Tumour Biol. 2015 Jul 10.
Seo AN, Park KU, Choe G, Kim WH, Kim DW, Kang SB, Lee HS.
PMID: 26159851
We aimed to explore the clinical and prognostic influence of numeric alterations of MET gene copy number (GCN) and chromosome 7 (CEP7) CN in colorectal cancer (CRC) patients. MET GCN and CEP7 CN were investigated in tissue arrayed tumors from 170 CRC patients using silver in situ hybridization (SISH). MET GCN gain was defined as ≥4 copies of MET, and CEP7 polysomy was prespecified as ≥3 copies of CEP7. Additionally, MET messenger RNA (mRNA) transcription was evaluated using mRNA ISH and compared with MET GCN. MET GCN gain was observed in 14.7 % (25/170), which correlated with advanced stage (P = 0.037), presence of distant metastasis (P = 0.006), and short overall survival (OS) (P = 0.009). In contrast, CEP7 polysomy was found in 6.5 % (11/170), which was related to tumor location in the left colon (P = 0.027) and poor OS (P = 0.029). MET GCN positively correlated with CEP7 CN (R = 0.659, P < 0.001) and mRNA transcription (R = 0.239, P = 0.002). Of note, MET GCN gain and CEP7 polysomy were also associated with poor OS (P = 0.016 and P < 0.001, respectively) in stage II/III CRC patients (n = 123). In multivariate analysis, CEP7 polysomy was an independent prognostic factor for poor OS in all patients (P = 0.009; hazard ratio [HR], 2.220; 95 % confidence interval [CI], 1.233-3.997) and in stage II/III CRC patients (P < 0.001; HR, 20.781; 95 % CI, 4.600-93.882). MET GCN gain and CEP7 polysomy could predict a poor outcome in CRC patients, especially CEP7 polysomy has the most powerful prognostic impact in stage II/III CRC patients
Ramberg, I;Vieira, FG;Toft, PB;von Buchwald, C;Heegaard, S;
PMID: 35626161 | DOI: 10.3390/cancers14102558
The pathogenesis of squamous cell neoplasms arising in the lacrimal drainage system is poorly understood, and the underlying genomic drivers for disease development remain unexplored. We aimed to investigate the genomic aberrations in carcinomas arising in the LDS and correlate the findings to human papillomavirus (HPV) status. The HPV analysis was performed using HPV DNA PCR, HPV E6/E7 mRNA in-situ hybridization, and p16 immunohistochemistry. The genomic characterization was performed by targeted DNA sequencing of 523 cancer-relevant genes. Patients with LDS papilloma (n = 17) and LDS carcinoma (n = 15) were included. There was a male predominance (68%) and a median age at diagnosis of 46.0 years (range 27.5-65.5 years) in patients with papilloma and 63.8 years (range 34.0-87.2 years) in patients with carcinoma. Transcriptional activity of the HPV E6/E7 oncogenes was detected in the whole tumor thickness in 12/15 (80%) papillomas (HPV6, 11, 16) and 10/15 (67%) squamous cell carcinomas (SCC) (HPV11: 3/15 (20%) and HPV16: 7/15 (47%)). Pathogenic variants in PIK3CA, FGFR3, AKT1, and PIK3R1, wildtype TP53, p16 overexpression, and deregulated high-risk E6/E7 transcription characterized the HPV16-positive SCC. The deregulated pattern of HPV E6/E7 expression, correlating with HPV DNA presence and p16 positivity, supports a causal role of HPV in a subset of LDS papillomas and carcinomas. The viral and molecular profile of LDS SCC resembles that of other HPV-driven SCC.
PLoS One. 2014 Nov 3;9(11):e111658.
Choi J, Lee HE, Kim MA, Jang BG, Lee HS, Kim WH.
