Khedoudja Nafa

Y-Chromosomal Diversity in Europe Is Clinal and Influenced Primarily by Geography, Rather than by Language

Clinal patterns of autosomal genetic diversity within Europe have been interpreted in previous studies in terms of a Neolithic demic diffusion model for the spread of agriculture; in contrast, studies using mtDNA have traced many founding lineages to the Paleolithic and have not shown strongly clinal variation. We have used 11 human Y-chromosomal biallelic polymorphisms, defining 10 haplogroups, to analyze a sample of 3,616 Y chromosomes belonging to 47 European and circum-European populations. Patterns of geographic differentiation are highly nonrandom, and, when they are assessed using spatial autocorrelation analysis, they show significant clines for five of six haplogroups analyzed. Clines for two haplogroups, representing 45% of the chromosomes, are continentwide and consistent with the demic diffusion hypothesis. Clines for three other haplogroups each have different foci and are more regionally restricted and are likely to reflect distinct population movements, including one from north of the Black Sea. Principal-components analysis suggests that populations are related primarily on the basis of geography, rather than on the basis of linguistic affinity. This is confirmed in Mantel tests, which show a strong and highly significant partial correlation between genetics and geography but a low, nonsignificant partial correlation between genetics and language. Genetic-barrier analysis also indicates the primacy of geography in the shaping of patterns of variation. These patterns retain a strong signal of expansion from the Near East but also suggest that the demographic history of Europe has been complex and influenced by other major population movements, as well as by linguistic and geographic heterogeneities and the effects of drift.

Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT): A Hybridization Capture-Based Next-Generation Sequencing Clinical Assay for Solid Tumor Molecular Oncology

The identification of specific genetic alterations as key oncogenic drivers and the development of targeted therapies are together transforming clinical oncology and creating a pressing need for increased breadth and throughput of clinical genotyping. Next-generation sequencing assays allow the efficient and unbiased detection of clinically actionable mutations. To enable precision oncology in patients with solid tumors, we developed Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT), a hybridization capture-based next-generation sequencing assay for targeted deep sequencing of all exons and selected introns of 341 key cancer genes in formalin-fixed, paraffin-embedded tumors. Barcoded libraries from patient-matched tumor and normal samples were captured, sequenced, and subjected to a custom analysis pipeline to identify somatic mutations. Sensitivity, specificity, reproducibility of MSK-IMPACT were assessed through extensive analytical validation. We tested 284 tumor samples with previously known point mutations and insertions/deletions in 47 exons of 19 cancer genes. All known variants were accurately detected, and there was high reproducibility of inter- and intrarun replicates. The detection limit for low-frequency variants was approximately 2% for hotspot mutations and 5% for nonhotspot mutations. Copy number alterations and structural rearrangements were also reliably detected. MSK-IMPACT profiles oncogenic DNA alterations in clinical solid tumor samples with high accuracy and sensitivity. Paired analysis of tumors and patient-matched normal samples enables unambiguous detection of somatic mutations to guide treatment decisions.

Rebiopsy of Lung Cancer Patients with Acquired Resistance to EGFR Inhibitors and Enhanced Detection of the T790M Mutation Using a Locked Nucleic Acid-Based Assay

Background: The epidermal growth factor receptor (EGFR) mutation T790M is reported in approximately 50% of lung cancers with acquired resistance to EGFR inhibitors and is a potential prognostic and predictive biomarker. Its assessment can be challenging due to limited tissue availability and underdetection at low mutant allele levels. Here, we sought to determine the feasibility of tumor rebiopsy and to more accurately assess the prevalence of the T790M using a highly sensitive locked nucleic acid (LNA) PCR/sequencing assay. MET amplification was also analyzed. Methods: Patients with acquired resistance were rebiopsied and samples were studied for sensitizing EGFR mutations. Positive cases were evaluated for T790M using standard PCR-based methods and a subset were re-evaluated with an LNA-PCR/sequencing method with an analytical sensitivity of approximately 0.1%. MET amplification was assessed by FISH. Results: Of 121 patients undergoing tissue sampling, 104 (86%) were successfully analyzed for sensitizing EGFR mutations. Most failures were related to low tumor content. All patients (61/61) with matched pretreatment and resistance specimens showed concordance for the original sensitizing EGFR mutation. Standard T790M mutation analysis on 99 patients detected 51(51%) mutants. Retesting of 30 negative patients by the LNA-based method detected 11 additional mutants for an estimated prevalence of 68%. MET was amplified in 11% of cases (4/37). Conclusions: The re-biopsy of lung cancer patients with acquired resistance is feasible and provides sufficient material for mutation analysis in most patients. Using high sensitivity methods, the T790M is detected in up to 68% of these patients. Clin Cancer Res; 17(5); 1169–80. ©2011 AACR.

