Amplification Of Chromosome 3q Research Articles

TNIK, a novel activator of FAK and YAP signaling, is a therapeutic target in Lung Squamous Cell Carcinoma

Distal chromosome 3q amplification (3q26-29, also known as the 3q amplicon) is the most frequent genomic alteration in lung squamous cell carcinoma (LSCC). Analysis of LSCC samples from the TCGA reveals that nearly 50% of LSCC patients harbor the 3q amplicon that includes the resident protein kinase gene TNIK. Recent studies have pinpointed TNIK as a potential oncogenic driver in cancer cells with distal 3q amplification; however, the therapeutic potential of TNIK remains unexplored. We found that TNIK was highly expressed in LSCC cells with the 3q amplicon, while its expression was modest in cells that lacked the 3q amplicon, consistent with data from the TCGA. To evaluate TNIK as a target in LSCC, we generated stable, doxycycline-inducible cells expressing shRNA to deplete TNIK from LSCC cells and conducted functional assays to measure cell proliferation and survival. TNIK knockdown or inhibition of its kinase activity with a small molecule inhibitor significantly diminished the viability of LSCC cells with 3q amplification in vitro and in cell line-derived xenograft mouse models. We also observed that TNIK inhibitors significantly abrogated the growth of LSCC patient-derived xenografts and showed that TNIK inhibition induced apoptotic cell death in LSCC cells that harbor the 3q amplicon. Importantly, TNIK depletion or catalytic inhibition in LSCC cells that lack the 3q amplicon had no significant effect on cell survival. Finally, we used a combination of bioinformatics and proteomic analysis (RPPA, peptide mapping, and mass spectrometry) to define the underlying mechanisms driving TNIK mediated cancer cell survival. We identified the tumor suppressor MERLIN as a novel TNIK substrate and determined that TNIK phosphorylates MERLIN at serine 13 and 315. We also show that TNIK is required to maintain FAK activation and stabilize the YAP transcription factor, two oncogenic pathways inhibited by MERLIN. In conclusion, our results demonstrate that TNIK maintains survival of LSCC cells through modulation of a novel TNIK-MERLIN-YAP/FAK signaling pathway and validate TNIK inhibitors in pre-clinical models of LSCC, including patient-derived xenografts. In summary, we have pinpointed the protein kinase TNIK as a promising therapeutic target for the treatment of LSCC patients with distal chromosome 3q amplification.

Tumor Analyses Reveal Squamous Transformation and Off-Target Alterations As Early Resistance Mechanisms to First-line Osimertinib in EGFR-Mutant Lung Cancer.

Patterns of resistance to first-line osimertinib are not well-established and have primarily been evaluated using plasma assays, which cannot detect histologic transformation and have differential sensitivity for copy number changes and chromosomal rearrangements. To characterize mechanisms of resistance to osimertinib, patients with metastatic EGFR-mutant lung cancers who received osimertinib at Memorial Sloan Kettering Cancer Center and had next-generation sequencing performed on tumor tissue before osimertinib initiation and after progression were identified. Among 62 patients who met eligibility criteria, histologic transformation, primarily squamous transformation, was identified in 15% of first-line osimertinib cases and 14% of later-line cases. Nineteen percent (5/27) of patients treated with first-line osimertinib had off-target genetic resistance (2 MET amplification, 1 KRAS mutation, 1 RET fusion, and 1 BRAF fusion) whereas 4% (1/27) had an acquired EGFR mutation (EGFR G724S). Patients with squamous transformation exhibited considerable genomic complexity; acquired PIK3CA mutation, chromosome 3q amplification, and FGF amplification were all seen. Patients with transformation had shorter time on osimertinib and shorter survival compared with patients with on-target resistance. Initial EGFR sensitizing mutation, time on osimertinib treatment, and line of therapy also influenced resistance mechanism that emerged. The compound mutation EGFR S768 + V769L and the mutation MET H1094Y were identified and validated as resistance mechanisms with potential treatment options. Histologic transformation and other off-target molecular alterations are frequent early emerging resistance mechanisms to osimertinib and are associated with poor clinical outcomes.See related commentary by Piotrowska and Hata, p. 2441.

