Loss of Chromatin-Remodeling Proteins and/or CDKN2A Associates With Metastasis of Pancreatic Neuroendocrine Tumors and Reduced Patient Survival Times.

Despite prognostic grading and staging systems, it is a challenge to predict outcomes for patients with pancreatic neuroendocrine tumors (PanNETs). Sequencing studies of PanNETs have identified alterations in death domain-associated protein (DAXX) and alpha-thalassemia/mental retardation X-linked chromatin remodeler (ATRX). In tumors, mutations in DAXX or ATRX and corresponding loss of protein expression correlate with shorter times of disease-free survival and disease-specific survival of patients. However, DAXX or ATRX proteins were lost in only 50% of distant metastases analyzed. We performed whole-exome sequencing analyses of 20 distant metastases from 20 patients with a single nonsyndrome, nonfunctional PanNET. We found distant metastases contained alterations in multiple endocrine neoplasia type 1 (MEN1) (n = 8), ATRX (n = 5), DAXX (n = 5), TSC2 (n = 3), and DEP domain containing 5 (DEPDC5) (n = 3). We found copy number loss of cyclin dependent kinase inhibitor 2A (CDKN2A) in 15 metastases (75%) and alterations in genes that regulate chromatin remodeling, including set domain containing 2 (SETD2) (n = 4), AT-rich interaction domain 1A (ARID1A) (n = 2), chromodomain helicase DNA binding protein 8 (CHD8) (n = 2), and DNA methyl transferase 1 (DNMT1) (n = 2). In a separate analysis of 347 primary PanNETs, we found loss or deletion of DAXX and ATRX, disruption of SETD2 function (based on loss of H3 lysine 36 trimethylation), loss of ARID1A expression or deletions in CDKN2A in 81% of primary PanNETs with distant metastases. Among patients with loss or deletion of at least 1 of these proteins or genes, 39% survived disease-free for 5 years and 44% had disease-specific survival times of 10 years. Among patients without any of these alterations, 98% survived disease-free for 5 years and 95% had disease-specific survival times of 10 years. Therefore, primary PanNETs with loss of DAXX, ATRX, H3 lysine 36 trimethylation, ARID1A, and/or CDKN2A associate with shorter survival times of patients. Our findings indicate that alterations in chromatin-remodeling genes and CDKN2A contribute to metastasis of PanNETs.

A comparison of ALK gene rearrangement and ALK protein expression in primary lung carcinoma and matched metastasis.

AIMS: The 2013 College of American Pathologists, the Association for Molecular Pathology and the International Association for the Study of Lung Cancer guideline for EGFR and ALK testing in lung carcinoma indicates that either the primary tumour or the metastasis is suitable for testing. The heterogeneity of gene mutations has been studied extensively, while similar reports on gene rearrangements are limited. The aim of this study was to determine if ALK status between primary tumour and matched metastasis differs. METHODS AND RESULTS: Fifteen ALK fluorescence in-situ hybridization (FISH) rearranged and 19 non-ALK FISH rearranged adenocarcinomas were collected retrospectively based on availability of tissue from a matched metastatic site. Sixty-eight samples were tested by ALK FISH (Vysis ALK break-apart FISH kit) and ALK immunohistochemistry (IHC) (Ventana ALK D5F3 CDx assay). Overall agreement of FISH and IHC was 88%, with IHC showing 100% specificity and 71% sensitivity. Concordance between primary site and metastasis by ALK FISH was seen in 30 cases (88%), and in 32 cases (94%) by ALK IHC. Five discordant cases were found (15%). Three ALK FISH discordant cases had low percentage of ALK FISH-positive tumour cells (average 23%, range: 18-31%) and all were negative by ALK IHC. One IHC discordant case had a high percentage of ALK FISH-positive tumour cells (67%), and was ALK IHC-negative. One FISH discordant case showed ALK FISH- and ALK IHC-positive primary tumour, but ALK FISH- and ALK IHC-negative metastasis. CONCLUSIONS: ALK FISH results show more frequent discordances between primary tumour and matched metastases than ALK IHC, due probably to technical challenges and sample quality. This observation indicates that the quality of sample and technical expertise of the laboratory should guide the decision about ALK testing in clinical practice.

The prognostic significance of BAP1, NF2, and CDKN2A in malignant peritoneal mesothelioma.

