Analysis Of Somatic Alterations Research Articles

A cancer-associated fibroblast gene signature predicts prognosis and therapy response in patients with pancreatic cancer.

Pancreatic cancer is a lethal malignancy with a 5-year survival rate of about 10% in the United States, and it is becoming an increasingly prominent cause of cancer death. Among pancreatic cancer patients, pancreatic ductal adenocarcinoma (PDAC) accounts for more than 90% of all cases and has a very poor prognosis with an average survival of only 1 year in about 18% of all tumor stages. In the past years, there has been an increasing interest in cancer-associated fibroblasts (CAFs) and their roles in PDAC. Recent data reveals that CAFs in PDAC are heterogeneous and various CAF subtypes have been demonstrated to promote tumor development while others hinder cancer proliferation. Furthermore, CAFs and other stromal populations can be potentially used as novel prognostic markers in cancer. In the present study, in order to evaluate the prognostic value of CAFs in PDAC, CAF infiltration rate was evaluated in 4 PDAC datasets of TCGA, GEO, and ArrayExpress databases and differentially expressed genes (DEGs) between CAF-high and CAF-low patients were identified. Subsequently, a CAF-based gene expression signature was developed and studied for its association with overall survival (OS). Additionally, functional enrichment analysis, somatic alteration analysis, and prognostic risk model construction was conducted on the identified DEGs. Finally, oncoPredict algorithm was implemented to assess drug sensitivity prediction between high- and low-risk cohorts. Our results revealed that CAF risk-high patients have a worse survival rate and increased CAF infiltration is a poor prognostic indicator in pancreatic cancer. Functional enrichment analysis also revealed that "extracellular matrix organization" and "vasculature development" were the top enriched pathways among the identified DEGs. We also developed a panel of 12 genes, which in additional to its prognostic value, could predict higher chemotherapy resistance rate. This CAF-based panel can be potentially utilized alone or in conjunction with other clinical parameters to make early predictions and prognosticate responsiveness to treatment in PDAC patients. Indeed, it is necessary to conduct extensive prospective investigations to confirm the clinical utility of these findings.

Abstract PS2-33: Investigating oncogenic signaling pathways in inflammatory metastatic breast cancer (MBC) though circulating tumor DNA (ctDNA) next-generation sequencing (NGS)