The deSUMOylase SENP2 coordinates homologous recombination and nonhomologous end joining by independent mechanisms.

SUMOylation (small ubiquitin-like modifier) in the DNA double-strand break (DSB) response regulates recruitment, activity, and clearance of repair factors. However, our understanding of a role for deSUMOylation in this process is limited. Here we identify different mechanistic roles for deSUMOylation in homologous recombination (HR) and nonhomologous end joining (NHEJ) through the investigation of the deSUMOylase SENP2. We found that regulated deSUMOylation of MDC1 prevents excessive SUMOylation and its RNF4-VCP mediated clearance from DSBs, thereby promoting NHEJ. In contrast, we show that HR is differentially sensitive to SUMO availability and SENP2 activity is needed to provide SUMO. SENP2 is amplified as part of the chromosome 3q amplification in many cancers. Increased SENP2 expression prolongs MDC1 focus retention and increases NHEJ and radioresistance. Collectively, our data reveal that deSUMOylation differentially primes cells for responding to DSBs and demonstrates the ability of SENP2 to tune DSB repair responses.

FOLFCIS Treatment and Genomic Correlates of Response in Advanced Anal Squamous Cell Cancer

BackgroundTreatment of advanced anal squamous cell cancer (SCC) is usually with the combination of cisplatin and 5-fluorouracil, which is associated with heterogeneous responses across patients and significant toxicity. We examined the safety and efficacy of a modified schedule, FOLFCIS (leucovorin, fluorouracil, and cisplatin), and performed an integrated clinical and genomic analysis of anal SCC. Patients and MethodsWe reviewed all patients with advanced anal SCC receiving first-line FOLFCIS chemotherapy – essentially a FOLFOX (leucovorin, fluorouracil, and oxaliplatin) schedule with cisplatin substituted for oxaliplatin – in our institution between 2007 and 2017, and performed deep sequencing to identify genomic markers of response and key genomic drivers. ResultsFifty-three patients with advanced anal SCC (48 metastatic; 5 unresectable, locally advanced) received first-line FOLFCIS during this period; all were platinum-naive. The response rate was 48% (95% confidence interval [CI], 32.6%-63%). With a median follow-up of 41.6 months, progression-free survival and overall survival were 7.1 months (95% CI, 4.4-8.6 months) and 22.1 months (95% CI, 16.9-28.1 months), respectively. Among all patients with advanced anal SCC that underwent sequencing during the study period, the most frequent genomic alterations consisted of chromosome 3q amplification (51%) and mutations in PIK3CA (29%) and KMT2D (22%). No genomic alteration correlated with response to platinum-containing treatment. Although there were few cases, patients with human papillomavirus-negative anal SCC did not appear to benefit from FOLFCIS, and all harbored distinct genomic profiles with TP53, TERT promoter, and CDKN2A mutations. ConclusionsFOLFCIS appears effective and safe as first-line chemotherapy in patients with advanced anal SCC and represents an alternative treatment option for these patients.

Abstract IA09: Comprehensive genomic characterization of head and neck squamous cell carcinomas

