Centromeric cohesion failure invokes a conserved choreography of chromosomal mis-segregations in pancreatic neuroendocrine tumor.

BACKGROUND: Pancreatic neuroendocrine tumors (PANETs) are rare, slow growing cancers that often present with local and distant metastasis upon detection. PANETS contain distinct karyotypes, epigenetic dysregulation, and recurrent mutations in MEN1, ATRX, and DAXX (MAD+); however, the molecular basis of disease progression remains uncharacterized. METHODS: We evaluated associations between aneuploidy and the MAD+ mutational state of 532 PANETs from 11 published genomic studies and 19 new cases using a combination of exome, targeted panel, shallow WGS, or RNA-seq. We mapped the molecular timing of MAD+ PANET progression using cellular fractions corrected for inferred tumor content. RESULTS: In 287 PANETs with mutational data, MAD+ tumors always exhibited a highly recurrent signature of loss of heterozygosity (LOH) and copy-number alterations affecting 11 chromosomes, typically followed by genome doubling upon metastasis. These LOH chromosomes substantially overlap with those that undergo non-random mis-segregation due to ectopic CENP-A localization to flanking centromeric regions in DAXX-depleted cell lines. Using expression data from 122 PANETs, we found decreased gene expression in the regions immediately adjacent to the centromere in MAD+ PANETs. Using 43 PANETs from AACR GENIE, we inferred this signature to be preceded by mutations in MEN1, ATRX, and DAXX. We conducted a meta-analysis on 226 PANETs from 8 CGH studies to show an association of this signature with metastatic incidence. Our study shows that MAD+ tumors are a genetically diverse and aggressive subtype of PANETs that display extensive chromosomal loss after MAD+ mutation, which is followed by genome doubling. CONCLUSIONS: We propose an evolutionary model for a subset of aggressive PANETs that is initiated by mutation of MEN1, ATRX, and DAXX, resulting in defects in centromere cohesion from ectopic CENP-A deposition that leads to selective loss of chromosomes and the LOH phenotype seen in late-stage metastatic PANETs. These insights aid in disease risk stratification and nominate potential therapeutic vulnerabilities to treat this disease.

Genomic profiling identifies GPC5 amplification in association with sarcomatous transformation in a subset of uterine carcinosarcomas.

Uterine carcinosarcoma, also known as Malignant Mixed Müllerian Tumour, is a high-grade biphasic neoplasm composed of sarcomatous elements thought to originate via transdifferentiation from high-grade endometrial carcinoma. To identify molecular factors contributing to the histogenesis of this tumour, we analyzed DNA extracted from matched carcinoma and sarcoma components from 12 cases of carcinosarcoma by a molecular inversion probe microarray to assess genomic copy number alterations (CNAs) and allelic imbalances. Widespread CNAs were identified in tumours with serous histology in the carcinoma component (9/12), while the remaining three cases with endometrioid carcinoma were near-diploid. Quantification of the extent of genomic aberrations revealed a significant increase in sarcoma relative to carcinoma in tumours with well-delineated histologic components. Focal amplification of 13q31.3 was identified in 6/12 profiled tumours, of which four harboured the aberration exclusively in the sarcoma component. This result was verified by fluorescence in situ hybridization against GPC5, the only gene situated within the minimal region of amplification. In a validation cohort composed of 97 carcinosarcomas and other uterine sarcomas, amplification of GPC5 (GPC5/CEP13 ratio ≥ 2.2) was identified in 11/97 (11.3%) cases (9/64 carcinosarcoma, 1/3 rhabdomyosarcoma, 1/21 leiomyosarcoma, 0/8 adenosarcoma, 0/1 undifferentiated endometrial sarcoma) and an additional 4 (2.8%) cases had low level gains (GPC5/CEP13 ratio ≥1.5 but <2.2). The functional relevance of Glypican-5, the gene product of GPC5, in regulating differentiation and lineage commitment was demonstrated in an endometrial carcinoma cell line in vitro. In conclusion, we identified GPC5 amplification as a molecular event mediating epithelial-mesenchymal transdifferentiation in a subset of uterine carcinosarcomas.

Analysis of papillary urothelial carcinomas of the bladder with grade heterogeneity: supportive evidence for an early role of CDKN2A deletions in the FGFR3 pathway.