PMID: 25364819 | DOI: 10.1371/journal.pone.0111658
We investigated MET mRNA expression status using RNA in situ hybridization (ISH) technique in primary and metastatic lesions of 535 surgically resected gastric carcinoma (GC) cases. We compared the results with those of immunohistochemistry and silver in situ hybridization, and examined the association with clinicopathologic characteristics and prognosis. Among 535 primary GCs, 391 (73.1%) were scored 0, 87 (16.3%) were scored 1, 38 (7.1%) were scored 2, 12 (2.2%) were scored 3 and 7 (1.3%) were scored 4 by RNA ISH. High MET mRNA expression (score ≥3) was associated with lymph node metastasis (P = .014), distant metastasis (P = .001), and higher TNM stage (P<.001). MET mRNA expression was correlated with protein expression (r = 0.398; P<.001) and gene copy number (r = 0.345; P<.001). The patients showing high-MET mRNA in primary or metastatic lesions had shorter overall survival than those showing low-MET mRNA (primary tumors, P = .002; metastatic lymph nodes, P<.001). The patients showing positive conversion of MET mRNA status in metastatic lymph node had shorter overall survival than those with no conversion (P = .011). Multivariate analysis demonstrated that high MET mRNA expression in metastatic lymph node was an independent prognostic factor for overall survival (P = .007). Therefore, this study suggests that MET mRNA expression assessed by RNA ISH could be useful as a potential marker to identify MET oncogene-addicted GC.
Bradley CA, Dunne PD, Bingham V, McQuaid S, Khawaja H, Craig S, James J, Moore WL, McArt DG, Lawler M, Dasgupta S, Johnston PG, Van Schaeybroeck S.
PMID: 27793046 | DOI: 10.18632/oncotarget.12933
c-MET and its ligand HGF are frequently overexpressed in colorectal cancer (CRC) and increased c-MET levels are found in CRC liver metastases. This study investigated the role of the HGF/c-MET axis in regulating migration/invasion in CRC, using pre-clinical models and clinical samples. Pre-clinically, we found marked upregulation of c-MET at both protein and mRNA levels in several invasive CRC cells. Down-regulation of c-MET using RNAi suppressed migration/invasion of parental and invasive CRC cells. Stimulation of CRC cells with rh-HGF or co-culture with HGF-expressing colonic myofibroblasts, resulted in significant increases in their migratory/invasive capacity. Importantly, HGF-induced c-MET activation promoted rapid downregulation of c-MET protein levels, while the MET transcript remained unaltered. Using RNA in situ hybridization (RNA ISH), we further showed that MET mRNA, but not protein levels, were significantly upregulated in tumor budding foci at the invasive front of a cohort of stage III CRC tumors (p < 0.001). Taken together, we show for the first time that transcriptional upregulation of MET is a key molecular event associated with CRC invasion and tumor budding. This data also indicates that RNA ISH, but not immunohistochemistry, provides a robust methodology to assess MET levels as a potential driving force of CRC tumor invasion and metastasis.
Tendron, A;Classe, M;Casiraghi, O;Pere, H;Even, C;Gorphe, P;Moya-Plana, A;
PMID: 35454782 | DOI: 10.3390/cancers14081874
Sinonasal squamous cell carcinoma (SNSCC) is a rare and aggressive malignancy with poor prognosis. Human papilloma virus (HPV) can induce SNSCC although its incidence and impact on patients' outcomes remains unclear. We performed a retrospective cohort study of patients with SNSCC treated consecutively in a comprehensive cancer center. HPV status was determined with p16 immunohistochemistry followed by RNA in situ hybridization (RNAscope). The incidence, clinical characteristics, and oncologic outcomes of HPV+SNSCC were assessed. P16 prognostic value was evaluated. Fifty-nine patients were included. Eleven (18.6%) SNSCC were p16+ with five (8.4%) doubtful cases. RNAscope was positive in nine cases (15.2%). Patients with HPV+SNSCC were younger (p = 0.0298) with a primary tumor originating mainly in nasal fossa (p < 10-4). Pathologic findings were not different according to HPV status. Among patients who were curatively treated, overall survival was better for HPV+SNSCC (p = 0.022). No prognostic value of p16 expression was reported. Patients with HPV+SNSCC have better oncologic outcomes, probably due to earlier tumor stage with primary location predominantly in the nasal fossa, a more suitable epicenter to perform a surgical resection with clear margins. P16 expression seems not to be a good surrogate of HPV status in SNSCC.
Schmid E, Klotz M, Steiner-Hahn K, Konen T, Frisk AL, Schatz C, Krahn T, von Ahsen O.