Cytoreductive surgery and hyperthermic intraperitoneal chemoperfusion for patients with malignant peritoneal mesothelioma has resulted in improved disease control and increased survival. Despite these results, there are significant perioperative risks associated with this aggressive procedure that necessitate consideration of prognostic markers during patient selection. The molecular pathogenesis of peritoneal mesothelioma remains relatively unknown, but extrapolation of findings from their pleural counterpart would suggest frequent alterations in CDKN2A, NF2, and BAP1. Homozygous deletions in CDKN2A portend a worse overall survival in peritoneal mesothelioma. However, the prevalence and prognostic significance of NF2 and BAP1 abnormalities has not been studied. Dual-color fluorescence in situ hybridization using CDKN2A and NF2 locus-specific probes and BAP1 immunohistochemistry identified homozygous CDKN2A deletions (n=25, 29%), hemizygous NF2 loss (n=30, 35%), and/or loss of BAP1 protein expression (n=49, 57%) in 68 of 86 (79%) peritoneal mesotheliomas. Homozygous CDKN2A deletions or hemizygous NF2 loss correlated with shorter progression-free survival (P<0.02) and poor overall survival (P<0.03). Moreover, the significance of these findings was cumulative. Patients harboring both homozygous CDKN2A deletions and hemizygous NF2 loss had a 2-year progression-free survival rate of 9% with a median of 6 months (P<0.01) and overall survival rate of 18% with a median of 8 months (P<0.01). By multivariate analysis, combined homozygous CDKN2A deletions and hemizygous NF2 loss was a negative prognostic factor for both progression-free survival and overall survival, independent of patient age, peritoneal cancer index, completeness of cytoreduction, and extent of invasion. In contrast, loss of BAP1 was not associated with clinical outcome. In summary, homozygous deletions in CDKN2A and hemizygous loss of NF2 as detected by fluorescence in situ hybridization would confer a poor clinical outcome and may guide future treatment decisions for patients with peritoneal mesothelioma.

Novel fusion transcripts associate with progressive prostate cancer.

The mechanisms underlying the potential for aggressive behavior of prostate cancer (PCa) remain elusive. In this study, whole genome and/or transcriptome sequencing was performed on 19 specimens of PCa, matched adjacent benign prostate tissues, matched blood specimens, and organ donor prostates. A set of novel fusion transcripts was discovered in PCa. Eight of these fusion transcripts were validated through multiple approaches. The occurrence of these fusion transcripts was then analyzed in 289 prostate samples from three institutes, with clinical follow-up ranging from 1 to 15 years. The analyses indicated that most patients [69 (91%) of 76] positive for any of these fusion transcripts (TRMT11-GRIK2, SLC45A2-AMACR, MTOR-TP53BP1, LRRC59-FLJ60017, TMEM135-CCDC67, KDM4-AC011523.2, MAN2A1-FER, and CCNH-C5orf30) experienced PCa recurrence, metastases, and/or PCa-specific death after radical prostatectomy. These outcomes occurred in only 37% (58/157) of patients without carrying those fusion transcripts. Three fusion transcripts occurred exclusively in PCa samples from patients who experienced recurrence or PCaerelated death. The formation of these fusion transcripts may be the result of genome recombination. A combination of these fusion transcripts in PCa with Gleason's grading or with nomogram significantly improves the prediction rate of PCa recurrence. Our analyses suggest that formation of these fusion transcripts may underlie the aggressive behavior of PCa.

PIK3CA, HRAS and PTEN in human papillomavirus positive oropharyngeal squamous cell carcinoma.

BACKGROUND: Recent genomic evidence suggests frequent phosphatidylinositide 3-kinase (PI3K) pathway activation in human papillomavirus (HPV) positive oropharyngeal squamous cell carcinoma. Mutations/amplification of the gene encoding p110α catalytic subunit of phosphoinositide 3-kinase (PIK3CA), loss of phosphatase and tensin homolog (PTEN) and HRAS mutations are known to activate PI3K pathway. METHODS AND RESULTS: PIK3CA mutations were identified by Sanger sequencing in 23 of 75 (31%) HPV-positive oropharyngeal carcinomas, including exon 9 (p.E545K [n = 10] and p.E542K [n = 5]) or exon 20 (p.H1047Y, n = 2) mutations. Five rare and one novel (p.R537Q) PIK3CA mutations were identified. HRAS mutation (p.Q61L) was detected in 1 of 62 tested cases. PIK3CA amplification by fluorescence in situ hybridization (FISH) was identified in 4 cases (4/21, 20%), while PTEN loss was seen in 7 (7/21, 33%) cases (chromosome 10 monosomy [n = 4], homozygous deletion [n = 3]). CONCLUSIONS: Overall, genetic alterations that likely lead to PI3K pathway activation were identified in 34 of 75 cases (45%) and did not correlate with disease specific survival. These findings offer a molecular rationale for therapeutic targeting of PI3K pathway in patients with HPV-positive oropharyngeal carcinoma.