AIMS: The dual pathway model of urothelial carcinogenesis does not fully explain grade and stage progression in patients with initial low-grade, non-muscle invasive urothelial carcinomas. Fibroblast growth factor receptor 3 (FGFR3) mutations are a hallmark of the low-grade pathway, with subsequent progression to muscle invasion occurring when FGFR3 mutant tumours exhibit a homozygous CDKN2A deletion. We hypothesized that grade heterogeneity represents the morphological manifestation of molecular changes associated with disease progression. METHODS AND RESULTS: We identified retrospectively 29 non-muscle invasive papillary urothelial carcinomas with grade heterogeneity (<20% high grade). Nineteen had sufficient material for immunohistochemistry, CDKN2A fluorescence in-situ hybridization and FGFR3 mutation analysis. Eight pure low-grade urothelial carcinomas (PLGUC) were also analysed. FGFR3 mutation was seen in 10 of 19 cases. A homozygous CDKN2A deletion was identified in the low-grade areas of eight of nine (88%) technically suitable FGFR3 mutant cases (including five pTa cancers), in five of nine FGFR3 wild-type carcinomas and in none of the PLGUC. Increased MIB-1 expression was seen in low-grade areas of 12 of 19, in high-grade areas of 17 of 19 cases with grade heterogeneity and in none of the PLGUC. p53 staining was increased in one of 19 low-grade and seven of 19 high-grade areas. CONCLUSION: Our findings show that grade heterogeneity in urothelial carcinoma is characterized by increased MIB-1 labelling, and particularly in the FGFR3 mutant pathway, with homozygous deletions of CDKN2A in low- and high-grade areas. This would suggest that CDKN2A deletion occurs prior to grade progression and supports the current convention to assign the highest grade to urothelial carcinomas with grade heterogeneity.

Spatial genomic heterogeneity within localized, multifocal prostate cancer.

Herein we provide a detailed molecular analysis of the spatial heterogeneity of clinically localized, multifocal prostate cancer to delineate new oncogenes or tumor suppressors. We initially determined the copy number aberration (CNA) profiles of 74 patients with index tumors of Gleason score 7. Of these, 5 patients were subjected to whole-genome sequencing using DNA quantities achievable in diagnostic biopsies, with detailed spatial sampling of 23 distinct tumor regions to assess intraprostatic heterogeneity in focal genomics. Multifocal tumors are highly heterogeneous for single-nucleotide variants (SNVs), CNAs and genomic rearrangements. We identified and validated a new recurrent amplification of MYCL, which is associated with TP53 deletion and unique profiles of DNA damage and transcriptional dysregulation. Moreover, we demonstrate divergent tumor evolution in multifocal cancer and, in some cases, tumors of independent clonal origin. These data represent the first systematic relation of intraprostatic genomic heterogeneity to predicted clinical outcome and inform the development of novel biomarkers that reflect individual prognosis.

Tumour genomic and microenvironmental heterogeneity for integrated prediction of 5-year biochemical recurrence of prostate cancer: a retrospective cohort study.

BACKGROUND: Clinical prognostic groupings for localised prostate cancers are imprecise, with 30-50% of patients recurring after image-guided radiotherapy or radical prostatectomy. We aimed to test combined genomic and microenvironmental indices in prostate cancer to improve risk stratification and complement clinical prognostic factors. METHODS: We used DNA-based indices alone or in combination with intra-prostatic hypoxia measurements to develop four prognostic indices in 126 low-risk to intermediate-risk patients (Toronto cohort) who will receive image-guided radiotherapy. We validated these indices in two independent cohorts of 154 (Memorial Sloan Kettering Cancer Center cohort [MSKCC] cohort) and 117 (Cambridge cohort) radical prostatectomy specimens from low-risk to high-risk patients. We applied unsupervised and supervised machine learning techniques to the copy-number profiles of 126 pre-image-guided radiotherapy diagnostic biopsies to develop prognostic signatures. Our primary endpoint was the development of a set of prognostic measures capable of stratifying patients for risk of biochemical relapse 5 years after primary treatment. FINDINGS: Biochemical relapse was associated with indices of tumour hypoxia, genomic instability, and genomic subtypes based on multivariate analyses. We identified four genomic subtypes for prostate cancer, which had different 5-year biochemical relapse-free survival. Genomic instability is prognostic for relapse in both image-guided radiotherapy (multivariate analysis hazard ratio [HR] 4·5 [95% CI 2·1-9·8]; p=0·00013; area under the receiver operator curve [AUC] 0·70 [95% CI 0·65-0·76]) and radical prostatectomy (4·0 [1·6-9·7]; p=0·0024; AUC 0·57 [0·52-0·61]) patients with prostate cancer, and its effect is magnified by intratumoral hypoxia (3·8 [1·2-12]; p=0·019; AUC 0·67 [0·61-0·73]). A novel 100-loci DNA signature accurately classified treatment outcome in the MSKCC low-risk to intermediate-risk cohort (multivariate analysis HR 6·1 [95% CI 2·0-19]; p=0·0015; AUC 0·74 [95% CI 0·65-0·83]). In the independent MSKCC and Cambridge cohorts, this signature identified low-risk to high-risk patients who were most likely to fail treatment within 18 months (combined cohorts multivariate analysis HR 2·9 [95% CI 1·4-6·0]; p=0·0039; AUC 0·68 [95% CI 0·63-0·73]), and was better at predicting biochemical relapse than 23 previously published RNA signatures. INTERPRETATION: This is the first study of cancer outcome to integrate DNA-based and microenvironment-based failure indices to predict patient outcome. Patients exhibiting these aggressive features after biopsy should be entered into treatment intensification trials. FUNDING: Movember Foundation, Prostate Cancer Canada, Ontario Institute for Cancer Research, Canadian Institute for Health Research, NIHR Cambridge Biomedical Research Centre, The University of Cambridge, Cancer Research UK, Cambridge Cancer Charity, Prostate Cancer UK, Hutchison Whampoa Limited, Terry Fox Research Institute, Princess Margaret Cancer Centre Foundation, PMH-Radiation Medicine Program Academic Enrichment Fund, Motorcycle Ride for Dad (Durham), Canadian Cancer Society.

