Genome-wide copy number analysis by Array CGH for the analysis of clonality of thoracic tumors
ABSTRACT: Multiple synchronous and metachronous lung tumors are frequently encountered in patients with lung cancer. For treatment purposes it is important to determine, whether or not tumors are clonally related. In other words, whether multiple tumors in a patient are either metastases or multiple primaries. Previous reports show considerable discordance between histopathological and molecular comparison of tumor pairs. The purpose of this study is to compare genome-wide copy number analysis to the classical histological and clinicopathological routine for clonality analysis in a prospective cohort of patients with synchronous or metachronous tumors, of which at least one site occurred in the thorax. Overall design: Two-colour experiment experiment,tumor vs reference. Multiple cases with 2 or more tumors per case.
INSTRUMENT(S): Agilent-014950 Human Genome CGH Microarray 4x44K (Probe Name version)
Project description:Multiple tumors in patients are frequently diagnosed, either synchronous or metachronous. The distinction between a second primary and a metastasis is important for treatment. Chromosomal DNA copy number aberrations (CNA) patterns are highly unique to specific tumors. The aim of this study was to assess genome-wide CNA-patterns as method to identify clonally related tumors in a prospective cohort of patients with synchronous or metachronous tumors, with at least one intrapulmonary tumor. In total, 139 tumor pairs from 90 patients were examined: 35 synchronous and 104 metachronous pairs. Results of CNA were compared to histological type, clinicopathological methods (Martini-Melamed-classification (MM) and ACCP-2013-criteria), and, if available, EGFR- and KRAS-mutation analysis. CNA-results were clonal in 74 pairs (53%), non-clonal in 33 pairs (24%), and inconclusive in 32 pairs (23%). Histological similarity was found in 130 pairs (94%). Concordance between histology and conclusive CNA-results was 69% (74 of 107 pairs: 72 clonal and two non-clonal). In 31 of 103 pairs with similar histology, genetics revealed non-clonality. In two out of four pairs with non-matching histology, genetics revealed clonality. The subgroups of synchronous and metachronous pairs showed similar outcome for the comparison of histological versus CNA-results. MM-classification and ACCP-2013-criteria, applicable on 34 pairs, and CNA-results were concordant in 50% and 62% respectively. Concordance between mutation matching and conclusive CNA-results was 89% (8 of 9 pairs: six clonal and two non-clonal). Interestingly, in one patient both tumors had the same KRAS mutation, but the CNA result was non-clonal. In conclusion, although some concordance between histological comparison and CNA profiling is present, arguments exist to prefer extensive molecular testing to determine whether a second tumor is a metastasis or a second primary.
Project description:In cases of multiple non-small cell lung cancer, clinicians must decide whether patients have independent tumors or metastases and tailor treatment accordingly. Decisions are currently made using the Martini and Melamed criteria, which are mostly based on tumor location and histologic type. New genomic tools could improve the ability to assess tumor clonality.We obtained fresh-frozen tumors specimens from patients who underwent surgery on at least two occasions for presumptively independent NSCLC. We did array comparative genomic hybridization (aCGH), mutational profiling of select genes, and detailed clinicopathologic review.We analyzed a total of 42 tumors from 20 patients (6 patients with synchronous tumors, 14 patients with metachronous tumors, 24 potential tumor pair comparisons); 22 tumor pairs were evaluable by aCGH. Surprisingly, classification based on genomic profiling contradicted the clinicopathologic diagnosis in four (18%) of the comparisons, identifying independent primaries in one case diagnosed as metastasis and metastases in three cases diagnosed as independent primaries. Matching somatic point mutations were observed in these latter three cases. Another four tumor pairings were assigned an "equivocal" result based on aCGH; however, matching somatic point mutations were also found in these tumor pairs. None of the tumor pairs deemed independent primaries by aCGH harbored matching mutations.Genomic analysis can help distinguish clonal tumors from independent primaries. The development of rapid, inexpensive, and reliable molecular tools may allow for refinement of clinicopathologic criteria currently used in this setting.
