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Urovysion multitarget fluorescence in situ hybridization (FISH) assay is a promising tool for detection of bladder cancer, however, there is still no consensus regarding abnormal signal pattern and cut-off level, and the recommended targeting carries limitations similar to urine cytology. Aim of this study was to explore diagnostic benefits of a recently introduced method featuring target specific genotyping, as well as to investigate the feasibility of locally and statistically determined cut-off, compared with conventional evaluation scheme. Histology, cytology, and comparative FISH approaches were performed on 42 patients with high clinical suspicion for urothelial carcinoma (UC). FISH parallels were (1) Urovysion-alone (according to manufacturer's instruction); (2) Targeted-Urovysion (cytokeratin7 immunophenotyping followed by Urovysion), both of which evaluated by both conventional and statistical evaluation scheme. For statistical evaluation cut-offs and sufficient sample size were determined on controls and ratio of positive cells was recorded, whereas conventional evaluation relied on manufacturer's recommendations. The specificity of cytology, Urovysion-alone in general and targeted-Urovysion in general appeared 86%, 86%, and 100%, respectively. In the same comparison, overall sensitivity was 60%, 80%, and 93%, respectively. In superficial cases sensitivity was 48% for cytology, 72% for Urovysion-alone and 91% for targeted-Urovysion, while no prominent differences were seen in muscle invasive cases. The ratio of FISH positive cells was proportionate with both stage and grade, however, targeted genotyping could separate high grade/high stage cases more effectively. In conclusion, CK7 targeting raises diagnostic efficiency of Urovysion, and could be an ideal tool for identifying tumor cells in ambiguous cases or when other tumors are present. Statistical evaluation produces accuracy comparable with results of conventional evaluation, and with laboratories setting cut-offs individually but according harmonized protocol, it could aid method standardization. Furthermore, by providing additional quantitative information about tumor characteristics, is likely to have therapy relevant value in the future. © 2011 International Society for Advancement of Cytometry

Introduction: Bruton tyrosine kinase inhibitor ibrutinib has altered the therapeutic landscape of chronic lymphocytic leukemia (CLL) with remarkable responses in relapsed or refractory CLL. Despite mostly durable responses, approximately 20% of patients experience disease progression with, in many cases, BTK or phospholipase Cg2 (PLCG2) resistance mutations predating the clinical progression by up to 15 months. Longitudinal studies have shed some light on the temporal aspects of clonal evolution processes leading to ibrutinib resistance, however, the spatial heterogeneity within the various environmental niches has not been described in detail yet. Here, we report the case of a CLL patient in which we are able to document, for the first time, an example of ibrutinib driven spatial convergent evolution leading to disease progression. The patient developed ibrutinib resistance after 21 months of treatment with simultaneous lymphadenomegaly and lymphocytosis. Sanger sequencing revealed a canonical BTK p.C481S mutation in peripherial blood and a PLCG2 p.D993H mutation in the lymph node with the absence of the BTK p.C481S mutation present in the peripherial blood suggesting convergent evolution in terms of the BTK and PLCG2 variants with both commonly associated with ibrutinib resistance. The patient was subsequently treated with venetoclax but due to further progression despite of high-dose salvage chemotherapy and autologous stem cell transplantation the patient died. Methods: Serial samples at ten different timepoints during the disease course were analysed. Genomic DNA was isolated from peripheral blood mononuclear cells and native lymph node tissue. Circulating cell-free DNA (ccfDNA) was isolated from peripheral blood samples. The fraction of tumor cells was determined by flow cytometry. During the Sanger sequencing BTK exons 11, 15, 16, and PLCG2 exon 12, 19, 20, 24, 27 and 30 were examined. Targeted ultra-deep next-generation sequencing (NGS) analysis of the BTK and PLCG2 genes was performed on a MiSeq platform (Illumina). Abundances of the PLCG2 p.P993H and BTK p.C481S variants were quantified using custom assays on a QX200 droplet digital PCR system (ddPCR, BioRad). Results: To dissect this spatial heterogeneity, BTK and PLCG2 mutations were screened by NGS which confirmed the Sanger finding and in addition to the dominant PLCG2 p.D993H variant, identified the BTK p.C481S mutation as a minor clone in the lymph node, with the BTK p.C481S mutation remaining the exclusive for the peripheral blood. Next, we tested the presence of these mutations in the circulating ccfDNA with ddPCR. Notably, both mutations were identified in the ccfDNA with a VAF of 1%. The ddPCR analysis revealed that both variants were absent in the pre-ibrutinib peripheral blood sample, however their emergence predated the clinical progression by 15 months. We observed a gradual expansion of these variants in the sequential samples, demonstrating clonal selection under the selective pressure of ibrutinib. Of note, the ddPCR assay identified the PLCG2 p.D993H mutation as a minor clone (VAF: 0.2%), previously unseen in the peripheral blood by NGS at the time of ibrutinib relapse. Intriguingly, the two subclones displayed differential sensitivity to the venetoclax therapy. In the peripheral blood, we observed a reduction of the BTK p.C481S positive subclone accompanied by expansion of the subclone harbouring PLCG2 p.D993H which was previously dominant in the lymph node. With these dynamic changes in the subclonal architecture, the disease progressed and ultimately led to death of the patient. Conclusions: Clonal evolution is a major driving force of disease progression in CLL. Several studies have traced the effect of treatment on temporal evolutionary trajectories in the context of standard and targeted therapies and identified subclonal heterogeneity and active clonal selection. However, the spatial aspect of subclonal dynamics has not been appreciated until recently. Here, we presented a unique case of CLL, developing ibrutinib resistance via multiple routes simultaneously, with the observed spatial heterogeneity leading to progression and aggressive transformation of the disease. This case highlights the importance of genetic profiling of multiple affected sites as investigations restricted to peripheral blood may underestimate the repertoire of clinically relevant genetic alterations. Disclosures No relevant conflicts of interest to declare.

