Distinct Effects of Alcohol Consumption and Smoking on Genetic Alterations in Head and Neck Carcinoma

Background

Tobacco and alcohol consumption are risk factors for head and neck squamous cell carcinoma (HNSCC). Recently, whole-exome sequencing clarified that smoking increased TP53 and other mutations in HNSCC; however, the effects of alcohol consumption on these genetic alterations remain unknown. We explored the association between alcohol consumption and somatic copy-number alterations (SCNAs) across the whole genome in human papillomavirus (HPV)-negative HNSCCs, and compared with the effects of smoking on genetic alterations.

Methods

SCNA and TP53 mutations in tumor samples were examined by high-resolution comparative genomic hybridization microarray 180K and by direct sequencing, respectively, and statistically analyzed for associations with alcohol consumption and smoking during the 20 years preceding diagnosis of HNSCC. Probes with a corrected p-value (=q-value) less than 0.05 and fold change greater than 1.2 or less than -1.2 were considered statistically significant.

Results

A total of 248 patients with HNSCC were enrolled. In the HPV-negative patients (n=221), heavy alcohol consumption was significantly associated with SCNAs of oncogenes/oncosuppressors that were previously reported to occur frequently in HNSCCs: CDKN2A (q=0.005), FHIT (q=0.005), 11q13 region including CCND1, FADD and CTTN (q=0.005), ERBB2 (HER2) (q=0.009), 3q25-qter including CCNL1, TP63, DCUN1D1 and PIK3CA (q=0.014), and CSMD1 (q=0.019). But, TP53 mutations were not affected. In contrast, smoking was associated with increased risk of TP53 mutations, but did not induce any significant SCNAs of oncogenes/oncosuppressors.

Conclusion

These results suggest that both alcohol consumption and smoking had distinct effects on genetic alterations in HNSCCs. Heavy alcohol consumption may trigger previously known and unknown SCNAs, but may not induce TP53 mutation. In contrast, smoking may induce TP53 mutation, but may not trigger any SCNAs.     

Squamousness: Next-generation sequencing reveals shared molecular features across squamous tumor types

In order to gain a better understanding of the underlying biology of squamous cell carcinoma (SCC), we tested the hypothesis that SCC originating from different organs may possess common molecular alterations. SCC samples (N = 361) were examined using clinical-grade targeted next-generation sequencing (NGS). The most frequent SCC tumor types were head and neck, lung, cutaneous, gastrointestinal and gynecologic cancers. The most common gene alterations were TP53 (64.5% of patients), PIK3CA (28.5%), CDKN2A (24.4%), SOX2 (17.7%), and CCND1 (15.8%). By comparing NGS results of our SCC cohort to a non-SCC cohort (N = 277), we found that CDKN2A, SOX2, NOTCH1, TP53, PIK3CA, CCND1, and FBXW7 were significantly more frequently altered, unlike KRAS, which was less frequently altered in SCC specimens (all P < 0.05; multivariable analysis). Therefore, we identified “squamousness” gene signatures (TP53, PIK3CA, CCND1, CDKN2A, SOX2, NOTCH 1, and FBXW7 aberrations, and absence of KRAS alterations) that were significantly more frequent in SCC versus non-SCC histologies. A multivariable co-alteration analysis established 2 SCC subgroups: (i) patients in whom TP53 and cyclin pathway (CDKN2A and CCND1) alterations strongly correlated but in whom PIK3CA aberrations were less frequent; and (ii) patients with PIK3CA alterations in whom TP53 mutations were less frequent (all P ≤ 0 .001, multivariable analysis). In conclusion, we identified a set of 8 genes altered with significantly different frequencies when SCC and non-SCC were compared, suggesting the existence of patterns for “squamousness.” Targeting the PI3K-AKT-mTOR and/or cyclin pathway components in SCC may be warranted.     