Epithelial-Myoepithelial Carcinoma: Frequent Morphologic and Molecular Evidence of Preexisting Pleomorphic Adenoma, Common HRAS Mutations in PLAG1-intact and HMGA2-intact Cases, and Occasional TP53, FBXW7, and SMARCB1 Alterations in High-grade Cases.

We hypothesized that there is a relationship between the preexisting pleomorphic adenoma [PA]), histologic grade of epithelial-myoepithelial carcinomas (EMCAs), and genetic alterations. EMCAs (n=39) were analyzed for morphologic and molecular evidence of preexisting PA (PLAG1, HMGA2 status by fluorescence in situ hybridization, FISH, and FGFR1-PLAG1 fusion by next-generation sequencing, NGS). Twenty-three EMCAs were further analyzed by NGS for mutations and copy number variation in 50 cancer-related genes. On the basis of combined morphologic and molecular evidence of PA, the following subsets of EMCA emerged: (a) EMCAs with morphologic evidence of preexisting PA, but intact PLAG1 and HMGA2 (12/39, 31%), (b) Carcinomas with PLAG1 alterations (9/39, 23%), or (c) HMGA2 alterations (10/39, 26%), and (d) de novo carcinomas, without morphologic or molecular evidence of PA (8/39, 21%). Twelve high-grade EMCAs (12/39, 31%) occurred across all subsets. The median disease-free survival was 80 months (95% confidence interval, 77-84 mo). Disease-free survival and other clinicopathologic parameters did not differ by the above defined subsets. HRAS mutations were more common in EMCAs with intact PLAG1 and HMGA2 (7/9 vs. 1/14, P<0.001). Other genetic abnormalities (TP53 [n=2], FBXW7 [n=1], SMARCB1 deletion [n=1]) were seen only in high-grade EMCAs with intact PLAG1 and HMGA2. We conclude that most EMCAs arose ex PA (31/39, 80%) and the genetic profile of EMCA varies with the absence or presence of preexisting PA and its cytogenetic signature. Progression to higher grade EMCA with intact PLAG1 and HMGA2 correlates with the presence of TP53, FBXW7 mutations, or SMARCB1 deletion.

Molecular characterization of apocrine salivary duct carcinoma.

Contemporary classification and treatment of salivary duct carcinoma (SDC) require its thorough molecular characterization. Thirty apocrine SDCs were analyzed by the Ion Ampliseq Cancer HotSpot panel v2 for mutations in 50 cancer-related genes. Mutational findings were corroborated by immunohistochemistry (eg, TP53, BRAF, ß-catenin, estrogen, and androgen receptors) or Sanger sequencing/SNaPshot polymerase chain reaction. ERBB2 (HER2), PTEN, FGFR1, CDKN2A/P16, CMET, EGFR, MDM2, and PIK3CA copy number changes were studied by fluorescence in situ hybridization. TP53 mutations (15/27, 56%), PTEN loss (11/29, 38%, including 2 cases with PTEN mutation), PIK3CA hotspot mutations (10/30, 33%), HRAS hotspot mutations (10/29; 34%), and ERBB2 amplification (9/29, 31%, including 1 case with mutation) represented the 5 most common abnormalities. There was no correlation between genetic changes and clinicopathologic parameters. There was substantial overlap between genetic changes: 8 of 9 cases with ERBB2 amplification also harbored a PIK3CA, HRAS, and TP53 mutation and/or PTEN loss. Six of 10 cases with PIK3CA mutation also had an HRAS mutation. These findings provide a molecular rationale for dual targeting of mitogen-activated protein kinase and phosphoinositide 3-kinase pathways in SDC. FGFR1 amplification (3/29, 10%) represents a new potential target. On the basis of studies of breast carcinomas, the efficacy of anti-ERBB2 therapy will likely be decreased in SDC with ERBB2 amplification co-occurring with PIK3CA mutation or PTEN loss. Therefore, isolated ERBB2 testing is insufficient for theranostic stratification of apocrine SDC. On the basis of the prevalence and type of genetic changes, apocrine SDC appears to resemble one subtype of breast carcinoma-"luminal androgen receptor positive/molecular apocrine."

RET rearrangements in lung adenocarcinoma and radiation.