Frequency and Distinctive Spectrum of KRAS Mutations in Never Smokers with Lung Adenocarcinoma

Purpose:KRAS mutations are found in ∼25% of lung adenocarcinomas in Western countries and, as a group, have been strongly associated with cigarette smoking. These mutations are predictive of poor prognosis in resected disease as well as resistance to treatment with erlotinib or gefitinib. Experimental Design: We determined the frequency and type of KRAS codon 12 and 13 mutations and characterized their association with cigarette smoking history in patients with lung adenocarcinomas. Results:KRAS mutational analysis was done on 482 lung adenocarcinomas, 81 (17%) of which were obtained from patients who had never smoked cigarettes. KRAS mutations were found in 15% (12 of 81; 95% confidence intervals, 8-24%) of tumors from never smokers. Similarly, 22% (69 of 316; 95% confidence intervals, 17-27%) of tumors from former smokers, and 25% (21 of 85; 95% confidence intervals, 16-35%) of tumors from current smokers had KRAS mutations. The frequency of KRAS mutation was not associated with age, gender, or smoking history. The number of pack years of cigarette smoking did not predict an increased likelihood of KRAS mutations. Never smokers were significantly more likely than former or current smokers to have a transition mutation (G→A) rather than the transversion mutations known to be smoking-related (G→T or G→C; P < 0.0001). Conclusions: Based on our data, KRAS mutations are not rare among never smokers with lung adenocarcinoma and such patients have a distinct KRAS mutation profile. The etiologic and biological heterogeneity of KRAS mutant lung adenocarcinomas is worthy of further study.

EWS-CREB1: A Recurrent Variant Fusion in Clear Cell Sarcoma—Association with Gastrointestinal Location and Absence of Melanocytic Differentiation

Purpose: Clear cell sarcoma (CCS) usually arises in the lower extremities of young adults and is typically associated with a t(12;22) translocation resulting in the fusion of EWS (EWSR1) with ATF1, a gene encoding a member of the cyclic AMP–responsive element binding protein (CREB) family of transcription factors. CCS arising in the gastrointestinal tract is rare and its pathologic and molecular features are not well defined. Experimental Design: We report a novel variant fusion of EWS to CREB1, a gene at 2q32 encoding another CREB family member highly related to ATF1, detected in three women with gastrointestinal CCS. All three cases contained an identical EWS-CREB1 fusion transcript that was shown by reverse transcription-PCR. In two of the cases tested, EWS gene rearrangement was also confirmed by fluorescence in situ hybridization and the EWS-CREB1 genomic junction fragments were isolated by long-range DNA PCR. Results: Morphologically, all three tumors lacked melanin pigmentation. By immunohistochemistry, there was a strong and diffuse S100 protein reactivity, whereas all melanocytic markers were negative. Ultrastructurally, two of the cases lacked melanosomes. The melanocyte-specific transcript of MITF was absent in two cases, and only weakly expressed in the third case. The Affymetrix gene expression data available in one case showed lower expression of the melanocytic genes MITF, TYR, and TYRP1, compared with four EWS-ATF1-positive CCSs of non-gastrointestinal origin. Conclusions:EWS-CREB1 may define a novel subset of CCS that occurs preferentially in the gastrointestinal tract and shows little or no melanocytic differentiation. Thus, evidence of melanocytic lineage or differentiation is not a necessary feature of sarcomas with gene fusions involving CREB family members.

Mutational landscape of metastatic cancer revealed from prospective clinical sequencing of 10,000 patients

Tumor molecular profiling is a fundamental component of precision oncology, enabling the identification of genomic alterations in genes and pathways that can be targeted therapeutically. The existence of recurrent targetable alterations across distinct histologically defined tumor types, coupled with an expanding portfolio of molecularly targeted therapies, demands flexible and comprehensive approaches to profile clinically relevant genes across the full spectrum of cancers. We established a large-scale, prospective clinical sequencing initiative using a comprehensive assay, MSK-IMPACT, through which we have compiled tumor and matched normal sequence data from a unique cohort of more than 10,000 patients with advanced cancer and available pathological and clinical annotations. Using these data, we identified clinically relevant somatic mutations, novel noncoding alterations, and mutational signatures that were shared by common and rare tumor types. Patients were enrolled on genomically matched clinical trials at a rate of 11%. To enable discovery of novel biomarkers and deeper investigation into rare alterations and tumor types, all results are publicly accessible.