Mutational Profiling of Malignant Mesothelioma Revealed Potential Therapeutic Targets in EGFR and NRAS

Pemetrexed and platinum (PP) combination chemotherapy is the current standard first-line therapy for treatment of malignant mesothelioma (MM). However, a useful predictive biomarker for PP therapy is yet to be found. Here, we performed targeted exome sequencing to profile somatic mutations and copy number variations in 12 MM patients treated with PP therapy. We identified 187 somatic mutations in 12 patients (65 synonymous, 102 missense, 2 nonsense, 5 splice site, and 13 small coding insertions/deletions). We identified somatic mutations in 23 genes including BAP1, TP53, NRAS, and EGFR. Interestingly, rare NRAS p.Q61K and EGFR exon 19 deletions were observed in 2 patients. We also found somatic chromosomal copy number deletions in CDKN2A and CDKN2B genes. Genetic alteration related to response after PP therapy was not found. Somatic mutation profiling in MM patients receiving PP therapy revealed genetic alterations in potential therapeutic targets such as NRAS and EGFR. No alterations in genes with potential predictive role for PP therapy were found.     

TP53 mutations,tetraploidy and homologous recombination repair defects in early stage high-grade serous ovarian cancer

To determine early somatic changes in high-grade serous ovarian cancer (HGSOC), we performed whole genome sequencing on a rare collection of 16 low stage HGSOCs. The majority showed extensive structural alterations (one had an ultramutated profile), exhibited high levels of p53 immunoreactivity, and harboured a TP53 mutation, deletion or inactivation. BRCA1 and BRCA2 mutations were observed in two tumors, with nine showing evidence of a homologous recombination (HR) defect. Combined Analysis with The Cancer Genome Atlas (TCGA) indicated that low and late stage HGSOCs have similar mutation and copy number profiles. We also found evidence that deleterious TP53 mutations are the earliest events, followed by deletions or loss of heterozygosity (LOH) of chromosomes carrying TP53, BRCA1 or BRCA2. Inactivation of HR appears to be an early event, as 62.5% of tumours showed a LOH pattern suggestive of HR defects. Three tumours with the highest ploidy had little genome-wide LOH, yet one of these had a homozygous somatic frame-shift BRCA2 mutation, suggesting that some carcinomas begin as tetraploid then descend into diploidy accompanied by genome-wide LOH. Lastly, we found evidence that structural variants (SV) cluster in HGSOC, but are absent in one ultramutated tumor, providing insights into the pathogenesis of low stage HGSOC.     

Integrated genomic and gene expression profiling identifies two major genomic circuits in urothelial carcinoma

Similar to other malignancies, urothelial carcinoma (UC) is characterized by specific recurrent chromosomal aberrations and gene mutations. However, the interconnection between specific genomic alterations, and how patterns of chromosomal alterations adhere to different molecular subgroups of UC, is less clear. We applied tiling resolution array CGH to 146 cases of UC and identified a number of regions harboring recurrent focal genomic amplifications and deletions. Several potential oncogenes were included in the amplified regions, including known oncogenes like E2F3, CCND1, and CCNE1, as well as new candidate genes, such as SETDB1 (1q21), and BCL2L1 (20q11). We next combined genome profiling with global gene expression, gene mutation, and protein expression data and identified two major genomic circuits operating in urothelial carcinoma. The first circuit was characterized by FGFR3 alterations, overexpression of CCND1, and 9q and CDKN2A deletions. The second circuit was defined by E3F3 amplifications and RB1 deletions, as well as gains of 5p, deletions at PTEN and 2q36, 16q, 20q, and elevated CDKN2A levels. TP53/MDM2 alterations were common for advanced tumors within the two circuits. Our data also suggest a possible RAS/RAF circuit. The tumors with worst prognosis showed a gene expression profile that indicated a keratinized phenotype. Taken together, our integrative approach revealed at least two separate networks of genomic alterations linked to the molecular diversity seen in UC, and that these circuits may reflect distinct pathways of tumor development.     