4FISH-IF, a four-color dual-gene FISH combined with p63 immunofluorescence to evaluate NKX3.1 and MYC status in prostate cancer.

NKX3.1 allelic loss and MYC amplification are common events during prostate cancer progression and have been recognized as potential prognostic factors in prostate cancer after radical prostatectomy or precision radiotherapy. We have developed a 4FISH-IF assay (a dual-gene fluorescence in situ hybridization combined with immunofluorescence) to measure both NKX3.1 and MYC status on the same slide. The 4FISH-IF assay contains four probes complementary to chromosome 8 centromere, 8p telomere, 8p21, and 8q24, as well as an antibody targeting the basal cell marker p63 visualized by immunofluorescence. The major advantages of the 4FISH-IF include the distinction between benign and malignant glands directly on the 4FISH-IF slide and the control of truncation artifact. Importantly, this specialized and innovative combined multiprobe and immunofluorescence technique can be performed on diagnostic biopsy specimens, increasing its clinical relevance. Moreover, the assay can be easily performed in a standard clinical molecular pathology laboratory. Globally, the use of 4FISH-IF decreases analytic time, increases confidence in obtained results, and maintains the tissue morphology of the diagnostic specimen.

Immunohistochemistry is a reliable screening tool for identification of ALK rearrangement in non-small-cell lung carcinoma and is antibody dependent.

INTRODUCTION: Fluorescence in situ hybridization (FISH) is the standard procedure for the detection of anaplastic lymphoma receptor tyrosine kinase (ALK) rearrangement in non-small-cell lung carcinoma (NSCLC) but is expensive and time consuming. We tested three antibodies to ALK, using various detection systems, and hypothesized that ALK immunohistochemistry (IHC) may represent a cost-effective and efficient means of screening for ALK rearrangement in NSCLC. METHODS: We screened 377 stage I or II NSCLC cases in a tissue microarray by FISH and IHC (5A4 [Leica Biosystems Newcastle Ltd, Newcastle upon Tyne, UYnited Kingdom] by Nichirei's N-Histofine ALK detection kit [Nichirei Biosciences inc., Tokyo, Japan], 5A4 by Novocastra with ADVANCE [Dako Canada inc., Burlington, Ontario, Canada], D5F3 by Cell Signaling Technology with ADVANCE [Cell Signalling Technologies inc., Danvers, MA], and DAKO clone ALK1 with FLEX [Dako Canada inc., Burlington, Ontario, Canada] and ADVANCE). IHC was scored as 0, 1+, 2+, or 3+. Possibly positive or positive cases were further analyzed by IHC and FISH on whole section. RESULTS: Tissue microarray results were available on 377 cases by IHC and 273 cases by FISH. Eleven cases were positive or possibly positive by either IHC or FISH, and three cases were positive or possibly positive by both methods. Three cases were ALK-positive by FISH on whole section validation. There was no correlation between semiquantitative IHC score (1+, 2+, 3+) and ALK rearrangement by FISH. D5F3 (Cell Signaling by ADVANCE) and 5A4 (Novocastra by ADVANCE) showed the greatest combination of sensitivity (100%) and specificity (87.5% for 5A4 by Novocastra and 75% for D5F3 by Cell Signaling), and produced no false-negative results. CONCLUSIONS: IHC is a reliable screening tool for identification of ALK rearrangement in NSCLC and is antibody dependent. D5F3 (Cell Signaling) and 5A4 (Novocastra) can be used with FISH for identification of IHC-positive cases to reduce screening costs.