There is a steady search for procedure which could replace or at least reduce the frequency of the invasive cystoscopy in the surveillance of heterogeneous superficial transitional cell carcinoma (TCC) of the bladder. Recently, UroVysion FISH assay has been shown to provide with better sensitivity than the urine cytology except for the lowest stage pTa and grade I-II TCCs. Data indicate that this failure of the sensitive FISH might be due to mistargeting. Therefore, our aim was to elaborate a procedure enabling FISH analysis in phenotypically preselected urothelial cells, only. Cytokeratin 7 (CK-7) chromogenic immunolabeling was applied to various mixtures of negative and positive control cells as well as voided urine specimens. Cellular targets and CK-7 positive cells were identified by morphometric and pixel intensity indices using an automated microscope workstation. UroVysion FISH pattern was analyzed only in the subsequently relocalized CK-7 positive events. Automated phenotypical preselection of urothelial cells proved to have 97.3% sensitivity, 96.1% specificity, and 99.0% accuracy, whereas combined pheno- and genotyping revealed 93.3% sensitivity and 99.8% specificity, respectively. In clinical samples, the overall 20.4% FISH positivity gained by traditional target identification contrasted with the 55.6% positivity obtained by the combined method, by which the efficiency of identifying chromosomally aberrant cells proved to be two to threefold higher even in grade I lesions. FISH analysis of phenotypically preselected urothelial cells might represent a reliable asset in surveillance of low stage–low grade TCCs. © 2008 International Society for Analytical Cytology

Chronic lymphocytic leukemia (CLL) is the most frequent mature B-cell non-Hodgkin's lymphoma in the Western countries. The recent next-generation sequencing (NGS) studies lead to an exponential increase in our knowledge of the pathogenesis and progression of CLL. Whole genome and exome sequencing studies revealed a remarkable inter- and intra-patient genetic heterogeneity with a significant therapy-induced clonal evolution in the majority of the patients. Driver mutations were identified in components of various signalling pathways and cellular processes with notable prognostic and therapeutic relevance. Interestingly, these studies revealed only a few genes mutated in at least 15-20% of the patients with a larger number of genes mutated in a smaller proportion of patients. This improved understanding of the genomic landscape of CLL has opened new avenues for a more precise patient stratification and rational application of novel, more effective targeted therapies.A krónikus limfocitás leukémia (CLL) a fejlett országokban a leggyakoribb érett B-sejtes non-Hodgkin-limfóma. Az újgenerációs szekvenálás megjelenésével az elmúlt években exponenciálisan növekedtek ismereteink a betegség kialakulásáról és progressziójáról. A teljes genom és exom vizsgálata során nagyfokú betegek közötti genetikai heterogenitás igazolódott. A legkülönbözõbb jelátviteli utakhoz tartozó CLL driver géneket azonosítottak, melyeknek prognosztikai és terápiás szerepük lehet. Csupán néhány gént írtak le, melyek az esetek legalább 15-20%-ában hordoztak mutációt, viszont számos alacsony frekvenciával mutálódott gént és nagyfokú betegen belüli genetikai heterogenitást figyeltek meg. A terápiaindukált klonális evolúció jelenségét is kimutatták a betegség lefolyása során, ami szerepet játszhat a kemorezisztencia kialakulásában. A CLL genetikai alapjainak alaposabb megismerésével lehetõség nyílik a betegek pontosabb rizikóbecslésére és az új terápiás lehetõségek hatékonyabb alkalmazására.