PMID: 28836864 | DOI: 10.1080/10520295.2017.1339913
Determination of predictive biomarkers by immunohistochemistry (IHC) relies on antibodies with high selectivity. RNA in situ hybridization (RNA ISH) may be used to confirm IHC and may potentially replace it if suitable antibodies are not available or are insufficiently selective to discriminate closely related protein isoforms. We validated RNA ISH as specificity control for IHC and as a potential alternative method for selecting patients for treatment with MET inhibitors. MET, the HGF receptor, is encoded by the MET proto-oncogene that may be activated by mutation or amplification. MET expression and activity were tested in a panel of control cell lines. MET could be detected in formalin fixed paraffin, embedded (FFPE) samples by IHC and RNA ISH, and this was confirmed by sandwich immunoassays of fresh frozen samples. Gastric cancer cell lines with high MET expression and phosphorylation of tyrosine-1349 respond to the MET inhibitor, BAY-853474. High expression and phosphorylation of MET is a predictive biomarker for response to MET inhibitors. We then analyzed MET expression and activity in a matched set of FFPE vs. fresh frozen tumor samples consisting of 20 cases of gastric cancer. Two of 20 clinical samples investigated exhibited high MET expression with RNA ISH and IHC. Both cases were shown by sandwich immunoassays to exhibits strong functional activity. Expression levels and functional activity in these two cases were in a range that predicted response to treatment. Our findings indicate that owing to its high selectivity, RNA ISH can be used to confirm findings obtained by IHC and potentially may replace IHC for certain targets if no suitable antibodies are available. RNA ISH is a valid platform for testing predictive biomarkers for patient selection.
Abstract LB190: DNAscopeTM: A novel chromogenic in-situ hybridization technology for high-resolution detection of DNA copy number and structural variations
Molecular and Cellular Biology/Genetics
Wang, L;Tondnevis, F;Todorov, C;Gaspar, J;Sahajan, A;Murlidhar, V;Zhang, B;Ma, X;
| DOI: 10.1158/1538-7445.am2021-lb190
Genomic DNA anomalies such as copy number variations (gene duplication, amplification, deletion) and gene rearrangements are important biomarkers and drug targets in many cancer types. DNA in-situ hybridization (ISH) is the gold standard method to directly visualize these molecular alterations in formalin-fixed paraffin-embedded (FFPE) tumor tissues at single-cell resolution within a histological section. However, currently available fluorescent ISH (FISH) assays provide limited morphological detail due to the use of fluorescent nuclear staining compared to chromogenic staining. Furthermore, FISH techniques rely on expensive fluorescence microscopes, risk loss of fluorescent signal over time and involve tedious imaging at high magnifications (100X). There is thus an unmet need for a sensitive and robust chromogenic DNA-ISH assay that can enable high-resolution detection of genomic DNA targets with the ease of bright-field microscopy. We present here DNAscope - a novel chromogenic DNA-ISH assay - for detecting and visualizing genomic DNA targets under a standard light microscope. DNAscope is based on the widely used RNAscope double-Z probe design and signal amplification technology and provides unparalleled sensitivity and specificity with large signal dots readily visualized at 40X magnification and with full morphological context. Furthermore, DNAscope ensures specific DNA detection without interference from RNA due to the use of a novel RNA removal method. Using a duplex chromogenic detection assay in red and blue, we demonstrate highly specific and efficient detection of gene rearrangements (ALK, ROS1, RET and NTRK1), gene amplification (ERBB2, EGFR, MET) and deletion (TP53 and CDKN2A). The DNAscope assay has been carefully optimized for probe signal size and color contrast to enable easy interpretation of signal patterns under conventional light microscopy or digital pathology. Compared to conventional FISH assays, DNAscope probes are standard oligos that are designed in silico to be free of any repetitive sequences and can be rapidly synthesized for any DNA target. In conclusion, the DNAscope assay provides a powerful and convenient alternative to commonly used FISH assays in many cancer research applications.