BACKGROUND: RET rearrangement, a hallmark of radiation-induced thyroid cancer, has been reported to occur in 1% of lung adenocarcinoma patients. Patients with this rearrangement tend to be younger and never smokers, raising a possibility of other causes, such as radiation. We hypothesized that RET chromosomal rearrangement may represent a genetic mechanism of radiation-induced lung cancer. METHODS: Two hundred forty-five consecutive primary lung adenocarcinomas without history of radiation and 38 lung adenocarcinoma patients with a history of therapeutic radiation for breast carcinoma or mediastinal Hodkgin lymphoma were tested for RET rearrangement by fluorescence in situ hybridization. Human lung adenocarcinoma cells (201T) were subjected to γ radiation and tested for RET gene fusions by reverse transcriptase-polymerase chain reaction and Southern blot hybridization. RESULTS: We identified one case with RET rearrangement in the group without history of radiation (1 of 240; 0.4%) and two cases in the group with history of radiation (2 of 37; 5.4%; P=0.0436). Both these patients were women, who were former smokers with a history of breast carcinoma treated with surgery and radiation. Furthermore, we found that RET fusions could be directly induced in 201T human lung cells by exposure to 1 Gy of γ radiation. All fusions identified were between RET and KIF5B genes, and no RET fusions to CCDC6 or NCOA4 genes, characteristic for thyroid cancer, were identified in the irradiated lung cells. CONCLUSION: RET fusions may represent a genetic mechanism of radiation-induced lung adenocarcinoma.

Sex-determining region Y-box 2 amplification in preneoplastic squamous lesions of the lung.

Sex-determining region Y-box 2 gene at 3q26.33 has been identified as oncogene in squamous cell carcinoma occurring at different anatomical sites including the lung. Sex-determining region Y-box 2 protein expression and gene amplification have been found in preinvasive squamous cell lesions such as dysplasia and carcinoma in situ. We sought to evaluate sex-determining region Y-box 2 expression and amplification in a spectrum of premalignant squamous lesions ranging from squamous metaplasia to low- and high-grade dysplasia to in situ and invasive squamous cell carcinoma of the lung. Each lesion was taken from 1 of 3 study groups: 18 patients with concurrent squamous cell carcinoma, 17 patients with prior squamous cell carcinoma undergoing surveillance biopsies, and 11 patients with no history of squamous cell carcinoma. Sex-determining region Y-box 2 amplification occurred only in a subset of invasive squamous cell carcinoma (3/5; 60%) and their associated high-grade dysplasia (3/4; 75%), but not in any of the low-grade dysplasias (0/1; 0%) or metaplasias (0/9; 0%). No sex-determining region Y-box 2 amplification was observed in squamous preneoplastic lesions in surveillance biopsies without current evidence of squamous cell carcinoma or in benign lungs. Sex-determining region Y-box 2 protein expression was seen in all squamous lesions regardless of presence or degree of dysplasia. Our results suggest that sex-determining region Y-box 2 amplification is not an early event in squamous carcinogenesis and is important for progression in a subset of squamous cell carcinoma. It appears that sex-determining region Y-box 2 gene amplification in lung squamous carcinogenesis is not the only regulator of sex-determining region Y-box 2 protein expression.

PIK3CA mutations and PTEN loss in salivary duct carcinomas.

Salivary duct carcinoma (SDC) is an aggressive malignancy that frequently presents at an advanced stage. Mutations/amplification of the gene encoding the p110α catalytic subunit of phosphoinositide 3-kinase (PIK3CA) and/or loss of the phosphatase and tensin homolog (PTEN) are known to activate the phosphoinositide 3-kinase (PI3K) pathway and may represent a therapeutic target. In 7 of 34 SDCs (20.5%) a SNaPshot polymerase chain reaction detected PIK3CA exon 9 [p.E545K (n=3) and p.E542K (n=2)] or exon 20 [p.H1047R (n=2)] mutations. PIK3CA p.E545K mutation was identified in 3 de novo SDCs with conventional morphology. The only case of SDC with anaplastic transformation showed PIK3CA p.H1047R mutation, whereas 1 of 2 PIK3CA p.E542K mutations was identified in SDC arising in a pleomorphic adenoma. None of the 16 tested SDCs showed PIK3CA amplification by fluorescence in situ hybridization. Fluorescence in situ hybridization identified PTEN loss in 8 of 16 tested SDCs (50%) [homozygous deletion (n=3), chromosome 10 monosomy (n=3), hemizygous deletion (n=2)]. Two cases showed both PIK3CA mutation and PTEN loss, suggesting that these events are not mutually exclusive. These findings offer a molecular rationale for therapeutic targeting of the PI3K pathway in patients with SDC.