Prevalence, clinicopathologic associations and molecular spectrum of ERBB2 (HER2) tyrosine kinase mutations in lung adenocarcinomas

Purpose: Activating mutations in the tyrosine kinase domain of HER2 (ERBB2) have been described in a subset of lung adenocarcinomas (ADCs) and are mutually exclusive with EGFR and KRAS mutations. The prevalence, clinicopathologic characteristics, prognostic implications, and molecular heterogeneity of HER2-mutated lung ADCs are not well established in U.S. patients. Experimental Design: Lung ADC samples (N = 1,478) were first screened for mutations in EGFR (exons 19 and 21) and KRAS (exon 2), and negative cases were then assessed for HER2 mutations (exons 19–20) using a sizing assay and mass spectrometry. Testing for additional recurrent point mutations in EGFR, KRAS, BRAF, NRAS, PIK3CA, MEK1, and AKT was conducted by mass spectrometry. ALK rearrangements and HER2 amplification were assessed by FISH. Results: We identified 25 cases with HER2 mutations, representing 6% of EGFR/KRAS/ALK-negative specimens. Small insertions in exon 20 accounted for 96% (24/25) of the cases. Compared with insertions in EGFR exon 20, there was less variability, with 83% (20/24) being a 12 bp insertion causing duplication of amino acids YVMA at codon 775. Morphologically, 92% (23/25) were moderately or poorly differentiated ADC. HER2 mutation was not associated with concurrent HER2 amplification in 11 cases tested for both. HER2 mutations were more frequent among never-smokers (P < 0.0001) but there were no associations with sex, race, or stage. Conclusions: HER2 mutations identify a distinct subset of lung ADCs. Given the high prevalence of lung cancer worldwide and the availability of standard and investigational therapies targeting HER2, routine clinical genotyping of lung ADC should include HER2. Clin Cancer Res; 18(18); 4910–8. ©2012 AACR.

EWSR1-CREB1 is the predominant gene fusion in angiomatoid fibrous histiocytoma

The molecular hallmark of angiomatoid fibrous histiocytoma (AFH) is not well defined, with only six cases with specific gene fusions reported to date, consisting of either FUS‐ATF1 or EWSR1‐ATF1. To address this, we investigated the presence of FUS‐ATF1, EWSR1‐ATF1, and the highly related EWSR1‐CREB1 fusion in a group of nine AFHs. All cases were subjected to RT‐PCR for EWSR1‐ATF1 and EWSR1‐CREB1. FISH for EWSR1 and FUS rearrangements was performed in most cases. Transcriptional profiling was performed in three tumors and their gene expression was compared to five clear cell sarcomas expressing either the EWSR1‐ATF1 or EWSR1‐CREB1 fusion. By RT‐PCR, eight out of nine tumors showed the presence of the EWSR1‐CREB1 fusion, while one had an EWSR1‐ATF1 transcript. FISH showed evidence of EWSR1 rearrangement in seven out of eight cases. Karyotypic analysis performed in one tumor showed a t(2;22)(q33;q12). High transcript levels were noted for TFE3 in AFH tumors, while overexpression of genes involved in melanogenesis, such as MITF, GP100, and MET was noted in somatic clear cell sarcomas. We report for the first time the presence of EWSR1‐CREB1 in AFH, which now appears to be the most frequent gene fusion in this tumor. EWSR1‐CREB1 is a novel translocation recently described in clear cell sarcoma of the GI tract. EWSR1‐ATF1, identified in some AFH cases, is the most common genetic abnormality in soft tissue clear cell sarcoma. Thus, identical fusions involving ATF1 and CREB1 are found in two distinct sarcomas, which may be able to transform two different types of mesenchymal precursor cells, unlike most other sarcoma gene fusions.