Genomic sequencing of key genes in mouse pancreatic cancer cells

Pancreatic cancer is a multiple genetic disorder with many mutations identified during the progression. Two mouse pancreatic cancer cell lines were established which showed different phenotype in vivo: a non-metastatic cell line, Panc02, and a highly metastatic cell line, Panc02-H7, a derivative of Panc02. In order to investigate whether the genetic mutations of key genes in pancreatic cancer such as KRAS, TP53 (p53), CDKN2A (p16), SMAD4, ZIP4, and PDX-1 contribute to the phenotypic difference of these two mouse pancreatic cancer cells, we sequenced the exonic regions of these key genes in both cell lines and in the normal syngeneic mouse pancreas and compared them with the reference mouse genome sequence. The exons of KRAS, SMAD4, CDKN2A (p16), TP53 (p53), ZIP4, and PDX-1 genes were amplified and the genotype of these genes was determined by Sanger sequencing. The sequences were analyzed with Sequencher software. A mutation in SMAD4 was identified in both cell lines. This homozygote G to T mutation in the first position of codon 174 (GAA) generated a stop codon resulting in the translation of a truncated protein. Further functional analysis indicates that different TGF-β/SMAD signaling pathways were involved in those two mouse cell lines, which may explain the phonotypic difference between the two cells. A single nucleotide polymorphism (SNP) in KRAS gene (TAT to TAC at codon 32) was also identified in the normal pancreas DNA of the syngenic mouse and in both derived tumoral Panc02 and Panc02-H7 cells. No mutation or SNP was found in CDKN2A (p16), TP53 (p53), ZIP4, and PDX-1 genes in these two cell lines. The absence of mutations in genes such as KRAS, TP53, and CDKN2A, which are considered as key genes in the development of human pancreatic cancer suggests that SMAD4 might play a central and decisive role in mouse pancreatic cancer. These results also suggest that other mechanisms are involved in the substantial phenotypic difference between these two mouse pancreatic cancer cell lines. Further studies are warranted to elucidate the molecular pathways that lead to the aggressive metastatic potential of Panc02-H7.     

Association of TP53 mutation, p53 overexpression, and p53 codon 72 polymorphism with susceptibility to apoptosis in adult patients with diffuse astrocytomas

Clarification of TP53 alterations is important to understand the mechanisms underlying the development of diffuse astrocytomas. It has been suggested that the alleles of TP53 at codon 72 differ in their ability to induce apoptosis in human cancers. The aim of this study was to analyze the possible association of TP53 mutation, p53 overexpression, and p53 codon 72 polymorphism with susceptibility to apoptosis in adult Brazilian patients with diffuse astrocytomas. We analyzed 56 surgical specimens of diffuse astrocytomas for alterations of TP53, using polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) direct sequencing. p53 and cleaved caspase 3 protein expression were assessed by immunohistochemistry. We found TP53 mutations in 19.6% (11 out of 56) of tumors tested, with the lowest mutation rate found in the cases of glioblastomas (8.8%) (p = 0.03). Only 16.1% of tumors tested showed cleaved caspase 3-positive staining, demonstrating that apoptosis is very inhibited in these tumors. All tumors having TP53 mutation and p53 accumulation had no expression of cleaved caspase 3. Additionally, no association was observed in tumors having proline and arginine alleles and expression of cleaved caspase 3. We concluded that clarification of the TP53 alterations allows a better understanding of the mechanisms involved in the progression of diffuse astrocytomas, and the allele status at codon 72 was not associated with apoptosis in these tumors.     