NKX3.1 haploinsufficiency is prognostic for prostate cancer relapse following surgery or image-guided radiotherapy.

BACKGROUND: Despite the use of prostate specific antigen (PSA), Gleason-score, and T-category as prognostic factors, up to 40% of patients with intermediate-risk prostate cancer will fail radical prostatectomy or precision image-guided radiotherapy (IGRT). Additional genetic prognosticators are needed to triage these patients toward intensified combination therapy with novel targeted therapeutics. We tested the role of the NKX3.1 gene as a determinant of treatment outcome given its reported roles in tumor initiating cell (TIC) renewal, the DNA damage response, and cooperation with c-MYC during prostate cancer progression. METHODS: Using high-resolution array comparative genomic hybridization (aCGH), we profiled the copy number alterations in TIC genes using tumor DNA from frozen needle biopsies derived from 126 intermediate-risk patients who underwent IGRT. These data were correlated to biochemical relapse-free rate (bRFR) by the Kaplan-Meier method and Cox proportional hazards models. RESULTS: A screen of the aCGH-IGRT data for TIC genes showed frequent copy number alterations for NKX3.1, PSCA, and c-MYC. NKX3.1 haploinsufficiency was associated with increased genomic instability independent of PSA, T-category, and Gleason-score. After adjusting for clinical factors in a multivariate model, NKX3.1 haploinsufficiency was associated with bRFR when tested alone (HR = 3.05, 95% CI: 1.46-6.39, P = 0.0030) or when combined with c-MYC gain (HR = 3.88, 95% CI: 1.78-8.49, P = 0.00067). A similar association was observed for patients following radical prostatectomy with a public aCGH database. NKX3.1 status was associated with positive biopsies post-IGRT and increased clonogen radioresistance in vitro. CONCLUSIONS: Our results support the use of genomic predictors, such as NKX3.1 status, in needle biopsies for personalized approaches to prostate cancer management.

EWSR1-ATF1 fusion is a novel and consistent finding in hyalinizing clear-cell carcinoma of salivary gland.

Hyalinizing clear-cell carcinoma (HCCC) is a rare, low-grade salivary gland tumor with distinctive clear-cell morphology and pattern of hyalinization as well as focal mucinous differentiation. However, histological overlap exists with other salivary gland tumors, such as epithelial-myoepithelial carcinoma (EMCa), salivary myoepithelial carcinoma, and mucoepidermoid carcinoma (MEC). The potential relationship between HCCC and its morphological mimics has not been yet investigated at the genetic level. In this study, we conducted a molecular analysis for the presence of rearrangements in MAML2, commonly seen in MECs, and EWSR1, involved in "soft tissue myoepithelial tumors" (SMET) by fusion with POU5F1, PBX1, or ZNF444. Fluorescence in situ hybridization (FISH) was performed on 23 HCCC cases for abnormalities in MAML2, EWSR1, FUS, POU5F1, PBX1, and ZNF444. FISH for MAML2 was negative in all cases (0 of 14), including those with mucinous differentiation (0 of 7). An EWSR1 rearrangement was identified in 18 of 22 HCCCs (82%), while no break-apart signals were seen in FUS, POU5F1, PBX1, or ZNF444. 3'RACE on an EWSR1 rearranged HCCC identified an EWSR1-ATF1 fusion, which was confirmed by RT-PCR. ATF1 involvement was further confirmed by FISH analysis in 13 of 14 EWSR1-rearranged HCCC cases (93%). In contrast, all control cases tested, including among others 5 EMCa and 3 MEC with clear cells, were negative for EWSR1 and ATF1 rearrangements. The presence of EWSR1-ATF1 fusion in most HCCCs reliably separates these tumors from its histological mimics. The distinction from MEC is particularly important, as conventional MEC grading schemes overgrade these indolent HCCCs, potentially impacting on treatment.

Targeted use of fluorescence in situ hybridization (FISH) in cytospin preparations: results of 298 fine needle aspirates of B-cell non-Hodgkin lymphoma.

