A Targeted Next‐Generation Sequencing Assay Detects a High Frequency of Therapeutically Targetable Alterations in Primary and Metastatic Breast Cancers: Implications for Clinical Practice

Background.

The aim of this study was to assess the frequency of potentially actionable genomic alterations in breast cancer that could be targeted with approved agents or investigational drugs in clinical trials using a next-generation sequencing-based genomic profiling assay performed in a Clinical Laboratory Improvement Amendments-certified and College of American Pathologists-accredited commercial laboratory.

Methods.

Fifty-one breast cancers were analyzed, including primary tumor biopsies of 33 stage I–II and 18 stage IV cancers (13 soft tissue, 3 liver, and 2 bone metastases). We assessed 3,230 exons in 182 cancer-related genes and 37 introns in 14 genes often rearranged in cancer for base substitutions, indels, copy number alterations, and gene fusions. The average median sequencing depth was 1,154×.

Results.

We observed 158 genomic alterations in 55 genes in 48 of 51 (94%) tumors (mean 3.1, range 0–9). The average number of potentially therapeutically relevant alterations was similar in primary (1.6, range 0–4) and in heavily pretreated metastatic cancers (2.0, range 0–4) (p = .24). The most common actionable alterations were in PIK3CA (n = 9, phosphatidylinositol 3-kinase [PI3K]/mammalian target of rapamycin [mTOR] inhibitors), NF1 (n = 7, PI3K/mTOR/mitogen-activated protein kinase inhibitors), v-akt murine thymoma viral oncogene homolog 1-3 (n = 7, PI3K/mTOR/AKT inhibitors), BRCA1/2 (n = 6, poly[ADP-ribose] polymerase inhibitors), and CCND1,2 and CCNE (n = 8)/cycline dependent kinase (CDK)6 (n = 1) (CDK4/6 inhibitors), KIT (n = 1, imatinib/sunitinib), ALK (n = 1, crizotinib), FGFR1,2 (n = 5, fibroblast growth factor receptor inhibitors), and EGFR (n = 2, epidermal growth factor receptor inhibitors). Our sequencing assay also correctly identified all six cases with HER2 (ERBB2) amplification by fluorescence in situ hybridization when tumor content was adequate. In addition, two known activating HER2 mutations were identified, both in unamplified cases.

Conclusion.

Overall, 84% of cancers harbored at least one genomic alteration linked to potential treatment options. Systematic evaluation of the predictive value of these genomic alterations is critically important for further progress in this field.     

Profiling Cancer Gene Mutations in Clinical Formalin‐Fixed,Paraffin‐Embedded Colorectal Tumor Specimens Using Targeted Next‐Generation Sequencing

Purpose.

The success of precision oncology relies on accurate and sensitive molecular profiling. The Ion AmpliSeq Cancer Panel, a targeted enrichment method for next-generation sequencing (NGS) using the Ion Torrent platform, provides a fast, easy, and cost-effective sequencing workflow for detecting genomic “hotspot” regions that are frequently mutated in human cancer genes. Most recently, the U.K. has launched the AmpliSeq sequencing test in its National Health Service. This study aimed to evaluate the clinical application of the AmpliSeq methodology.

Methods.

We used 10 ng of genomic DNA from formalin-fixed, paraffin-embedded human colorectal cancer (CRC) tumor specimens to sequence 46 cancer genes using the AmpliSeq platform. In a validation study, we developed an orthogonal NGS-based resequencing approach (SimpliSeq) to assess the AmpliSeq variant calls.

Results.

Validated mutational analyses revealed that AmpliSeq was effective in profiling gene mutations, and that the method correctly pinpointed “true-positive” gene mutations with variant frequency >5% and demonstrated high-level molecular heterogeneity in CRC. However, AmpliSeq enrichment and NGS also produced several recurrent “false-positive” calls in clinically druggable oncogenes such as PIK3CA.

Conclusion.