Owing to our rapidly expanding knowledge on the genetic background of various oncohematologic diseases and the introduction of novel targeted therapies, molecular genetic techniques have been playing an increasingly important role in the diagnostics and follow-up of hematological malignancies. The various DNA- and RNA-based in situ hybridization, polymerase chain reaction and sequencing technologies are of key significance in diagnostics, classification and prognostic assessment of these diseases, as well as in the monitoring of minimal residual disease and selection of the most appropriate targeted therapy. This review provides an overview on the background and applications of the molecular methods most commonly used in oncohematological diagnostics.A különbözõ onkohematológiai megbetegedések genetikai hátterérõl való ismereteink bõvülésének és az új célzott terápiák megjelenésének köszönhetõen a molekuláris genetikai eljárások mára egyre fontosabb szerepet töltenek be az onkohematológiai betegségek diagnosztikájában és követésében. A DNS- és RNS-vizsgálatokon alapuló, in situ hibridizációs, polimeráz láncreakciós és szekvenálási eljárások a különbözõ entitások diagnosztikájában, klasszifikációjában és prognosztikai besorolásában, valamint a minimális reziduális betegség követésében és a megfelelõ célzott terápia kiválasztásában is kiemelt szereppel bírnak. A jelen összefoglaló az onkohematológiai diagnosztikában alkalmazott molekuláris módszerek alapjait és alkalmazási területeit tekinti át.

Advances in genetic analysis technologies revealed a previously underestimated level of intratumor heterogeneity (ITH) across many different cancer types. Genetic ITH, increasingly found to arise through branched evolution of multiple cancer subclones, may confer spatially heterogeneous tumor structures and functionally distinct cell populations. Hence, ITH presents major challenges to personalized medicine and biomarker development due to sampling biases and the simultaneous presence of cancer subclones differing in their clinical aggressiveness and drug sensitivities. Ongoing evolution continues to re-shape subclonal genomic structures of cancers over time, further complicating precision medicine genetic profiling. This review provides insights into mechanisms underlying ITH generation through new mutations, the evolutionary principles governing clonal selection and cancer evolution and the resulting heterogeneous genomic landscapes observed in cancers to date. The clinical implications of ITH and strategies to improve treatment efficacy in heterogeneous cancers will be discussed, highlighting the potential role of epigenetic therapeutic interventions.

GEAs are aggressive tumors in which several targeted therapy trials have failed. We assessed intratumor heterogeneity (ITH) and its impact on progression and therapy failure by applying an 81-gene NGS panel and SNP array copy number aberration (CNA) analysis to multiple primary tumor (T) regions and lymph node (LN) metastases from 9 GEAs. Analysis of 39 samples found ITH in all cases. 8 chromosomally instable (CIN) GEAs predominantly evolved through CNAs, with 17-76% of the genome affected by heterogeneous CNAs. A microsatellite instable GEA showed parallel evolution and diversified exclusively through point mutations (58% ITH). This demonstrates ongoing genomic instability rather than punctuated evolution and that specific instability mechanisms impact evolutionary trajectories. LN metastases contributed more to ITH (p To assess the phenotypes established by MAPK-activating amp evolution, we analyzed 135 published primary CIN subtype GEAs. Cytolytic activity (CYT), estimating tumor immune recognition from RNA expression data, correlated with the mutation load in GEAs with EGFR, ERBB2 or MET amp (p=0.04). In contrast, CYT did not correlate with mutation load in GEAs with KRAS or ERBB3 amp (p=0.22, NRAS/ERK2: insufficient data), indicating that these specific alterations, that also recurrently evolved in LN, may enable immune evasion. Downregulation of TAP and Class I MHC genes (p Moreover, ITH of MAPK-activating amp is likely to confer resistance to upstream tyrosine kinase inhibition. We used GEA cell lines with various MAPK-activating amp (ERBB2, MET, NRAS) to investigate downstream MAPK-pw inhibition as a novel strategy to broadly target heterogeneous subclones. Growth control was incomplete with ERK- and MEK-inhibitors but the panRAF/SRC inhibitor CCT196969 was effective in all lines, suggesting that it can effectively intercept subclonal heterogeneity in GEAs. In conclusion, we identified ITH with parallel and convergent evolution in 9/9 metastatic GEAs. Distinct selection pressures in LN foster the evolution of subclonal MAPK-activating amp that decrease immunogenicity and drive evolutionary pre-adaptation to future targeted drugs that can be intercepted by panRAF/SRC inhibitors. Citation Format: Matthew N. Davies, Louise J. Barber, Georgia Spain, Filipa Lopes, Katharina von Loga, Beatrice Griffiths, Andrew Woolston, Donat Alpar, Marta Gomez, Kamil A. Lipinski, Kerry Fenwick, Zakaria Eltahir, Stefano Lise, Emese I. Agoston, Laszlo Harsanyi, Richard Marais, Andrew Wotherspoon, A Szasz, Caroline Springer, Marco Gerlinger. Lymph node metastasis evolution drives immune evasion and targeted therapy resistance in gastro-esophageal adenocarcinomas (GEAs) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 422. doi:10.1158/1538-7445.AM2017-422