Abstract Large and comprehensive genomic analyses of head and neck squamous cell carcinomas (HNSCC) are greatly increasing our understanding of the heterogeneity of this disease and identifying the key genomic changes that drive these tumors. HNSCC tumors associated with HPV are distinct from HNSCC disease linked to tobacco exposure (HPV-positive versus HPV-negative). HPV- tumors are characterized by: 1) amplification of receptor tyrosine kinases (RTK); 2) genomic loss of TP53 and loss of CKDN2A/RB1 by a variety of mechanisms including truncating mutation, deletion, and/or alternative splicing; 3) amplification of CCND1/CDK4/CDK6; 4) activation of NFE2L2/KEAP1/CUL3; and 5) loss of NOTCH1/FAT1/AJUBA. In contrast, HPV-positive HNSCCs harbor: 1) RTK amplification; 2) FGFR2/3 mutations and/or FGFR3-TACC3 fusions; 3) CDKN2A loss; and 4) E2F1 amplification. Interestingly, activating mutations in the PIK3CA oncogene are found in approximately one third of HPV-negative HNSCC and in over fifty percent of HPV-positive tumors. Mutually exclusive mutations in RAS family genes, specifically RHOA, KRAS, and HRAS, are present but infrequently found in HNSCC (6% of TCGA cases). Amplification of chromosome 3q, a region containing the TP63, SOX2, and PIK3CA genes, is seen in the majority of both HPV-negative and HPV-positive HNSCC. HPV-negative HNSCCs demonstrate clear evidence of molecular heterogeneity, as demonstrated by expression profiling studies indicating diverse biologic subclasses within HPV-negative disease, including a class of tumors without EGFR amplification and/or overexpression, previously termed “atypical” HNSCCs, which consist of approximately 20% of HPV-negative cases and the majority of HPV-positive HNSCCs. An intriguing mutational pattern identified by TCGA was a subset of HPV-negative HNSCC originating in the oral cavity with few to no copy-number alterations statistically enriched for HRAS, CASP8, and PIK3CA mutations and lack of TP53 mutation. With increasing genetic profiling of HNSCC cell lines and patient-derived xenografts, there is increasing opportunity to rationally select preclinical models to test specific precision medicine hypotheses. Citation Format: Jennifer R. Grandis. Comprehensive genomic characterization of head and neck squamous cell carcinomas [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr IA09.

Abstract 3847: Oral cavity squamous cell carcinoma xenografts display conservation of primary tumor genomic heterogeneity

Abstract Purpose: Patient derived xenograft (PDX) models represent a platform for defining therapeutic opportunities based on their ability to maintain the genomic and molecular features of their respective tumors. We used a comprehensive genomics approach to analyze PDXs for oral squamous cell carcinoma (OSCC), which had not been previously described. This involved confirming that the PDXs conserve the heterogeneous somatic landscape of the primary tumors from which they were derived. Methods: We established a large panel of OSCC PDX models by engrafting fresh, primary tumor samples into NOD scid gamma (NSG) mice. We performed whole genome sequencing (WGS; n=10), whole exome sequencing (WES; n=15), and transcriptome sequencing (RNAseq; n=15) on matched primary and first passage xenografts. Somatic events, including mutations, copy number alterations (CNAs), loss-of-heterozygosity (LOH) regions, and structural variation (SV), were detected independently in tumors and PDXs. Conservation was defined by the correlation of variant allele frequency (VAF) distributions and gene expression patterns, presence of larger somatic events, and predictions made about the subclonal architecture of the tumor and xenograft. Results: PDXs were established with an overall success rate of 47% (56/118). The somatic landscape of primary tumors was representative of the published genomic data on OSCC, including amplifications of CCND1 and EGFR family members, inactivating mutations and loss of TP53 and CDKN2A, and chromosome 3q amplification. These somatic events were also strongly conserved in matched xenograft samples. Mutations that were detected in either the xenograft or primary tumor, but not its matched sample, consisted primarily of low-frequency mutations. The predicted clonal architecture, however, revealed correlative subclonal heterogeneity between matched xenograft and primary tumor. This includes maintenance of founding clone mutations and consistent VAF distributions, indicating that a heterogeneous tumor population engrafted. Conclusions: This comprehensive approach establishes early-passage PDXs as high fidelity models for OSCC. Their ability to maintain the genomic heterogeneity detected in primary OSCC tumors presents a clinically relevant platform for understanding tumor biology and identifying novel therapeutic strategies. Citation Format: Katie M. Campbell, Tianxiang Lin, Ashley E. Winkler, Paul Zolkind, Zachary L. Skidmore, Erica K. Barnell, Ian Hagemann, Elaine R. Mardis, Malachi Griffith, Rebecca D. Chernock, Obi L. Griffith, Ravindra Uppaluri. Oral cavity squamous cell carcinoma xenografts display conservation of primary tumor genomic heterogeneity [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 3847. doi:10.1158/1538-7445.AM2017-3847