AmpliSeq provided highly sensitive and quantitative mutation detection for most of the genes on its cancer panel using limited DNA quantities from formalin-fixed, paraffin-embedded samples. For those genes with recurrent “false-positive” variant calls, caution should be used in data interpretation, and orthogonal verification of mutations is recommended for clinical decision making.     

Enabling a Genetically Informed Approach to Cancer Medicine: A Retrospective Evaluation of the Impact of Comprehensive Tumor Profiling Using a Targeted Next‐Generation Sequencing Panel

Background.

Oncogenic genetic alterations “drive” neoplastic cell proliferation. Small molecule inhibitors and antibodies are being developed that target an increasing number of these altered gene products. Next-generation sequencing (NGS) is a powerful tool to identify tumor-specific genetic changes. To determine the clinical impact of extensive genetic analysis, we reviewed our experience using a targeted NGS platform (FoundationOne) in advanced cancer patients.

Patients and Methods.

We retrospectively assessed demographics, NGS results, and therapies received for patients undergoing targeted NGS (exonic sequencing of 236 genes and selective intronic sequencing from 19 genes) between April 2012 and August 2013. Coprimary endpoints were the percentage of patients with targeted therapy options uncovered by mutational profiling and the percentage who received genotype-directed therapy.

Results.

Samples from 103 patients were tested, most frequently breast carcinoma (26%), head and neck cancers (23%), and melanoma (10%). Most patients (83%) were found to harbor potentially actionable genetic alterations, involving cell-cycle regulation (44%), phosphatidylinositol 3-kinase-AKT (31%), and mitogen-activated protein kinase (19%) pathways. With median follow-up of 4.1 months, 21% received genotype-directed treatments, most in clinical trials (61%), leading to significant benefit in several cases. The most common reasons for not receiving genotype-directed therapy were selection of standard therapy (35%) and clinical deterioration (13%).

Conclusion.

Mutational profiling using a targeted NGS panel identified potentially actionable alterations in a majority of advanced cancer patients. The assay identified additional therapeutic options and facilitated clinical trial enrollment. As time progresses, NGS results will be used to guide therapy in an increasing proportion of patients.     

Fluorescence In Situ Hybridization,Immunohistochemistry, and Next‐Generation Sequencing for Detection of EML4‐ALK Rearrangement in Lung Cancer

Background.

The U.S. Food and Drug Administration-approved method for detecting EML4-ALK rearrangement is fluorescence in situ hybridization (FISH); however, data supporting the use of immunohistochemistry (IHC) for that purpose are accumulating. Previous studies that compared FISH and IHC considered FISH the gold standard, but none compared data with the results of next-generation sequencing (NGS) analysis.

Materials and Methods.

We studied FISH and IHC (D5F3 antibody) systematically for EML4-ALK rearrangement in 51 lung adenocarcinoma patients, followed by NGS in case of discordance.

Results.

Of 51 patients, 4 were positive with FISH (7.8%), and 8 were positive with IHC (15.7%). Three were positive with both. NGS confirmed that four of the five patients who were positive with IHC and negative with FISH were positive for ALK. Two were treated by crizotinib, with progression-free survival of 18 and 6 months. Considering NGS as the most accurate test, the sensitivity and specificity were 42.9% and 97.7%, respectively, for FISH and 100% and 97.7%, respectively, for IHC.

Conclusion.

The FISH-based method of detecting EML4-ALK rearrangement in lung cancer may miss a significant number of patients who could benefit from targeted ALK therapy. Screening for EML4-ALK rearrangement by IHC should be strongly considered, and NGS is recommended in borderline cases. Two patients who were negative with FISH and positive with IHC were treated with crizotinib and responded to therapy.     