Abstract Background: Inflammatory breast cancer (IBC) has a distinctive and aggressive clinical behavior but its underlying biological characteristics have not been fully elucidated. The extended analysis of somatic alterations in The Cancer Genome Atlas (TCGA) highlighted canonical oncogenic pathways that were consistently represented across different tumor subtypes. The aim of this study was to translate such pathway-based characterization to the clinical setting through ctDNA NGS to dissect IBC’s biology and prognosis. Methods: The study retrospectively analyzed 255 metastatic breast cancer (MBC) patients (pts) treated and characterized for ctDNA at Northwestern University (Chicago, IL). ctDNA was analyzed using the Guardant360 NGS assay (Guardant Health). Only non-synonymous alterations were analyzed. Pathway classification was defined based on prior work (Sanchez-Vega F et al, Cell. 2018). Associations among clinical characteristics, pathway classification, and IBC were explored through uni- and multivariate logistic regression; survival was tested though uni- and multivariate Cox regression both for progression-free survival (PFS) and overall survival (OS). Results: Of 255 enrolled pts, 124 (48%) were diagnosed with hormone receptor positive (HR pos) MBC, 75 (30%) with HER2-positive (HER2_pos) MBC and 56 (22%) with triple negative (TNBC) MBC. IBC was diagnosed in 74 pts (30%). Receptor-tyrosine kinase, RTK (130 pts, 51%), p53 (130 pts, 51%), PI3K/Akt (116 pts, 46%), and cell cycle (91 pts, 36%) were the most often altered pathways. The multivariate model highlighted the association of IBC with HER2_pos (OR: 2.19; 95%CI: 1.09 - 4.38; P=0.0276), an increased number of alterations in the p53 pathway (OR: 2.05; 95%CI: 1.12 - 3.75; P=0.0197) and a decreased number of alterations in the RAS pathway (OR: 0.34; 95%CI: 0.14 - 0.80; P=0.0137). Decreased alterations in the ER pathway were borderline significant (OR: 0.48; 95%CI: 0.22 - 1.03; P=0.0584). Only cell cycle alterations had an impact on PFS for IBC (HR: 2.20; 95%CI: 1.18 - 4.08; P=0.0127), while p53 and Wnt had an impact on nonIBC (respectively HR: 2.00; 95%CI: 1.23 - 3.25; P=0.0052 and HR: 3.40; 95%CI: 1.20 - 9.64; P=0.0212). The univariate model showed a significant impact on OS RAF, ER, and cell cycle pathways alterations for IBC, the role of ER and cell cycle pathways alterations was confirmed in the multivariate model (respectively HR: 6.19; 95%CI: 1.63 - 23.48; P=0.0073 and HR: 3.79; 95%CI: 1.04 - 13.75; P=0.0431). The multivariate model showed a prognostic impact only for p53 in the nonIBC subgroup (HR: 2.20; 95%CI: 1.11 - 4.36; P=0.0237) Conclusion: The ctDNA-based oncogenic signaling pathway characterization showed different biological and prognostic features across IBC and nonIBC MBC patients. Alterations of the p53 pathway were more likely to be present in IBC pts, while alterations in the RAS pathway were less represented in this cohort. ER and cell cycle pathways’ alterations impacted the OS of IBC MBC patients. Although preliminary, these results suggest a more comprehensive biological characterization based on ctDNA for treatment selection and clinical decision-making. Main alterations and pathwayGeneNumber of alterations%PathwayTP5318815.58p53PIK3CA14111.68PI3K/AktERBB2725.97RTKESR1705.8ERMYC574.72MycFGFR1463.81RTKEGFR453.73RTKCCNE1383.15Cell cycleMET322.65RTKNF1322.65RAS Citation Format: Lorenzo Gerratana, Andrew A Davis, Marco Mina, Saya L Jacob, Qiang Zhang, Ami N Shah, Paolo D’Amico, Neelima Katam, Firas Wehbe, Elena Vagia, Lisa Flaum, Leonidas Platanias, Amir Behdad, Fabio Puglisi, William J Gradishar, Giovanni Ciriello, Massimo Cristofanilli. Investigating oncogenic signaling pathways in inflammatory metastatic breast cancer (MBC) though circulating tumor DNA (ctDNA) next-generation sequencing (NGS) [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS2-33.

120P Predictive biomarkers for hyper progression in response to immune checkpoint inhibitors therapy: Analysis of somatic alterations by NGS

120P Predictive biomarkers for hyper progression in response to immune checkpoint inhibitors therapy: Analysis of somatic alterations by NGS

Abstract 2512: Pan-cancer characterization of the tumor and immune microenvironment facilitates identification of cancer-specific biological signatures