True 3q Chromosomal Amplification in Squamous Cell Lung Carcinoma by FISH and aCGH Molecular Analysis: Impact on Targeted Drugs

Squamous lung carcinoma lacks specific “ad hoc” therapies. Amplification of chromosome 3q is the most common genomic aberration and this region harbours genes having role as novel targets for therapeutics. There is no standard definition on how to score and report 3q amplification. False versus true 3q chromosomal amplification in squamous cell lung carcinoma may have tremendous impact on trials involving drugs which target DNA zones mapping on 3q. Forty squamous lung carcinomas were analyzed by FISH to assess chromosome 3q amplification. aCGH was performed as gold-standard to avoid false positive amplifications. Three clustered patterns of fluorescent signals were observed. Eight cases out of 40 (20%) showed ≥8 3q signals. Twenty out of 40 (50%) showed from 3 to 7 signals. The remaining showed two fluorescent signals (30%). When corrected by whole chromosome 3 signals, only cases with ≥8 signals maintained a LSI 3q/CEP3 ratio >2. Only the cases showing 3q amplification by aCGH (+3q25.3−3q27.3) showed ≥8 fluorescent signals at FISH evidencing a 3q/3 ratio >2. The remaining cases showed flat genomic portrait at aCGH on chromosome 3. We concluded that: 1) absolute copy number of 3q chromosomal region may harbour false positive interpretation of 3q amplification in squamous cell carcinoma; 2) a case results truly “amplified for chromosome 3q” when showing ≥8 fluorescent 3q signals; 3) trials involving drugs targeting loci on chromosome 3q in squamous lung carcinoma therapy have to consider false versus true 3q chromosomal amplification.

Genomic Amplification of hTERC in Paraffin-embedded Tissues of Cervical Intraepithelial Neoplasia and Invasive Cancer

Cervical lesions and invasive cervical carcinomas are frequently associated with the amplification of chromosome 3q, which harbors the human telomerase gene hTERC. The differential diagnosis between low-grade and high-grade cervical lesions has been a clinical challenge in cytopathology. In the current study, we evaluated hTERC amplification by fluorescence in situ hybridization in cervical lesions with distinct histopathological changes and assessed the potential application of this technology in diagnosis. We showed that the frequency of genomic amplification of hTERC increased with the severity of the disease, with no marked difference between disease-free cervical squamous epithelium and Grade I cervical intraepithelial neoplasia(CIN)1, but that there were significant differences between these 2 stages and the CIN2, CIN3, and squamous cell carcinoma lesions. The difference between CIN1 and CIN2 would be of great importance for differential diagnosis in the clinic. In addition, hTERC amplification status exhibited a sensitivity of 59.42%, a specificity of 85.29%, an accuracy of 67.96%, a positive prediction value of 89.13%, and a negative prediction value of 68.42% in distinguishing CIN1 from CIN2/3 lesions. Therefore, hTERC amplification within cervical lesions can be accurately assessed by fluorescence in situ hybridization analysis on paraffin tissue microarrays. This technique may be clinically applied to differentiate between CIN1 and CIN2/3 lesions in paraffin-embedded biopsy tissues.