Project description:Purpose:To investigate differences in clinical characteristics and mutational patterns between synchronous and metachronous colorectal liver metastases (CLMs). Patients and methods:From June 2008 to December 2014, patients with RAS wild-type CLMs treated at Zhongshan Hospital, Fudan University were included. DNA extracted from formalin-fixed paraffin-embedded tissue of primary tumors was sequenced with next-generation sequencing for single-nucleotide polymorphism of 96 genes according to custom panel. Mutations were compared between synchronous and metachronous liver metastases and correlated with clinical characteristics. Results:A total of 161 patients were included: 93 patients with synchronous CLM and 68 patients with metachronous CLM. Patients with metachronous CLM were obviously elder. For pathology of primary tumors, synchronous CLMs were larger in size, poorly differentiated, and more frequently local advanced and lymph node positive. For evaluation of liver metastases, synchronous CLM had more and larger metastatic lesions. The median number of mutations in synchronous CLMs was significantly higher than in metachronous group (22 vs. 18, p<0.001). EGFR rs2227983 is the most prevalent mutation in both groups and only a part of prevalent mutations is shared in both groups. Prevalent mutations were correlated with many clinical characteristics. EGFR rs2227983, RBMXL3 rs12399211, and PTCH1 rs357564 were prognostic for latency of metachronous CLM. Conclusion:Clinically, synchronous CLMs, compared with metachronous CLMs, were younger and showed heavier tumor burden for both primaries and liver metastases. Genetically, we identified different mutational patterns between synchronous and metachronous CLMs and several correlations between mutations and clinical characteristics. Further researches were needed to confirm these potential key mutations of CLMs.
Project description:<h4>Introduction</h4>Discordance in hormone receptor status has been observed between two breast tumors of the same patients; however, the degree of heterogeneity is debatable with regard to whether it reflects true biological difference or the limited accuracy of receptor assays.<h4>Methods</h4>A Bayesian misclassification correction method was applied to data on hormone receptor status of two primary breast cancers from the Surveillance, Epidemiology, and End Results database between 1990 and 2010 and to data on primary breast cancer and paired recurrent/metastatic disease assembled from a meta-analysis of the literature published between 1979 and 2014.<h4>Results</h4>The sensitivity and specificity of the estrogen receptor (ER) assay were estimated to be 0.971 and 0.920, respectively. After correcting for misclassification, the discordance in ER between two primary breast cancers was estimated to be 1.2% for synchronous ipsilateral pairs, 5.0% for synchronous contralateral pairs, 14.6% for metachronous ipsilateral pairs, and 25.0% for metachronous contralateral pairs. Technical misclassification accounted for 53%-83% of the ER discordance between synchronous primary cancers and 11%-25% of the ER discordance between metachronous cancers. The corrected discordance in ER between primary tumors and recurrent or metastatic lesions was 12.4%, and there were more positive-to-negative changes (10.1%) than negative-to-positive changes (2.3%). Similar patterns were observed for progesterone receptor (PR), although the overall discordance in PR was higher.<h4>Conclusion</h4>A considerable proportion of discordance in hormone receptor status can be attributed to misclassification in receptor assessment, although the accuracy of receptor assays was excellent. Biopsy of recurrent tumors for receptor retesting should be conducted after considering feasibility, cost, and previous ER/PR status.