Abstract Background: A better understanding of the characteristics of cancer across different indications is required to drive the development of personalized treatments, inform therapy decisions, and improve outcomes. Integrating data from the tumor and the immune system can enable the identification of comprehensive biological signatures and composite biomarkers for the improved stratification of responders/progressors. Here, we describe a pan cancer study, including an enhanced whole-exome and transcriptome sequencing approach, across over 500 samples representing 13 tumor types, analyzed at high depth using the ImmunoID NeXT platform. Methods: We sequenced paired tumor-normal samples on the ImmunoID NeXT platform, an enhanced exome/transcriptome-based diagnostic platform that can simultaneously profile the tumor and immune microenvironment from a single FFPE sample, across all of the approximately 20,000 genes. For each sample, we analyzed a broad set of features focused on both the tumor and immune system. From DNA, we profiled small variants, CNAs, MSI status, oncoviruses, HLA LOH, and neoantigens. From RNA, we profiled gene expression, small variants, fusions, TILs, TCR, BCR, and immune signatures. Integrated analyses assessing the impact of each feature, both within and across tumor types, were performed across the cohort. Results: Through immunogenomic analysis we identified striking differences in both tumor and TME profiles across cancer types. In addition to mutation and neoantigen burden, by. we also computed a composite neoantigen score for each sample, which we have shown in a separate melanoma study can be a stronger predictor of response to immunotherapy. The composite neoantigen score integrates neoantigen prediction with mechanisms of tumor escape that can affect neoantigen presentation, providing a more accurate model of the antigen presentation biology. We also looked at the distribution of HLA LOH using our DASH algorithm and found differences in LOH frequency between tumor types. For example, we found HLA LOH to be five timesmore common in lung cancer than breast cancer. Further, we profiled immune gene signatures, including Gejewski and Ribas signatures, highlighting varied immune activation across cancer types. Analysis of somatic alterations in pathways controlling cell growth, PI3K/AKT signaling, apoptosis, and other canonical pathways revealed malignancy-specific alteration frequencies. The varying frequency, and combination of these alterations is indicative of a complex hierarchy of cross-talk between pathways, which operates in a cancer specific manner. Conclusions: We performed a broad integrated analysis of the tumor and immune microenvironment for over 500 samples across 13 different tumor types using the ImmunoID NeXT platform. This comprehensive profiling revealed significant differences between cancer types beyond mutational burden, including neoantigen burden, immune microenvironment differences, and incidence of putative tumor escape mechanisms Citation Format: Sean Michael Boyle, Charles Abbott, Eric Levy, Rachel Marty Pyke, Dattatreya Mellacheruvu, Simo Zhang, Mengyao Tan, Rena McClory, John West, Richard Chen. Pan-cancer characterization of the tumor and immune microenvironment facilitates identification of cancer-specific biological signatures [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2512.

Combined recombinant humanized anti-PD-1 monoclonal antibody (JS001) and nab-paclitaxel/gemcitabine (AG) as the first-line therapy for patients with advanced pancreatic adenocarcinoma: Preliminary results of an open-label, phase Ib/II clinical study.

e16792 Background: To determine evaluate the safety and efficacy of nab-paclitaxe, gemcitabine (AG) and Recombinant Humanized Anti-PD-1 Monoclonal Antibody (JS001) for patients with advanced pancreatic ductal adenocarcinoma (PDAC). Methods: This was a single-arm, open-label, phase 1b/2 clinical trial of nab-paclitaxel plus gemcitabine plus Anti-PD-1 Monoclonal Antibody in which 11 patients with previously untreated advanced PDAC were enrolled from December 2018. Patients were treated with nab-paclitaxel 125 mg/m2 on days 1 and 8 plus gemcitabine 1000 mg/m2 on days 1 and 8 plus fixed dose of JS001 240mg on day 1 of a 21-day cycle until progression or toxicity or receiving maintance therapy. Safety monitoring, RECIST 1.1, and irRECIST assessments were conducted. Results: 11 chemotherapy naïve PDAC patients with a median age of 52 years old were treated. 5 were men and all patients had an ECOG of 0 or 1. Grade 3/4 events were observed in 27.3% of patients (2 patients presented hematological toxicity ), and JS001 was discontinued because of asymptomatic elevated cardiac biomarkers and hypothyroidism as immunotherapy-related toxicities in a female patient, no patient was discontinued AG therapy because of chemotherapy-related adverse events. Of the 11 evaluable patients, the disease control rate (partial response [PR] + stable disease[SD]) was 81.8%. There were 2 patients with PR and 1 patient with complete response respectively, and overall response rate (ORR) was 27.3%. The median progression-free survival (PFS) was 7 months and all patients survive at the last following-up in Jan-2020. All patients were proficient mismatch repair (pMMR) status. Whole-exome sequencing was performed on 7 patients, no immunotherapy-associated gene was detected, and next comprehensive analysis of somatic alterations was ongoing. Conclusions: Combined JS001 and AG can be safely given to chemotherapy naïve PDAC patients. Efficacy appears to be promising over previously reported results for standard AG chemotherapy every 28 days gemcitabine and nab-paclitaxel dosing. Comprehensive data available was ongoing to analysis the potential effect to efficacy of the combination.