Sec62 Bridges the Gap from 3q Amplification to Molecular Cell Biology in Non–Small Cell Lung Cancer

The molecular carcinogenesis of lung cancer has yet to be clearly elucidated. We investigated the possible oncogenic function of SEC62 in lung cancer, which was predicted based on our previous findings that lung and thyroid cancer tissue samples exhibited increased Sec62 protein levels. The SEC62 gene locus is at 3q26.2, and 3q amplification is reportedly the most common genomic alteration in non-small cell lung cancer. We analyzed SEC62 mRNA and protein levels in tissue samples from lung cancer patients by real-time quantitative PCR, Western blot, and IHC and found significantly increased SEC62 mRNA and protein levels in tumors compared with tumor-free tissue samples from the same patients. Correlation analyses revealed significantly higher Sec62 levels in tumors with lymph node metastases compared with nonmetastatic tumors, as well as in poorly compared with moderately differentiated tumors. On the basis of these promising results, we examined the role of Sec62 in cancer cell biology in vitro. Cell migration assays with lung and thyroid cancer cells showed distinct stimulation of migration in SEC62-overexpressing cells and inhibition of migration in Sec62-depleted cells. Moreover, we found that SEC62 silencing sensitized the cells to thapsigargin-induced endoplasmic reticulum stress. Thus, our results indicate that SEC62 represents a potential candidate oncogene in the amplified 3q region in cases of non-small cell lung cancer and harbors various functions in cancer cell biology.

Overexpression of eIF5A‐2 is an adverse prognostic marker of survival in stage I non–small cell lung cancer patients

We have previously isolated an oncogene EIF5A2 (eukaryotic initiation factor 5A2) from a frequently amplified region at 3q of a primary ovarian cancer cell line, and demonstrated its impact on prognosis in human ovarian cancer. Amplification of chromosome 3q has also been detected frequently in non-small cell lung cancer (NSCLC), however, abnormalities of EIF5A2 and its clinicopathologic significance in NSCLC haven't been studied. In our study, the methods of immunohistochemistry and fluorescence in situ hybridization were utilized to examine protein expression and amplification of EIF5A2 in 248 surgically resected NSCLCs (learning cohort) and another validation cohort of 120 stage I NSCLC patients. Overexpression and amplification of EIF5A2 was detected informatively in 48.7% and 13.7% of NSCLCs in learning cohort, 33.3% and 6.0% of NSCLCs in validation cohort. Overexpression of eIF5A-2 was found to correlate with gene amplification, increased cell proliferation and advanced T stage. In learning cohort, eIF5A-2 expression was evaluated as a strong prognostic factor on disease-specific survival, but in subgroup analyses, it only retained its stratified significance in stage I set (Hazards ratio = 2.799, p = 0.001). In validation cohort, the impact of eIF5A-2 expression on survival in stage I NSCLC patients was also observed (Hazard ratio = 2.097, p = 0.014). Our findings suggested that overexpression of eIF5A-2 correlates with local invasion of NSCLC, and might serve as an adverse prognostic marker of survival for stage I NSCLC patients.

Role of Chromosome 3q Amplification in Lung Cancer

Chromosome abnormalities often correlate with molecular abnormalities and provide a starting point for gene discovery and characterization in the context of a specific disorder. In cancer biology, chromosomal abnormalities carry diagnosis, prognostic, and predictive value of response to treatment. Recently, methodologies such as array comparative genomic hybridization and oligonucleotide microarrays allow discovery of regions of frequent alterations with high resolution. Genomic gain at chromosome 3q location has been recognized as one the most prevalent and significant alterations in lung cancer. Emerging data suggests that regions of amplification have profound effect on tumor development and house candidate biomarkers of disease progression, response to therapy and prognosis. This review examines how genome-wide analysis of lung cancer lead to the evaluation of a specific genomic alteration on chromosome 3q, the study of candidate driver genes and their potential clinical implications.