Molecular Characterization of an Intact p53 Pathway Subtype in High-Grade Serous Ovarian Cancer

High-grade serous ovarian cancer (HGSOC) is the most aggressive histological type of epithelial ovarian cancer, which is characterized by a high frequency of somatic TP53 mutations. We performed exome analyses of tumors and matched normal tissues of 34 Japanese patients with HGSOC and observed a substantial number of patients without TP53 mutation (24%, 8/34). Combined with the results of copy number variation analyses, we subdivided the 34 patients with HGSOC into subtypes designated ST1 and ST2. ST1 showed intact p53 pathway and was characterized by fewer somatic mutations and copy number alterations. In contrast, the p53 pathway was impaired in ST2, which is characterized by abundant somatic mutations and copy number alterations. Gene expression profiles combined with analyses using the Gene Ontology resource indicate the involvement of specific biological processes (mitosis and DNA helicase) that are relevant to genomic stability and cancer etiology. In particular we demonstrate the presence of a novel subtype of patients with HGSOC that is characterized by an intact p53 pathway, with limited genomic alterations and specific gene expression profiles.     

Extremely high Tp53 mutation load in esophageal squamous cell carcinoma in Golestan Province, Iran

Background

Golestan Province in northeastern Iran has one of the highest incidences of esophageal squamous cell carcinoma (ESCC) in the world with rates over 50 per 100,000 person-years in both sexes. We have analyzed TP53 mutation patterns in tumors from this high-risk geographic area in search of clues to the mutagenic processes involved in causing ESCC.

Methodology/Principal Findings

Biopsies of 119 confirmed ESCC tumor tissue from subjects enrolled in a case-control study conducted in Golestan Province were analyzed by direct sequencing of TP53 exons 2 through 11. Immunohistochemical staining for p53 was carried out using two monoclonal antibodies, DO7 and 1801. A total of 120 TP53 mutations were detected in 107/119 cases (89.9%), including 11 patients with double or triple mutations. The mutation pattern was heterogeneous with infrequent mutations at common TP53 “hotspots” but frequent transversions potentially attributable to environmental carcinogens forming bulky DNA adducts, including 40% at bases known as site of mutagenesis by polycyclic aromatic hydrocarbons (PAHs). Mutations showed different patterns according to the reported temperature of tea consumption, but no variation was observed in relation to ethnicity, tobacco or opium use, and alcoholic beverage consumption or urban versus rural residence.

Conclusion/Significance

ESCC tumors in people from Golestan Province show the highest rate of TP53 mutations ever reported in any cancer anywhere. The heterogeneous mutation pattern is highly suggestive of a causative role for multiple environmental carcinogens, including PAHs. The temperature and composition of tea may also influence mutagenesis.     

TP53 mutations in colorectal cancer from Tunisia: relationships with site of tumor origin,microsatellite instability and KRAS mutations

Loss of TP53 function through gene mutation is a critical event in the development and progression of colorectal cancer (CRC). Here we examined 51 primary CRC tumors from Tunisia for mutations in TP53 exons 4–9 using PCR-direct sequencing. TP53 status and mutation site/type were than correlated with nuclear protein accumulation, familial and clinicopathologic variables and data on KRAS mutations and microsatellite instability (MSI-H). The TP53 mutation analysis was possible in the tumor of 47 patients and a deleterious somatic mutation has been detected in 59.6 % of the patients (28/47) including 20 (71.4 %) missense mutations, 7 nonsense mutations (25 %) and 1 (3.6 %) frameshift mutation. 89.3 % (25/28) of the detected mutations were in exons 5–8, whereas 10.7 % (3/28) were in exon 4. Among the 27 non frameshift mutations, 89 % (24/27) were transitions and 11 % (3/27) were transversions. 64.3 % (18/27) of the altered amino acids corresponded to arginine. 74 % (20/27) were G>C to A>T transitions, and more than half (14/27) occur at hotspots codons with CpG sites. TP53 mutations correlated closely with TP53 accumulation (p = 0.0090) and inversely with MSI phenotype (p = 0.0658). A KRAS somatic mutation was identified in 25 % (7/28) of the TP53 mutated tumors. All these mutations were G>A transitions in codon 12 and all the tumors with combined alterations but one were distally located and MSS. In conclusion, frequency and types of TP53 mutations and correlations with TP53 protein accumulation, and MSI were as reported for non-Tunisian patients. However, no significant associations have been detected between TP53 mutations and clinicopathological data in Tunisian patients as previously reported.     