Comprehensive Genomic Review of TCGA Head and Neck Squamous Cell Carcinomas (HNSCC).

The aim of this present study was to comprehensively describe somatic DNA alterations and transcriptional alterations in the last extension of the HNSCC subsets in TCGA, encompassing a total of 528 tumours. In order to achieve this goal, transcriptional analysis, functional enrichment assays, survival analysis, somatic copy number alteration analysis and somatic alteration analysis were carried out. A total of 3491 deregulated genes were found in HNSCC patients, and the functional analysis carried out determined that tissue development and cell differentiation were the most relevant signalling pathways in upregulated and downregulated genes, respectively. Somatic copy number alteration analysis showed a “top five” altered HNSCC genes: CDKN2A (deleted in 32.03% of patients), CDKN2B (deleted in 28.34% of patients), PPFIA1 (amplified in 26.02% of patients), FADD (amplified in 25.63% of patients) and ANO1 (amplified in 25.44% of patients). Somatic mutations analysis revealed TP53 mutation in 72% of the tumour samples followed by TTN (39%), FAT1 (23%) and MUC16 (19%). Another interesting result is the mutual exclusivity pattern that was discovered between the TP53 and PIK3CA mutations, and the co-occurrence of CDKN2A with the TP53 and FAT1 alterations. On analysis to relate differential expression genes and somatic copy number alterations, some genes were overexpressed and amplified, for example, FOXL2, but other deleted genes also showed overexpression, such as CDKN2A. Survival analysis revealed that overexpression of some oncogenes, such as EGFR, CDK6 or CDK4 were associated with poorer prognosis tumours. These new findings help us to develop new therapies and programs for the prevention of HNSCC.

1140PD - Predictive biomarkers for hyper-progression (HP) in response to immune checkpoint inhibitors (ICI) – analysis of somatic alterations (SAs)

1140PD - Predictive biomarkers for hyper-progression (HP) in response to immune checkpoint inhibitors (ICI) – analysis of somatic alterations (SAs)

Abstract 137: Development and application of a 62-gene panel for assessment of somatic sequence and structural variants in tumor DNA derived from non-Hodgkin lymphoma patients treated in a phase 1 clinical trial with the EZH2 inhibitor tazemetostat