Project description:The pathologic classification of nonsmall cell lung cancer (NSCLC) is evolving. Lung adenocarcinoma is morphologically heterogeneous, with mixtures of acinar, papillary, bronchioloalveolar, and solid patterns in more than 80% of cases. In case of synchronous or metachronous multiple NSCLC, the distinction of intrapulmonary metastases from independent primary tumors is of great clinical importance as it influences staging and potentially the therapeutic strategy. Here we took advantage of a cohort of 20 patients with 42 multiple NSCLC tumors (24 potential pair comparisons) that were annotated molecularly using genomic and mutational profiling to evaluate the value of comprehensive histologic assessment in this setting. Using the Martini-Melamed criteria, paired tumors were characterized as multiple primary NSCLCs in 21 cases and as intrapulmonary metastases in 3 cases. Genomic and mutational data led to a diagnosis of multiple primaries in 14 cases and of metastases in 8 cases; 2 cases could not be assessed. This molecular characterization contradicted the Martini-Melamed diagnosis in 7 (32%) of the 22 assessable comparisons. Adenocarcinoma was found in 32 (76%) of the 42 tumors. After review in a blinded fashion, semiquantitative comprehensive histologic assessment of paired tumors was different in 16 and similar in 8 paired tumors. We found that comparing adenocarcinomas is a complex issue that requires assessment not only of percentages of the histologic subtypes, but also the recording of additional histologic details such as cytologic features, patterns of stroma, necrosis, discrete nodularity versus miliary growth and variants such as clear cell, signet ring, mucinous, and fetal patterns. We also found that paired squamous cell carcinomas could be compared based on histologic subtyping in addition to cytologic and stromal characteristics. Considering histologically different tumors as multiple primaries, and similar tumors as metastases, comprehensive histologic subtyping was consistent with the molecular characterization in 20 (91%) of the 22 pairs comparisons. In summary, based on a well characterized cohort with detailed clinical, pathologic and molecular data, we found comprehensive histologic assessment is a powerful tool that seems to be a promising way to determine whether multiple lung adenocarcinomas or squamous cell carcinomas are metastatic or multiple primaries. This has great clinical implications for staging and therapeutic management of lung cancer patients with multiple tumors. Given its high correlation with molecular characterization of such tumors, it may provide a much cheaper and faster method to address this problem.
Project description:Multiple tumours from the same patient were analysed for copy number alterations to assess tumour clonality. Seventy-four tumours corresponding to 37 patients were stratified into four groups based on the anatomic location of the multiple breast cancers (ipsilateral or bilateral) and time interval between the diagnoses (synchronous or metachronous). Ipsilateral was defined as tumours occurring in the same breast while bilateral was defined as the occurrence of tumours in both breasts. Metachronicity was defined as a time interval greater than six months between the diagnoses of the first and second tumours, while synchronicity specified that the two tumours occurred concurrently (BM: bilateral-metachronous; BS: bilateral-synchronous; IM: ipsilateral-metachronous; IS: ipsilateral-synchronous). Overall design: Genomic DNA was isolated from fresh-frozen tissue samples
Project description:PURPOSE:To evaluate the rate of discordance of epidermal growth factor receptor (EGFR) mutation between primary lung tumor and paired distant metastases in non-small-cell lung cancer (NSCLC). METHODS:We performed a meta-analysis of 17 studies (518 cases) assessing discordance rates of EGFR mutation in primary tumors and paired distant metastases. We performed subgroup analyses based on EGFR mutation status in primary tumor (mutant or wildtype), site of distant metastasis (bone, central nervous system (CNS) or lung/ pleural), methods of testing (direct sequencing or allele-specific testing) and timing of metastasis (synchronous or metachronous). RESULTS:The overall discordance rate in EGFR mutation was low at 10.36% (95% CI = 4.23% to 18.79%) and varied widely between studies (I2 = 83.18%). The EGFR discordance rate was statistically significantly higher in bone metastases (45.49%, 95% CI = 14.13 to 79.02) than CNS (17.26%, 95% CI = 7.64 to 29.74; P = 0.002) and lung/ pleural metastases (8.17%, 95% CI = 3.35 to 14.85; P < 0.001). Subgroup analyses did not demonstrate any significant effect modification on the discordance rates by the EGFR mutation status in primary lung tumor, methods of testing and timing of metastasis. CONCLUSION:The overall discordance rate in EGFR mutation between primary lung tumor and paired distant metastases in NSCLC is low, although higher discordance rates were observed in bone metastases compared with CNS and lung/pleural metastases. Future studies assessing the impact of EGFR mutation discordance on treatment outcomes are required.