Detection of somatic mutations by high-resolution DNA melting (HRM) analysis in multiple cancers

Identification of somatic mutations in cancer is a major goal for understanding and monitoring the events related to cancer initiation and progression. High resolution melting (HRM) curve analysis represents a fast, post-PCR high-throughput method for scanning somatic sequence alterations in target genes. The aim of this study was to assess the sensitivity and specificity of HRM analysis for tumor mutation screening in a range of tumor samples, which included 216 frozen pediatric small rounded blue-cell tumors as well as 180 paraffin-embedded tumors from breast, endometrial and ovarian cancers (60 of each). HRM analysis was performed in exons of the following candidate genes known to harbor established commonly observed mutations: PIK3CA, ERBB2, KRAS, TP53, EGFR, BRAF, GATA3, and FGFR3. Bi-directional sequencing analysis was used to determine the accuracy of the HRM analysis. For the 39 mutations observed in frozen samples, the sensitivity and specificity of HRM analysis were 97% and 87%, respectively. There were 67 mutation/variants in the paraffin-embedded samples, and the sensitivity and specificity for the HRM analysis were 88% and 80%, respectively. Paraffin-embedded samples require higher quantity of purified DNA for high performance. In summary, HRM analysis is a promising moderate-throughput screening test for mutations among known candidate genomic regions. Although the overall accuracy appears to be better in frozen specimens, somatic alterations were detected in DNA extracted from paraffin-embedded samples.     

Co-localization of mutant p53 and amyloid-like protein aggregates in breast tumors

P53 is one of the most important tumor suppressor proteins in human cancers. Mutations in the TP53 gene are common features of malignant tumors and normally correlate to a more aggressive disease. In breast cancer, these gene alterations are present in approximately 20% of cases and are characteristically of missense type. In the present work we describe TP53 mutations in breast cancer biopsies and investigate whether wild and mutant p53 participate in protein aggregates formation in these breast cancer cases. We analyzed 88 biopsies from patients residing in the metropolitan area of Rio de Janeiro, and performed TP53 mutation screening using direct sequencing of exons 5-10. Seventeen mutations were detected, 12 of them were of missense type, 2 nonsenses, 2 deletions and 1 insertion. The presence of TP53 mutation was highly statistically associated to tumor aggressiveness of IDC cases, indicated here by Elston Grade III (p<0.0001). Paraffin embedded breast cancer tissues were analyzed for the presence of p53 aggregates through immunofluorescence co-localization assay, using anti-aggregate primary antibody A11, and anti-p53. Our results show that mutant p53 co-localizes with amyloid-like protein aggregates, depending on mutation type, suggesting that mutant p53 may form aggregates in breast cancer cells, in vivo.     

KRAS mutations in colorectal cancer from Tunisia: relationships with clinicopathologic variables and data on TP53 mutations and microsatellite instability

Mutations in KRAS gene are among the critical transforming alterations occurring during CRC tumorigenesis. Here we screened 51 primary CRC tumors from Tunisia for mutations in KRAS (codons 12 and 13) using PCR-direct sequencing. Our aim was to analyze tumor mutation frequencies and spectra in Tunisian patients with CRC. KRAS status and mutation site/type were than correlated with familial and clinicopathologic variables and data on TP53 mutations and nuclear protein accumulation and microsatellite instability (MSI). A KRAS somatic mutation has been detected in the CRC tumor of 31.5 % (16/51) of the patients. 81.2 % had a single mutation at codon 12 and 23 % had a single mutation at codon 13. The most common single mutation (50 %) was a G>A transition in codon 12 (c.35G>A; p.Gly12Asp). 81.25 % of the KRAS mutations were transitions and 23 % were transversions. All the mutations in codon 13 were a c.38G>A transition, whereas both G>A transitions and G>T and G>C transversions were found in codon 12. The mutation spectrum was different between MSS and MSI-H tumors and more varied mutations have been detected in MSS tumors. Some amino acid changes were detected only in MSS tumors, i.e. p.Gly12Ser, p.Gly12Cys and p.Gly12Ala. Whereas, the KRAS mutation p.Gly13Asp have been detected only in MSI-H. 43.75 % of the patients harboured combined mutations in KRAS and TP53 genes and the tumor of 71.42 % of them showed TP53 overexpression. In conclusion, the frequency and types of KRAS mutations were as reported for non-Tunisian patients. However, no significant associations have been detected between KRAS mutations and clinicopathologic variables and MSI in Tunisian patients as previously reported.     