Identification of a Novel EHBP1-MET Fusion in an Intrahepatic Cholangiocarcinoma Responding to Crizotinib

As an aggressive tumor, intrahepatic cholangiocarcinoma (ICC) originates in the epithelium of the bile duct and has a poor prognosis. The therapeutic options for ICC are challenging and limited because of poor response to chemotherapy and the lack of targeted therapy. Here we report on a 41‐year‐old female patient with ICC with EHBP1‐MET fusion and multiple intrahepatic metastases responding to crizotinib. Next‐generation sequencing–based tumor mutation profiling was performed on the tumor biopsy and circulating tumor DNA from plasma. A novel EHBP1‐MET fusion was identified and confirmed by Sanger sequencing. Immunohistochemistry of biopsy sample also revealed c‐MET positivity. Subsequently, the patient started treatment with MET inhibitor crizotinib. Magnetic resonance imaging scan demonstrated a partial response for 8 months. To the best of our knowledge, this is the first clinical case report of a patient with MET‐rearranged ICC successfully treated with crizotinib. This case suggests that crizotinib may be a promising treatment option for patients with ICC with MET fusion, warranting further clinical investigation.Key Points

• To the authors'' knowledge, this is the first reported case of EHBP1‐MET fusion.
• This is also the first clinical case report of clinical benefit from crizotinib treatment in an intrahepatic cholangiocarcinoma (ICC) with MET fusion.
• MET fusion is rare in ICC, and inhibition of MET could be a viable option for ICC that warrants further clinical investigation.

     

Implementation of a Molecular Tumor Board: The Impact on Treatment Decisions for 35 Patients Evaluated at Dartmouth‐Hitchcock Medical Center

Background.

Although genetic profiling of tumors is a potentially powerful tool to predict drug sensitivity and resistance, its routine use has been limited because clinicians are often unfamiliar with interpretation and incorporation of the information into practice. We established a Molecular Tumor Board (MTB) to interpret individual patients’ tumor genetic profiles and provide treatment recommendations.

Patients and Methods.

DNA from tumor specimens was sequenced in a Clinical Laboratory Improvement Amendments-certified laboratory to identify coding mutations in a 50-gene panel (n = 34) or a 255-gene panel (n = 1). Cases were evaluated by a multidisciplinary MTB that included pathologists, oncologists, hematologists, basic scientists, and genetic counselors.

Results.

During the first year, 35 cases were evaluated by the MTB, with 32 presented for recommendations on targeted therapies, and 3 referred for potential germline mutations. In 56.3% of cases, MTB recommended treatment with a targeted agent based on evaluation of tumor genetic profile and treatment history. Four patients (12.5%) were subsequently treated with a MTB-recommended targeted therapy; 3 of the 4 patients remain on therapy, 2 of whom experienced clinical benefit lasting >10 months.

Conclusion.

For the majority of cases evaluated, the MTB was able to provide treatment recommendations based on targetable genetic alterations. The most common reasons that MTB-recommended therapy was not administered stemmed from patient preferences and genetic profiling at either very early or very late stages of disease; lack of drug access was rarely encountered. Increasing awareness of molecular profiling and targeted therapies by both clinicians and patients will improve acceptance and adherence to treatments that could significantly improve outcomes.

Implications for Practice:

Case evaluation by a multidisciplinary Molecular Tumor Board (MTB) is critical to benefit from individualized genetic data and maximize clinical impact. MTB recommendations shaped treatment options for the majority of cases evaluated. In the few patients treated with MTB-recommended therapy, disease outcomes were positive and support genetically informed treatment.     

Molecular predictive markers in tumors of the gastrointestinal tract

Gastrointestinal malignancies are among the leading causes of cancer-related deaths worldwide. Like all human malignancies they are characterized by accumulation of mutations which lead to inactivation of tumor suppressor genes or activation of oncogenes. Advances in Molecular Biology techniques have allowed for more accurate analysis of tumors’ genetic profiling using new breakthrough technologies such as next generation sequencing (NGS), leading to the development of targeted therapeutical approaches based upon biomarker-selection. During the last 10 years tremendous advances in the development of targeted therapies for patients with advanced cancer have been made, thus various targeted agents, associated with predictive biomarkers, have been developed or are in development for the treatment of patients with gastrointestinal cancer patients. This review summarizes the advances in the field of molecular biomarkers in tumors of the gastrointestinal tract, with focus on the available NGS platforms that enable comprehensive tumor molecular profile analysis.     