Biology of HPV in HIV Infection

HIV-positive men and women are at increased risk of anogenital and oral HPV infection. The risks for HPV-associated high-grade intra-epithelial neoplasia (IN) and cancer are also increased. The prevalence of oral, anal, and cervical HPV infection in HIV-positive individuals compared with HIV-negative individuals increases with progressively lower CD4+ levels, as does incident high-grade IN. In contrast to IN, development of cancer is not related to lower CD4+ level. With increasing grades of IN and cancer, the proportion of tissues with copy-number abnormalities (CNA) increases, with one of the most common genetic changes being amplification of chromosome 3q. The presence of CNA is associated with the integration of HPV DNA into the host genome, with loss of HPV E2 and/or E2 rearrangement. This suggests a link between CNA and increased HPV-induced chromosomal instability mediated through de-repressed E6 and E7 expression consequent to loss of functional E2 protein. In addition, epigenetic changes occur with increasing frequency in high-grade IN and cancer, such as hypermethylation leading to down-regulation of potential tumor suppressor genes. Analysis of these data together suggests that immune suppression plays a more prominent role in the earlier stages of HPV-associated disease, up to and including incident high-grade IN. Persistent high-grade IN and development of cancer may be more strongly related to the cumulative effect of HPV-associated genetic instability and the resulting host genetic changes. There are few data to suggest a direct role for HIV in the pathogenesis of HPV-associated neoplasia, but HIV-associated attenuation of HPV-specific immune responses may allow for persistence of high-grade IN and sufficient time for accumulation of genetic changes that are important in progression to cancer.

Large cell neuroendocrine carcinoma of the uterine cervix with cytogenetic analysis by comparative genomic hybridization: a case study

Large cell neuroendocrine carcinoma (LCNEC) of the uterine cervix is a newly introduced category of the revised World Health Organization classification. We reported a case of cervical LCNEC with cytogenetic analysis by comparative genomic hybridization (CGH). The cervical tumor showed moderately increased mitotic activity (8-14 mitotic figures per 10 high-power fields) and focal necrosis, which made it problematic to differentiate from atypical carcinoid. CGH analysis failed to detect chromosome 11q loss that has been reported to be characteristic of pulmonary atypical carcinoids. Furthermore, chromosome 3q amplification, which has been detected frequently in pulmonary small cell carcinomas and LCNECs but not in pulmonary typical and atypical carcinoids, was the most remarkable chromosomal aberration. Although CGH reports are extremely rare in neuroendocrine tumors of the uterine cervix, specific chromosomal aberrations may be useful in their distinction.

Fragment analysis of chromosomal region 3q25.31–3q28 in HNSCC

Problem: Amplification of chromosome 3q has been implicated to contribute to the genesis of head and neck squamous cell carcinoma (HNSCC). Here we targeted the question of whether early (small) chromosomal changes are detectable in the tumor and macroscopically normal surrounding mucosa by fragment analysis. Therefore we conducted fragment analysis of chromosomal region 3q26 and surrounding regions. Methods: From 20 patients biopsies of the primary tumors and adjacent mucosa and at 1 cm and 2 cm from the tumor border were collected. Eleven microsatellite markers at loci 3q25.31–3q28 were amplified and analyzed by capillary electrophoresis. Results: We confirmed high ratios of gains in the samples as previously observed by Comparative Genomic Hybridization (CGH), which we called in analogy to losses, GOH (gain of heterozygosity or amplification of 1 microsatellite allele). We found GOH in tumor samples as well as high ratios of GOH (up to one third of all informative allelotypes) in adjacent macro- and microscopically normal mucosa. Conclusion: The results indicate that the term “molecular dysplasia” should be introduced for preneoplastic processes that are not detectable by conventional histopathological examination. Significance: As we detected by fragment analysis small aberrations in noncancerous (dysplastic or normal) mucosa adjacent to the tumor, we propose that these loci harbor one or more oncogenes that might be relevant in early carcinogenesis in HNSCC. Our data suggest further molecular studies to identify mechanisms of early carcinogenesis in HNSCC. Support: None reported.