Bone marrow specimens from 185 patients with plasma cell disorders (PCD) were investigated by fluorescence in situ hybridization (FISH) in order to determine the temporal sequence of cytogenetic aberrations. In 25 cases combined FISH analysis has also been performed at single cell level. Clonal evolution was observed in 16% of cases. The Δ13 was preceded by t(4;14)(p16;q32) and t(14;16)(q32;q23) translocations. Deletion of p53 gene was a secondary aberration compared to Δ13 and t(11;14)(q13;q32) translocation. In 22% of all cases with recurrent IGH translocation, this aberration was presented only in a subset of purified plasma cells questioning its initiating role.

Identical twins have provided unique insights on timing or sequence of genetic events in acute lymphoblastic leukaemia (ALL). To date, this has mainly focused on ALL with MLL or ETV6-RUNX1 fusions, with hyperdiploid ALL remaining less well characterised. We examined three pairs of monozygotic twins, two concordant and one discordant for hyperdiploid ALL, for single-nucleotide polymorphism (SNP)-defined copy number alterations (CNAs), IGH/L plus TCR gene rearrangements and mutations in NRAS, KRAS, FLT3 and PTPN11 genes. We performed whole exome sequencing in one concordant twin pair. Potential 'driver' CNAs were low, 0-3 per case, and all were different within a pair. One patient had an NRAS mutation that was lacking from leukaemic cells of the twin sibling. By exome sequencing, there were 12 nonsynonymous mutations found in one twin and 5 in the other, one of which in SCL44A2 was shared or identical. Concordant pairs had some identical IGH/L and TCR rearrangements. In the twin pair with discordant hyperdiploid ALL, the healthy co-twin had persistent low level hyperdiploid CD19+ cells that lacked a CNA present in the ALL cells of her sibling. From these data, we propose that hyperdiploid ALL arises in a pre-B cell in utero and mutational changes necessary for clinical ALL accumulate subclonally and postnatally.

DNA-, RNA-, and cell-based methods provide different biologic information for determining the presence of minimal residual disease (MRD). We monitored the responses of patients with pediatric acute lymphoblastic leukemia (pALL) using DNA markers, TEL/AML1 expression, and scanning fluorescence microscopy (SFM). Using SFM, 36% of patients exhibited 1.5–3.1 log and 2.9–4.2 log higher MRD levels compared with those based on DNA and RNA markers, respectively. CD10+ ancestor cells with germline antigen receptors, but silent TEL/AML1 expression, may reside in the lymphoid stem cell compartment of treated t(12;21)-positive patients and might act as a potential source of cells for late relapses. Pediatr Blood Cancer 2010; 54:158–160. © 2009 Wiley-Liss, Inc.