BACKGROUND: Fluorescence in situ hybridization (FISH) results from fine needle aspirates (FNA) of B-cell non-Hodgkin lymphomas (NHLs) were reviewed to 1) investigate the value added by using specific gene rearrangement probes to lymphoma diagnosis, prognosis, and subtyping; and 2) evaluate the prevalence of cytogenetic alterations other than specific translocations. METHODS: FISH results from assays performed on cytospin preparations from NHL FNAs over a 6-year period (2003-2009) were selected. Immunophenotyping, clinical data, and cytomorphologic data were reviewed according to the current World Health Organization (WHO) classification system. Hybridized probes, the purpose for the assay (subtyping or prognosis), and the cytogenetic abnormalities observed were retrieved from cytology reports. Data was categorized according to specific rearrangements and other chromosomal abnormalities. RESULTS: Successful results were obtained in 284 (95.3%) of 298 cases from 282 patients. Abnormalities were found in 216 (76%) cases and 68 (24%) did not show alteration. Among cases submitted for subtyping, 198 showed FISH-positive results, and specific gene rearrangements were found in 122 (61.6%) cases as follows: follicular 82, mantle cell 21, marginal zone 3, "dual hit" 13, and Burkitt lymphoma 3. In 21 cases, abnormalities were useful for prognosis. Nonspecific alterations alone or in combination with translocations were found in 98 cases. CONCLUSIONS: FISH performed on cytospin preparations was useful for confirmation of specific subclasses of NHL and may also provide valuable prognostic information. Cytogenetic abnormalities other than specific translocations were frequently found and could provide supportive evidence for a definitive diagnosis of lymphoma in FNA.

HER2 testing in a population-based study of patients with metastatic breast cancer treated with trastuzumab.

CONTEXT: The development of trastuzumab has led to new therapeutic strategies for patients with breast cancer. Trastuzumab has been shown to only be effective in those patients whose cancers are HER2 positive by either immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH). OBJECTIVE: To determine the accuracy of HER2 testing in 2 provinces in Canada. DESIGN: Patients with metastatic breast cancer diagnosed between 1999 and 2002 who were HER2 positive in the local laboratories were included in this study. All cases were retested centrally, by IHC (using 4 methods) and FISH. In addition, 205 locally reported HER2-negative metastatic breast cancer cases were retested centrally. RESULTS: Concordance between the 505 local IHC-positive cases with central IHC testing varied between 79.3% and 89.6% depending on the IHC method and scoring method used. HER2 gene amplification by FISH was present in 86.1% of local IHC-positive cases. Concordance between the 205 local IHC-negative tumors with central testing ranged between 94.8% and 100% for IHC and was 98.5% for FISH. CONCLUSIONS: This study demonstrated that locally reported HER2-negative results were highly accurate, but less accuracy was demonstrated with local HER2-positive results. These results emphasize the need for participation in a quality assurance program.

PGDS, a novel technique combining chromogenic in situ hybridization and immunohistochemistry for the assessment of ErbB2 (HER2/neu) status in breast cancer.

Given the important prognostic and predictive utility of v-erb-b2 erythroblastic leukemia viral oncogene homolog 2 (ErbB2) [human epidermal growth factor receptor-2 (HER2/neu)] in breast cancer, it is recommended that ErbB2 testing be performed on all invasive breast cancers at the time of diagnosis. A consensus, however, has not yet been reached as to the optimal method of evaluating ErbB2 status. Immunohistochemistry to detect protein overexpression and fluorescence in situ hybridization (FISH) to detect ErbB2 gene amplification are the most frequently used methods. As no one detection method fulfills all necessary requirements of reliability, reproducibility, and ease of use, we developed a novel approach in the form of a simple assay we refer to as protein and gene double staining (PGDS) which simultaneously evaluates protein overexpression and gene amplification by combining immunohistochemistry with chromogenic in situ hybridization (CISH). A total of 134 invasive breast carcinomas, including 81 cases with a full-face section and 53 cases included in a tissue microarray (TMA), were assessed by PGDS, and the results were correlated with ErbB2 gene amplification status as determined by FISH. ErbB2 gene copy number determined by CISH analysis in the PGDS assay showed excellent concordance with that of FISH (correlation coefficient 0.82; P<0.001 with full-face section cases, and 0.98; P<0.001 with cases in a TMA). The overall concordance rate for gene amplification status between PGDS and FISH was 90.12% in cases with a full-face section and 92.45% with TMA cases. Perfect correlation was seen between the PGDS assay and FISH in cases that were considered either nonamplified or highly amplified by the dual assay. Of the 17 cases that showed low amplification by PGDS, 5 were classified as nonamplified by FISH. Correction for chromosome 17 copy number in the FISH assessment contributed to the discordance between CISH and FISH results. This newly developed PGDS method represents a novel approach to ErbB2 status determination that combines the assessment of both protein overexpression and gene amplification in one simple assay. It is likely that this assay will aid in immunohistochemical calibration and will also increase the sensitivity and specificity of ErbB2 testing.