Project description:Multiple tumours from the same patient were analysed for DNA methylation to assess tumour clonality. Seventy-four tumours corresponding to 37 patients were stratified into four groups based on the anatomic location of the multiple breast cancers (ipsilateral or bilateral) and time interval between the diagnoses (synchronous or metachronous). Ipsilateral was defined as tumours occurring in the same breast while bilateral was defined as the occurrence of tumours in both breasts. Metachronicity was defined as a time interval greater than six months between the diagnoses of the first and second tumours, while synchronicity specified that the two tumours occurred concurrently (BM: bilateral-metachronous; BS: bilateral-synchronous; IM: ipsilateral-metachronous; IS: ipsilateral-synchronous). A subset of 16 samples was randomly selected to represent each clinical group with four samples corresponding to two patients per group and analysed for DNA methylation using Illumina Infinium Human MethylationEPIC BeadChips. Overall design: Genomic DNA was isolated from fresh-frozen tissue samples
Project description:Metachronous (MBC) and synchronous bilateral breast tumors (SBC) are mostly distinct primaries, whereas paired primaries and their local recurrences (LRC) share a common origin. Intra-pair gene expression variability in MBC, SBC, and LRC derives from time/tumor microenvironment-related and tumor genetic background-related factors and pairs represents an ideal model for trying to dissect tumor-related from microenvironment-related variability. Pairs of tumors derived from women with SBC (n = 18), MBC (n = 11), and LRC (n = 10) undergoing local-regional treatment were profiled for gene expression; similarity between pairs was measured using an intraclass correlation coefficient (ICC) computed for each gene and compared using analysis of variance (ANOVA). When considering biologically unselected genes, the highest correlations were found for primaries and paired LRC, and the lowest for MBC pairs. By instead limiting the analysis to the breast cancer intrinsic genes, correlations between primaries and paired LRC were enhanced, while lower similarities were observed for SBC and MBC. Focusing on stromal-related genes, the ICC values decreased for MBC and were significantly different from SBC. These findings indicate that it is possible to dissect intra-pair gene expression variability into components that are associated with genetic origin or with time and microenvironment by using specific gene subsets.
Project description:In patients presenting with synchronous or metachronous multiple lung cancer (MLC), it is important to distinguish between multiple primary lung cancer (MP) and intrapulmonary metastasis (IM). The present study was aimed at investigating the mutational profiles of synchronous/metachronous MLC and to compare the classification of paired tumors by multiplex gene mutation analysis with the histopathological evaluation. We carried out targeted sequencing of 20 lung cancer-related oncogenes using next-generation sequencing (NGS) in 82 tumors from 37 MLC patients who underwent surgical resection at our department. The patients were diagnosed as MP or IM cases based on the Martini and Melamed criteria, histopathological and gene mutational evaluations. Matching mutations between paired tumors was observed in 20 (54%) patients, who were diagnosed as IM cases by mutational evaluation. Patients who could not be clearly diagnosed by histopathological evaluation were classified as equivocal cases. Among the histopathological IM cases (n = 7), six (86%) were confirmed as IM cases also by mutational evaluation, and most of the paired tumors of these cases (n = 5) harbored multiple matching mutations. Among the histopathological MP cases (n = 17), mutational evaluation yielded a discordant diagnosis in eight (47%) cases. Of these, the paired tumors of four cases harbored multiple matching mutations, suggesting that the mutational diagnosis might be more suitable in these patients. Our findings suggest that multiplex mutational analysis could be a useful complementary tool for distinguishing between MP and IM in addition to histopathological evaluation.