Pathogenesis of the non-random accumulation of extra chromosomes in the low and high hyperdiploid (HeL, HeH) pre-B pediatric acute lymphoblastic leukemia (B-pALL) is largely unknown, and has been clarified with respect only to tetrasomic chromosomes. We analyzed the hierarchy of changes in chromosome number and chromosomal instability, as well as clonal heterogeneity and evolution, in the untreated bone marrow cell samples from 214 B-pALL patients.Applying relocation, 2 × 4 color interphase fluorescence in situ hybridization was used to detect copy number alterations (CNAs) of the most commonly involved chromosomes, 4, 6, 10, 14, 17, 18, 21, and X. This approach allowed us to acquire a dataset correlated for all eight parameters.Based on chromosome number, an average of 6.9 and 10.2, whereas according to unique constellation 15.3 and 26.7 subclones could be identified in the HeL and HeH subgroups, respectively. Cluster analysis revealed the order of CNAs to chromosomes was highly conserved, and network analysis indicated changes in chromosome number were sequential for 80-90% of all numerical aberrations. Significant chromosome instability was revealed in both subgroups of leukemia.Data generated using this new approach indicate that chromosomal instability, which causes heterogeneity in the subclonal landscape, and the sequential changes to chromosome numbers, are both determining factors in the pathomechanism of the hyperdiploid B-pALL. These new observations could prompt research into the mitotic machinery of leukemic cells to identify new therapeutic targets for treating this disease.

Copy number alterations (CNAs) at 58 different loci have been investigated in 95 bone marrow or peripheral blood samples from patients with chronic myeloid leukemia (CML) or pediatric acute lymphoblastic leukemia (pALL) using multiplex ligation-dependent probe amplification (MLPA). In all but one case, the CNA profile correctly distinguished patients with CML who were in chronic phase from those in lymphoblast crisis. Within the chronic phase group, we could not separate patients resistant to imatinib therapy from those who were good responders. In our investigation of patients with pALL, a panel of MLPA probes broader than ever before was applied. Paired diagnostic and relapse samples from patients with pALL demonstrated clonally related or independent dominant clones, suggesting the presence of a pre-leukemic cell group. Identification of the origin of cell populations dominating at relapse will have a great effect on future treatment strategies. In summary, we have demonstrated the versatility of MLPA by using this cost-effective technique for two new applications.

Cytogenetic aberrations are very important factors in risk assessment of childhood hematological malignancies. We report six childhood acute lymphoid leukemia (ALL) cases with rare cytogenetic aberrations: five with RUNX1, ABL1 or MLL proto-oncogene amplification and one case of multiple copies of ETV6/RUNX1 fusion genes. The simultaneous presence of two adverse genetic aberrations is of special interest: ETV6-RUNX1 fusion gene is associated with good prognosis and intrachromosomal amplification of the homologue RUNX1 gene is associated with poor prognosis. We also report a patient with MLL amplification, a unique finding in childhood T-ALL. Report of these subtle rearrangements contributes to our understanding of diagnostic and prognostic significance of these rare cytogenetic abnormalities.

Allogeneic bone marrow transplantation (allo-BMT) is the treatment of choice for many patients with poor prognosis or refractory leukemia. Chimerism and residual tumor load after allo-BMT are widely monitored to detect impending graft rejection and the early phase of relapse. In most cases, the malignant cell population during post-transplant relapse contains host-derived cells, but the leukemic clone can rarely be of donor-cell origin. Various genetic tests with different strategies, targets and sensitivities are available for donor-host discrimination. However, changes in the genomic material of the dominant host cell population as a result of clonal evolution and/or clonal selection can hamper the correct identification of the origin of aberrant cells after allo-BMT, thus confounding the assessment of the chimeric state. Consequently, a good knowledge of the techniques applied in clinical practice and careful interpretation of their results are essential. A lack of host-specific markers at the time of clinical relapse is not adequate for verifying the presence of donor cell leukemia, and unequivocal demonstration of donor-specific markers is also required.

A 12-year-old male with pre-B-cell acute lymphoblastic leukemia with cryptic BCR/ABL rearrangement underwent sex-mismatched allogeneic bone marrow transplantation (allo-BMT). Contradictory results were provided by various chimerism analyses 3 months later. Y-chromosome-specific quantitative polymerase chain reaction and sex chromosome-specific interphase fluorescence in situ hybridization (i-FISH) showed complete donor chimerism. Analysis of autosomal short tandem repeats (A-STR), BCR/ABL i-FISH test, and X-STR haplotype indicated relapse. Metaphase-FISH and combined BCR/ABL and sex chromosome-specific i-FISH patterns revealed loss of the Y-chromosome and duplication of the X-chromosome in the host cells. Sex chromosome changes after allo-BMT can cause significant difficulties in chimerism analysis. Pediatr Blood Cancer. 2010;55:1239–1242. © 2010 Wiley-Liss, Inc.