Rasmussen, SA;Lewis, JS;Mirabello, L;Bass, S;Yeager, M;Corsten, MJ;Bullock, MJ;
PMID: 35771403 | DOI: 10.1007/s12105-022-01463-4
Oropharyngeal squamous cell carcinoma is frequently associated with high-risk HPV infection, which confers a good prognosis. Immunohistochemistry for p16 is used as a surrogate for HPV status, but discrepant results are occasionally seen. Here, we report a case with a unique pattern of partial loss of p16.A 63 year old male presented with a base of tongue nonkeratinizing squamous cell carcinoma and a large metastatic neck mass. The primary lesion and multiple regions of the metastatic mass were assessed with p16 immunohistochemistry, RNA in situ hybridization for high-risk HPV, and HPV16 genome sequencing.The primary lesion was p16 negative, and the metastatic neck mass had large, confluent regions that were either strongly p16 positive or entirely p16 negative. All of these regions were positive for high-risk HPV with identical HPV16 genomes.This unusual case illustrates a potential diagnostic pitfall, and it raises important questions regarding molecular mechanisms and prognostic implications of p16 staining in oropharyngeal squamous cell carcinoma.
The American journal of surgical pathology
Hopkins, MR;Palsgrove, DN;Ronnett, BM;Vang, R;Lin, J;Murdock, TA;
PMID: 36069815 | DOI: 10.1097/PAS.0000000000001970
Human papillomavirus (HPV)-independent primary endometrial squamous cell carcinoma (PESCC) is a rare but aggressive subtype of endometrial carcinoma for which little is known about the genomic characteristics. Traditional criteria have restricted the diagnosis of PESCC to cases without any cervical involvement. However, given that modern ancillary techniques can detect HPV and characteristic genetic alterations that should identify the more common mimics in the differential diagnosis, including endometrial endometrioid carcinoma with extensive squamous differentiation and HPV-associated primary cervical squamous cell carcinoma, those criteria may benefit from revision. To further characterize PESCC, we identified 5 cases of pure squamous cell carcinoma dominantly involving the endometrium that had the potential to be PESCC: 1 case involving only the endometrium and 4 cases with some involvement of the cervix. Clinicopathologic features were assessed and immunohistochemical analysis (p16, estrogen receptor, progesterone receptor, and p53), HPV RNA in situ hybridization (high-risk and low-risk cocktails and targeted probes for 16 and 18), and molecular studies were performed. All tumors showed aberrant/mutation-type p53 expression, were negative for estrogen receptor, progesterone receptor, and p16, and had no detectable HPV. Per whole-exome sequencing, 4 of the 5 tumors demonstrated comutations in TP53 and CDKN2A (p16). Four patients died of disease within 20 months (range, 1 to 20 mo; mean, 9 mo), and 1 patient had no evidence of disease at 38 months. PESCC represents a unique, clinically aggressive subtype of endometrial cancer with TP53 and CDKN2A comutations. This characteristic profile, which is similar to HPV-independent squamous cell carcinoma of the vulva, is distinct from endometrioid carcinoma with extensive squamous differentiation and HPV-associated primary cervical squamous cell carcinoma and can be used to distinguish PESCC from those mimics even when cervical involvement is present. Diagnostic criteria for PESCC should be relaxed to allow for cervical involvement when other pathologic features are consistent with, and ancillary techniques are supportive of classification as such.
Oncogenic switch and single-agent MET inhibitor sensitivity in a subset of EGFR-mutant lung cancer
Science translational medicine
Eser, PÖ;Paranal, RM;Son, J;Ivanova, E;Kuang, Y;Haikala, HM;To, C;Okoro, JJ;Dholakia, KH;Choi, J;Eum, Y;Ogino, A;Missios, P;Ercan, D;Xu, M;Poitras, MJ;Wang, S;Ngo, K;Dills, M;Yanagita, M;Lopez, T;Lin, M;Tsai, J;Floch, N;Chambers, ES;Heng, J;Anjum, R;Santucci, AD;Michael, K;Schuller, AG;Cross, D;Smith, PD;Oxnard, GR;Barbie, DA;Sholl, LM;Bahcall, M;Palakurthi, S;Gokhale, PC;Paweletz, CP;Daley, GQ;Jänne, PA;
PMID: 34516823 | DOI: 10.1126/scitranslmed.abb3738
[Figure: see text].