Abstract Tazemetostat is a small molecule inhibitor of the histone methyltransferase EZH2 and is currently in phase 2 clinical trials in relapsed refractory Non-Hodgkin's Lymphomas (RR-NHL) including diffuse large B cell and follicular lymphoma. We report the development and application of a NHL targeted sequencing panel designed to identify molecular variants, including specific somatic sequence mutations (single base and insertion/deletion), amplifications and translocations in both tumor and cell free circulating tumor DNA (ctDNA). Analysis of somatic alterations in both tissue and ctDNA collected pre-dose enables determination of molecular variants that may correspond with clinical activity, while longitudinal analysis of ctDNA sampled on therapy could help monitor patients for minimal residual disease and emergence of acquired resistance. In collaboration with Personal Genome Diagnostics (PGDx) a panel of 62 NHL specific genes was designed to selectively analyze regions of the genome previously identified as somatically altered in NHL. DNA derived from matched tumor tissue and plasma were screened utilizing this panel using the Illumina HiSeq 2500 platform with 100 bp paired-end reads. Average target coverage for the tissue panel was 1,250-fold while coverage for the ctDNA was approximately 20,000-fold and 3,700-fold for sequence mutations and structural alterations, respectively. Data were analyzed using PGDx's validated cancer genome analysis algorithms that allow for reliable identification of mutations with high sensitivity and specificity. Validation of both the tumor and ctDNA panels was performed using tumor and plasma specimens previously characterized for sequence mutations, amplifications, translocations, and microsatellite instability. For archive tumor, analyses of cell line specimens with an experimental tumor purity of 20-100% using 50-100ng of DNA yielded sensitivity and specificity of 100% for detection of 358 previously characterized sequence mutations and structural variants. Similar ctDNA analyses using fragmented cell line and plasma derived DNA with an experimental tumor purity of 0.10%-25.0% using 9-167ng of DNA yielded a sensitivity of 100% for detection of over 100 genetic variants. Following successful validation of the panel we proceeded to sequence tumor tissue from 11 patients and ctDNA from 16 NHL patients enrolled in the tazemetostat phase 1 clinical trial. Tumor tissue from these patient samples had been previously sequenced using a smaller 39 gene panel. We observed high concordance with 100% of variants detected within the shared gene set of 33 genes between the historic data and our new 62 gene panel. We will report the landscape and concordance of genetic alterations identified through next-generation sequencing analyses of tumor and cell-free DNA. Citation Format: Scott R. Daigle, Samuel Angiuoli, Sian Jones, Scott Ribich, Heike Keilhack, Mark Sausen, Peter T. Ho, Stephen J. Blakemore. Development and application of a 62-gene panel for assessment of somatic sequence and structural variants in tumor DNA derived from non-Hodgkin lymphoma patients treated in a phase 1 clinical trial with the EZH2 inhibitor tazemetostat. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 137.

Molecular Biomarkers in the Personalized Treatment of Colorectal Cancer.

Colorectal cancer (CRC) is a disease in which pathogenesis is influenced by genetic and epigenetic events that occur with tumor initiation and progression. Large variation exists in individual patient prognosis and response to chemotherapy, caused by molecular heterogeneity. Certain biomarkers have been identified that can predict clinical outcome beyond tumor staging, and inform treatment selection. Molecular testing is routinely performed in clinical practice for the selection of patients for targeted biological agents or immunotherapy, and is advocated for prognostic stratification. Estimating prognosis can avoid undertreatment or overtreatment and also guide the intensity of patient follow-up. Classifiers of CRC have been developed that integrate genetic and/or epigenetic features. The mutational status of KRAS and BRAF(V600E) oncogenes combined with analysis of the DNA mismatch repair system with/without the CpG island methylator phenotype (CIMP) has been shown to identify colon cancer subtypes with distinct clinical features and prognoses. Gene expression profiling has also been used to subtype CRCs and can overcome the limitations of single/limited gene testing. A recent effort identified 4 consensus molecular subtypes of biological relevance that were associated with different patient outcomes. Efforts to validate and refine these subtypes to include additional genomic features are ongoing. The focus of this article is to highlight molecular markers that can inform clinical decision-making in patients with CRC.

Abstract 4322: The landscape of mitochondrial DNA mutations in human cancer

Abstract Recent sequencing studies have extensively explored the somatic alterations present in the nuclear genomes of human cancers. Although mitochondria in cancer cells have important roles in energy metabolism and survival, the origins and impact of somatic mutations in mitochondrial DNA (mtDNA) are unclear. Here, we provide a comprehensive analysis of somatic alterations in cancer mitochondria from 1,675 genome sequences across 31 tumor types. We identified 1,907 somatic substitutions which were mainly C:G>T:A and T:A>C:G. These mutations exhibited dramatic replicative strand bias, being almost all C>T and A>G on the mitochondrial heavy strand. This strand-asymmetric mitochondrial mutational signature is different from those found in nuclear cancer genomes but is similar to processes shaping human mtDNA sequence over evolutionary time. The number of mtDNA mutations showed considerable heterogeneity across tumor types, being significantly higher in tumors from gastrointestinal and hepatobiliary systems. With respect to selection, observed missense mutations were nearly neutral and often gradually drifted towards homoplasmy over time. In contrast, mutations resulting in protein truncation showed evidence of negative selection and were more frequently heteroplasmic. Our findings indicate that the endogenous mutational mechanism has far greater impact than any other external mutagens in mitochondria, and is fundamentally linked to mtDNA replication. Citation Format: Young Seok Ju, Ludmil Alexandrov, Moritz Gerstung, Inigo Martincorena, ICGC Breast Cancer Group; ICGC Chronic Myeloid Disorders Group; ICGC Prostate Cancer Group, Mike Stratton, Peter J. Campbell. The landscape of mitochondrial DNA mutations in human cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4322. doi:10.1158/1538-7445.AM2014-4322