The Genomic Landscape of TP53 and p53 Annotated High Grade Ovarian Serous Carcinomas from a Defined Founder Population Associated with Patient Outcome

High-grade ovarian serous carcinomas (HGSC) are characterized by TP53 mutations and non-random patterns of chromosomal anomalies, where the nature of the TP53 mutation may correlate with clinical outcome. However, the frequency of common somatic genomic events occurring in HGSCs from demographically defined populations has not been explored. Whole genome SNP array, and TP53 mutation, gene and protein expression analyses were assessed in 87 confirmed HGSC samples with clinical correlates from French Canadians, a population exhibiting strong founder effects, and results were compared with independent reports describing similar analyses from unselected populations. TP53 mutations were identified in 91% of HGSCs. Anomalies observed in more than 50% of TP53 mutation-positive HGSCs involved gains of 3q, 8q and 20q, and losses of 4q, 5q, 6q, 8p, 13q, 16q, 17p, 17q, 22q and Xp. Nearly 400 regions of non-overlapping amplification or deletion were identified, where 178 amplifications and 98 deletions involved known genes. The subgroup expressing mutant p53 protein exhibited significantly prolonged overall and disease-free survival as compared with the p53 protein null subgroup. Interestingly, a comparative analysis of genomic landscapes revealed a significant enrichment of gains involving 1q, 8q, and 12p intervals in the subgroup expressing mutant p53 protein as compared with the p53 protein null subgroup. Although the findings show that the frequency of TP53 mutations and the genomic landscapes observed in French Canadian samples were similar to those reported for samples from unselected populations, there were differences in the magnitude of global gains/losses of specific chromosomal arms and in the spectrum of amplifications and deletions involving focal regions in individual samples. The findings from our comparative genomic analyses also support the notion that there may be biological differences between HGSCs that could be related to the nature of the TP53 mutation.     

Precision medicine for patients with gastro-oesophageal cancer: A subset analysis of the ProfiLER program

BackgroundChemotherapy, anti-HER2 and PD-1 antibodies are standard treatments but only a minority of patients derive long-term benefit from these agents.MethodsIn this report we describe the mutational landscape and outcome of patients with gastroesophageal cancers enroled in the ProfiLER program.ResultsAdenocarcinoma (n = 86, 59%), signet-cell (n = 37, 25%) and squamous-cell (n = 21, 14%) were the dominant histology amongst 147 patients. Genomic analyses could be performed for 114 (78%) patients. The most common genomic alterations involved ERBB2 (15%), KRAS (12%), CCND1 (7%), FGFR1–3 (8%), EGFR (5%) and MET (3%), TP53 (51%) and CDKN2A/B (10%). ERBB2, MET and FGFR alterations were found exclusively in the adenocarcinoma and signet-cell subtypes, while CCND1 amplification, TP53 mutations and CDKN2A/B loss were found in both adenocarcinoma and squamous-cell subtypes. Nine patients (8%) received therapy matched to their genomic alteration, with 5 of them achieving disease control. In an exploratory analysis, patients with stage IV disease at diagnosis who had an actionable alteration had longer overall survival compared to those without.ConclusionGenomic profiling for patients with advanced gastroesophageal cancers allows the identification of actionable alterations in large proportion of patients. Increased accessibility to molecularly matched therapy may improve survival in this disease.