Value of histopathology in predicting microsatellite instability in hereditary nonpolyposis colorectal cancer and sporadic colorectal cancer

Identification of colorectal carcinomas with high levels of DNA microsatellite instability (MSI-H) is important because of the suggested prognostic and therapeutic significance associated with MSI. The role of histology in identifying MSI-H colorectal carcinomas has been suggested by some studies but not confirmed by others. Furthermore, previous studies assumed that hereditary nonpolyposis colorectal cancer (HNPCC)-associated MSI-H tumors and sporadic MSI-H tumors have similar histology. This assumption, however, has been challenged by more recent studies. In this report, we first analyzed the value of various histologic features in predicting MSI-H in a series of 218 colorectal carcinomas containing mixed HNPCC and sporadic cases [77 tumors (35%) were MSI-H by polymerase chain reaction (PCR) method]. Then, we evaluated the various histologic features comparatively in two groups extracted from the 218 cases. Group A was composed of 84 tumors from 82 patients obtained based on a strong family history (HNPCC/HNPCC-like group) (male to female ratio, 42:40; age range, 23–80 years, median, 53.5 years). Thirty-one of the 84 tumors (41.7%) were MSI-H by PCR, and all 31cases were HNPCC by Amsterdam criteria. Group B was composed of 109 patients with no family history of colorectal cancer or HNPCC-associated cancer, obtained from surgical clinics (sporadic group) (male to female ratio, 65:69; age range, 31–84 years, median, 65 years). Thirty-five of the 109 tumors (32.1%) were MSI-H by PCR. Our results showed that, overall, poor tumor differentiation, medullary type, mucinous type, signet-ring cell component, histologic heterogeneity, and increased tumor-infiltrating lymphocytes (TILs) were features more commonly seen in MSI-H tumors than in non-MSI-H tumors. Comparative analyses showed that the overall TIL count was significantly higher in HNPCC/HNPCC-like group, and mucinous type appeared to be more frequent in HNPCC MSI-H tumors than in sporadic MSI-H tumors. However, there was no significant difference in the odds ratio for predicting MSI-H status for any of the analyzed histologic features between HNPCC/HNPCC-like group and sporadic group, indicating that differences between HNPCC and sporadic MSI-H tumors did not significantly impact on the informative value of histology in predicting MSI in the two different clinical settings. TIL counts followed by histologic heterogeneity provided the greatest sensitivity and specificity in predicting MSI status in both HNPCC/HNPCC-like and sporadic cases. Using a stepwise logistic regression model, a formula was generated that could be used to calculate the probability of a colorectal carcinoma being MSI-H based on morphologic features.

EGFR Exon 20 Insertion Mutations in Lung Adenocarcinomas: Prevalence, Molecular Heterogeneity, and Clinicopathologic Characteristics

In contrast to other primary epidermal growth factor receptor (EGFR) mutations in lung adenocarcinomas, insertions in exon 20 of EGFR have been generally associated with resistance to EGFR-tyrosine kinase inhibitors. Their molecular spectrum, clinicopathologic characteristics, and prevalence are not well established. Tumors harboring EGFR exon 20 insertions were identified through an algorithmic screen of 1,500 lung adenocarcinomas. Cases were first tested for common mutations in EGFR (exons 19 and 21) and KRAS (exon 2) and, if negative, further analyzed for EGFR exon 20 insertions. All samples underwent extended genotyping for other driver mutations in EGFR, KRAS, BRAF, ERBB2/HER2, NRAS, PIK3CA, MEK1, and AKT by mass spectrometry; a subset was evaluated for ALK rearrangements. We identified 33 EGFR exon 20 insertion cases [2.2%, 95% confidence interval (CI), 1.6–3.1], all mutually exclusive with mutations in the other genes tested (except PIK3CA). They were more common among never-smokers (P < 0.0001). There was no association with age, sex, race, or stage. Morphologically, tumors were similar to those with common EGFR mutations but with frequent solid histology. Insertions were highly variable in position and size, ranging from 3 to 12 bp, resulting in 13 different insertions, which, by molecular modeling, are predicted to have potentially different effects on erlotinib binding. EGFR exon 20 insertion testing identifies a distinct subset of lung adenocarcinomas, accounting for at least 9% of all EGFR-mutated cases, representing the third most common type of EGFR mutation after exon 19 deletions and L858R. Insertions are structurally heterogeneous with potential implications for response to EGFR inhibitors. Mol Cancer Ther; 12(2); 220–9. ©2012 AACR.