Detecting balanced translocations using tissue sections plays an important diagnostic role in cases of hematological malignancies. Manual scoring is often problematic due to truncation and overlapping of nuclei. Reports have described automated analysis using primarily tile sampling. The aim of this study was to investigate an automated fluorescent in situ hybridization analysis method using grid sampling on tissue sections, and compare the performance of dual-fusion (DF) and break-apart (BA) probes in this setting. Ten follicular, 10 mantle cell lymphoma, and 10 translocation-negative samples were used to set the threshold of false positivity using IGH/CCND1, IGH/BCL-2 DF, and IGH BA probes. The cut-off distances of red and green signals to define fusion signals were 0.5, 1.0, and 1.2 mum for the IGH/CCND1, IGH/BCL-2 DF, and IGH BA probes, respectively. The mean false positivity of grid units was 5.3, 11.4, and 28.1%, respectively. Ten to 14 additional samples analyzed blindly and were correctly classified using each probe. Discriminating positive and negative samples using automated analysis and grid sampling was possible with each probe, although different definitions of fusion signals were required due to the different physical distances between the DNA probes. Using the DF probes resulted in lower false positivity, which was less affected by signal numbers per grid units.

Background: For chronic myeloid leukemia, the FISH detection of t(9;22)(q34;q11) in interphase nuclei of peripheral leukocytes is an alternative method to bone marrow karyotyping for monitoring treatment. With automation, several drawbacks of manual analysis may be circumvented. In this article, the capabilities of a commercially available automated image acquisition and analysis system were determined by detecting t(9;22)(q34;q11) in interphase nuclei of peripheral leukocytes. Methods: Three peripheral blood samples of normal adults, 21 samples of CML patients, and one sample of a t(9;22)(q34;q11) positive cell-line were used. Results: Single nuclei with correctly detected signals amounted to 99.6% of nuclei analyzed after exclusion of overlapping nuclei and nuclei with incorrect signal detection. A cut-off value of 0.84 μm was defined to discriminate between translocation positive and negative nuclei based on the shortest distance between signals. Using this value, the false positive rate of the automated analysis for negative samples was 7.0%, whereas that of the manual analysis was 5.8%. Automated and manual results showed strong correlation (R2 = 0.985), the mean difference of results was only 3.7%. Conclusions: A reliable and objective automated analysis of large numbers of cells is possible, avoiding interobserver variability and producing statistically more accurate results than manual evaluation. © 2006 International Society for Analytical Cytology

78 Background: DNA copy number aberrations (CNAs) are common in oesophageal and gastric adenocarcinomas (OGCs) and display extensive inter-tumour heterogeneity. CNA patterns define gastric cancer molecular subtypes and ERBB2 amplifications, present in a small fraction of patients with OGC, are predictive for ERBB2-targeted drug sensitivity. Together, this suggests a critical role of CNAs determining OGC tumour biology and clinical outcomes. Despite this, predictive and prognostic CNA biomarkers have not been identified for the majority of OGCs, precluding the development of effective personalized therapy approaches for these aggressive tumours. Intra-tumour heterogeneity, characterized by the presence of multiple subclones with distinct genetic profiles within an individual cancer, can hinder the accurate molecular analysis and classification of tumours. The aim of this pilot study was to assess whether chemotherapy-naïve localized OGCs display intra-tumour macroheterogeneity of CNA profiles. Methods: Tissue specimens from four tumour regions representing the macroscopic spatial extent of each of five OGCs were systematically collected after surgical resection. DNA extracted from these FFPE specimens was analysed by molecular inversion probe SNP arrays for high resolution CNA detection. Results: Comparison of genome wide copy number and B-allele frequency profiles suggested highly concordant CNA profiles across the regions from individual primary tumours. Eight driver CNAs leading to amplification of the MET, KRAS, ERBB2, PIK3CA or FGFR2 oncogenes were identified in 4/5 tumours. Only one out of these eight driver CNA’s, harbouring the KRAS oncogene, was heterogeneous within a tumour. Conclusions: Although chromosomal instability is thought to be common in this tumour type, this pilot study suggests that macroheterogeneity is limited and that CNA profiles assessed from a single tumour biopsy are likely to be representative of the dominant CNA profile of localized OGCs. Thus, clinical correlative CNA analyses may be possible from single biopsies of localized OGCs. Mutational heterogeneity and microheterogeneity in microdissected and single cells are currently being investigated.