Automation Highlights from the Literature

Automation Highlights from the Literature

Abstract A41: Somatic mutations in human cancer cell lines predict sorafenib response

Abstract It is now widely accepted that a spectrum of somatic alterations occur in human cancer and that particular alterations are associated with response or lack of response to anti-cancer therapies. Here, we undertook a systematic correlation analysis of somatic mutations, copy number aberrations and drug response across 300+ human cancer cell lines. We studied the RAF and VEGFR inhibitor, sorafenib, evaluating associations between sorafenib response and somatic mutations in 35 common cancer genes and high-level amplifications and homozygous deletions in more than 100 minimal common regions of gain and loss, which include many known oncogenes and tumor suppressor genes. Notably, we found that NRAS mutations, among other alterations, were significantly associated with sensitivity to sorafenib (odds ratio = 7, p-value = 0.003), whereas activating BRAF mutations were strongly associated with a lack of response (odds ratio = 4.9, p-value = 0.012). Specifically, 64% (7 of 11) of NRAS mutant cell lines were sensitive to sorafenib, while only 20% (34 of 170) of NRAS wild-type lines were sensitive. On the contrary, just 6.7% (2 of 30) of BRAF mutant lines were sensitive to sorafenib. Because NRAS mutations occur frequently in leukemia and no leukemia cell lines were included in the original analysis, we selected 5 NRAS mutant and 5 NRAS wild-type leukemia cell lines for further evaluation. Consistent with our previous observation, the four most sensitive leukemia cell lines were all NRAS mutant, each line exhibiting greater than 50% growth inhibition after 72 hours of exposure to 4μM sorafenib. These results confirm the original association and suggest that across both solid tumors and hematologic malignancies, NRAS mutations may identify a subset of cancers that are likely to respond sorafenib. In addition, this work demonstrates that systematic analysis of somatic alterations and drug response across a large collection of cancer cell lines is capable of discovering novel predictive biomarkers. Citation Information: Clin Cancer Res 2010;16(7 Suppl):A41

Abstract A47: Somatic mutations in human cancer cell lines predict sorafenib response

Abstract It is now widely accepted that a spectrum of somatic alterations occur in human cancer and that particular alterations are associated with response or lack of response to anti-cancer therapies. Here, we undertook a systematic correlation analysis of somatic mutations, copy number aberrations and drug response across 300+ human cancer cell lines. We studied the RAF and VEGFR inhibitor, sorafenib, evaluating associations between sorafenib response and somatic mutations in 35 common cancer genes and high-level amplifications and homozygous deletions in more than 100 minimal common regions of gain and loss, which include many known oncogenes and tumor suppressor genes. Notably, we found that NRAS mutations, among other alterations, were significantly associated with sensitivity to sorafenib (odds ratio = 7, p-value = 0.003), whereas activating BRAF mutations were strongly associated with a lack of response (odds ratio = 4.9, p-value = 0.012). Specifically, 64% (7 of 11) of NRAS mutant cell lines were sensitive to sorafenib, while only 20% (34 of 170) of NRAS wild-type lines were sensitive. On the contrary, just 6.7% (2 of 30) of BRAF mutant lines were sensitive to sorafenib. Because NRAS mutations occur frequently in leukemia and no leukemia cell lines were included in the original analysis, we selected 5 NRAS mutant and 5 NRAS wild-type leukemia cell lines for further evaluation. Consistent with our previous observation, the four most sensitive leukemia cell lines were all NRAS mutant, each line exhibiting greater than 50% growth inhibition after 72 hours of exposure to 4uM sorafenib. These results confirm the original association and suggest that across both solid tumors and hematologic malignancies, NRAS mutations may identify a subset of cancers that are likely to respond sorafenib. In addition, this work demonstrates that systematic analysis of somatic alterations and drug response across a large collection of cancer cell lines is capable of discovering novel predictive biomarkers. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A47.

