59) of the initial cohort. At diagnosis, MRD markers with sensitivity of at least 0.01% were identified by RQ-PCR detection of fusion gene transcripts, IGH/TRG rearrangements, and FC. Using a combined RQ-PCR and FC approach, the evaluation of 367 follow-up BM samples revealed that the detection of MRD >1% at Day 15 (P = 0.04), >0.01% at the end of induction (P = 0.02), >0.01% at the end of consolidation (P = 0.01), >0.01% prior to the first delayed intensification (P = 0.01), and >0.1% prior to the second delayed intensification and continued maintenance (P = 0.001) were all associated with relapse and, based on early time-points (end of induction and consolidation) a significant log-rank trend (P = 0.0091) was noted between survival curves for patients stratified into high, intermediate and low-risk MRD groups.

Array comparative genome hybridization analysis of acute lymphoblastic leukaemia and acute megakaryoblastic leukaemia in patients with Down syndrome.

Twenty-five cases of B-cell precursor acute lymphoblastic leukaemia (ALL) from Down syndrome (DS) patients were analyzed using array comparative genomic hybridization (aCGH) and compared with two other subgroups of non-DS patients with ALL; five cases with high-hyperdiploidy (HH) and nine cases with ETV6-RUNX1 positive clones. Seven cases of DS-acute megakaryoblastic leukaemia (AMKL) were also included, DS-ALL cases showed relatively stable karyotypes with cryptic losses and gains that most frequently involved chromosomes X, 1, 2, 9, 11, 16, and 17. The most consistent change involved a deletion in 2p, spanning region Chr2:88273220-91084234, which in some cases appeared to be homozygous. ALL from non-DS patients showed a similar overall karyotypic stability, although gains of chromosome 21 were infrequent in the ETV6-RUNX1 positive cases. The most consistent change in this group involved a 12p deletion, where Chr12:10383878-16017619 defined the common region of overlap. All HH-ALL karyotypes showed variable gains of chromosome 21. This overall analysis supports the suggestion that, although constitutional trisomy 21 predisposes to ALL/AMKL, the cytogenetic changes associated with DS-ALL in particular, are most similar to those found in non-DS ETV6-RUNX1 positive ALL. The HH-ALL group, however, undergoes distinct karyotypic evolution not dependent on chromosome translocation/deletion events.

Characterization of the t(17;19) translocation by gene-specific fluorescent in situ hybridization-based cytogenetics and detection of the E2A-HLF fusion transcript and protein in patients' cells.

This is the first report to comprehensively characterize the E2A-HLF fusion generated from the t(17;19)(q22;p13) translocation in childhood B-lineage acute lymphoblastic leukemia. E2A gene rearrangement and E2A-HLF transcript and protein expression were determined using conventional cytogenetics, fluorescent in situ hybridization, reverse transcriptase polymerase chain reaction and Western blotting in leukemic cells from three patients.

3'CBFbeta deletion associated with inv(16) in acute myeloid leukemia.

Recent reports have shown that concomitant submicroscopic deletions can occur in association with chromosomal translocations/inversions in several leukemia subtypes. Detectable by fluorescence in situ hybridization (FISH), these losses of sequence include deletion of the 5' region of the ABL gene and the 3' region of BCR in chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL), as well as the 5' region of ETO in acute myeloid leukemia (AML) French-American-British type M2 associated with t(8;21), 3'MLL in AML and ALL, and 3' core-binding factor beta (CBFbeta) in AML associated with inv(16). While it has been widely reported that submicroscopic deletions of the derivative 9 in CML have an adverse prognostic impact, the clinical significance, if any, of deletions associated with t(8;21), inv(16)/t(16;16), or MLL rearrangement is yet to be determined. We analyzed a series of 39 patients diagnosed with AML who had cytogenetically detectable inv(16)/t(16;16) by using a FISH probe for the CBFbeta region to determine the incidence of the 3'CBFbeta deletion. Deletions were detected in three patients (8%), all associated with inv(16), bringing the number of cases reported so far to seven. The prognostic significance of this finding remains unclear.

Development of a human acute myeloid leukaemia screening panel and consequent identification of novel gene mutation in FLT3 and CCND3.

A study was undertaken to develop an acute myeloid leukaemia (AML) screening panel to uncover novel recurring gene mutations. Analysis was performed on six genes known to be mutated in AML (RUNX1, FLT3, KIT, CEBPA, PTPN11 and NRAS) and an additional two candidate genes (CCND3 and FES) in a panel of 175 primary human AML samples that included all French-American-British types except M3, and all cytogenetic risk groups. One hundred and fifteen mutations were identified in 97 (55%) patients comprising 81 patients (46%) with one mutation, 14 patients (8%) with two mutations and two patients (1%) with three mutations. Fifty-five of 88 (63%) patients with normal karyotype AML had at least one mutation. Correlation was observed between KIT mutation and 'favourable risk' cytogenetics (P <0.001), CEBPA mutation and 'intermediate risk' cytogenetics (P=0.045), and PTPN11 mutation and 'poor risk' disease (P <0.001). The frequency of individual gene mutation was in accordance with previously published studies. Three novel mutations of FLT3 were detected (Y589D, D839G, Y842H) that would have been overlooked by conventional gel electrophoresis. A 51-bp deletion was detected in CCND3 in a patient with normal karyotype AML. This validated panel now provides an important tool to evaluate other candidate genes in the genesis of myeloid malignancy.

Mutations of CEBPA in acute myeloid leukemia FAB types M1 and M2.

CEBPA encodes the transcription factor C/EBPalpha and is specifically up-regulated during granulocytic differentiation. The gene is mutated in approximately 20% of patients with acute myeloid leukemia (AML) FAB type M2 and occurs in the absence of the t(8;21). In much the same way as specific translocations are associated with a particular AML FAB type, the identification of non-random associations of gene mutation with karyotype or FAB type may be helpful in elucidating the molecular basis of certain forms of leukemia. To confirm these initial findings, 99 patients with AML FAB type M1 or M2 were screened for CEBPA mutations by use of a PCR-single-strand conformational polymorphism and sequencing approach. Nine CEBPA mutations were identified in eight patients. The mutations were clustered toward the COOH terminal of the protein and occurred exclusively in the intermediate cytogenetic risk group (8/64, 12.5%). Two patients with biallelic mutation, one homozygous for 1137Ins (57 bp) and another with two CEBPA mutations, 1096Ins (27 bp) and 363Ins (GGCC), were observed. There was no evidence for deletion of this region in the other six mutated samples analyzed by fluorescence in situ hybridization with a BAC clone spanning the CEBPA locus. CEBPA mutation status was not demonstrated to be of prognostic importance in this patient group, although this may reflect the selection and size of the AML population studied. In conclusion, mutation of CEBPA is a recurrent finding in AML and appears specific to the intermediate cytogenetic risk group patients.