Clonality and Prognostic Implications of p53 and Epidermal Growth Factor Receptor Somatic Aberrations in Multiple Primary Lung Cancers

For treatment decision and prognostic applications, we evaluated p53/epidermal growth factor receptor (EGFR) somatic aberrations in multiple primary lung cancers to differentiate multifocal tumors from intrapulmonary metastasis. Fifty-eight multiple primary lung cancers of 1,037 patients in a 10-year period were identified to investigate somatic mutations and altered expression of p53 and EGFR for clonality assessment. Genomic DNA was extracted from microdissected cells of paraffin-embedded multiple primary lung cancer tissues. Overexpression and somatic mutations in exons of p53 (exons 5-8) and tyrosine kinase domain of EGFR (exons 18-22) were examined by immunohistochemical staining and DNA sequencing, respectively. High frequency of somatic mutations in p53 (33 of 58, 56.9%) and/or EGFR (44 of 58, 75.9%) resulted in high discrimination rate of tumor clonality (50 of 58, 86.2%) of multiple primary lung cancers. Twenty-two cases (37.9%) were assessed as having the same clonality and 28 cases (48.3%) were determined as having different clonality, which further supported the carcinogenic theory of field cancerization. Notably, the occurrence of lymph node metastasis was more commonly observed in tumors with the same clonality (P = 0.045) and was associated with poor patient 5-year survival rate (P = 0.001). However, no correlation was found between tumor clonality and patient survival (P = 0.630). The EGFR somatic aberrations in 58 multiple primary lung cancers, including vascular invasion associated with EGFR overexpression (P = 0.012) and mutation (P = 0.025), further suggested the potential benefits of target therapy of inoperable multiple primary lung cancers. Our results suggest that analysis of somatic alterations in p53 and EGFR can significantly improve the clonality assessment and impact management of multiple primary lung cancer patients.

Gastric cancers of the microsatellite mutator phenotype display characteristic genetic and clinical features

Background & Aims: Colon cancer of the microsatellite mutator phenotype (MMP) exhibits significant genotype differences from cancer without the MMP. Twenty-nine MMP-positive gastric cancers were analyzed to clarify if these genotype differences were also associated with distinctive clinicopathologic features. Methods: Alterations of p53, β 2-microglobulin ( β2M), hMLH1, and hMSH2 genes were analyzed by using polymerase chain reaction, single-strand conformational polymorphism, sequencing, microallelotyping, hypermethylation assays, and immunostaining. The results were contrasted with mutations in BAX, hMSH3, and hMSH6, genes target for the MMP. Results: Tumors with the MMP had a significantly lower incidence of p53 gene mutations than the other tumors and often contained β2M gene somatic mutations. Many tumors contained concomitant genetic and epigenetic alterations in DNA mismatch repair genes, hMLH1, hMSH2, hMSH3, and hMSH6. Gastric cancer of the MMP was associated with well/moderate differentiation, distal location, and better survival. Conclusions: Analysis of somatic alterations in microsatellite sequences and in cancer genes target for the MMP is useful for the classification of groups of gastric cancers with different prognosis. The results further support the concept that (gastric) cancer of the MMP represents a distinctive oncogenic pathway because the mutated cancer genes are usually different from those found in tumors without the MMP. GASTROENTEROLOGY 1999;116:1348-1357