“去重”在肺癌NGS数据分析中的重要作用

目的 探讨“不去重”和“去重”两种方法分析后各NGS相关指标间的差异,研究“去重”在靶向捕获NGS数据分析中的重要作用。方法 通过对58例肺癌患者的DNA样本进行靶向286基因探针杂交方法建库并进行NGS检测,每例NGS检测数据分别进行“去重”和“不去重”两种方法的生物信息学分析,比较两种方法分析得到的NGS相关指标Mapped Reads（可比对上reads比例）、On Target（靶向区域reads比例）、Mean Depth（平均测序深度）以及Uniformity（均一性）等数据之间的差异。结果 “不去重”和“去重”两种方法分析得到平均Mapped Reads、On Target、Mean Depth和Uniformity值,各组指标间差异均有统计学意义（P<0.001）。“去重”方法分析时,Mapped Reads、On Target和MeanDepth 3个指标均呈现出血浆样本与其他3种类型样本间（FFPE、穿刺和手术）差异有统计学意义,在Uniformity指标上则呈现出4种类型样本间差异均无统计学意义。而“不去重”方法分析后结果正好相反。结论 去除PCR扩增所致重复序列的“去重”步骤在NGS数据分析中具有重要的作用,能够改善结果均一性,真实反映所测样本的DNA模板数量及等位基因频率（AF值,allele frequency）。“去重”后结果更有助于临床决策。     

EGFR基因突变对靶向治疗非小细胞肺癌患者的疗效影响及生存分析

目的 探讨表皮生长因子受体(EGFR)基因不同染色体上外显子突变对靶向治疗非小细胞肺癌患者的疗效影响及生存分析.方法 选取76例EGFR基因敏感突变非小细胞肺癌患者,根据EGFR基因检测结果分为四组:A组,EGFR基因18号染色体上外显子突变型组(18例);B组,EGFR基因19号染色体上外显子突变型组(20例);C组,EGFR基因20号染色体上外显子突变型组(19例);D组,EGFR基因21号染色体上外显子突变型组(19例).同时选取同期住院治疗的20例EGFR基因野生型(EGFR基因未突变)非小细胞肺癌患者作为E组(对照组).各组患者均给予吉非替尼(250 mg/d)治疗.结果 与E组比较,A、B、D组的总有效率和疾病控制率均升高,差异均有统计学意义(P均<0.05);与E组比较,C组的总有效率和疾病控制率均降低,差异均有统计学意义(P均<0.05).与E组比较,A、B、D组PFS、OS和QOL均升高,差异均有统计学意义(P均<0.05);与E组比较,C组的PFS、OS和QOL均降低,差异均有统计学意义(P均<0.05).与E组比较,A、B、D组总不良反应发生率均降低,差异均有统计学意义(P均<0.05);与E组比较,C组的总不良反应发生率升高,差异有统计学意义(P<0.05).结论 采用靶向药物吉非替尼治疗EGFR基因18、19和21号染色体上外显子突变的非小细胞肺癌患者效果较好,同时延长患者生存时间,降低不良反应;相反,采用靶向药物吉非替尼治疗EGFR基因20号染色体上外显子突变的非小细胞肺癌患者因出现耐药而效果不佳.     

Picoliter‐Droplet Digital Polymerase Chain Reaction‐Based Analysis of Cell‐Free Plasma DNA to Assess EGFR Mutations in Lung Adenocarcinoma That Confer Resistance to Tyrosine‐Kinase Inhibitors

Purpose.

The objective of this study was to evaluate the utility of analyzing cell-free plasma DNA (cfDNA) by picoliter-droplet digital polymerase chain reaction (ddPCR) to detect EGFR mutations that confer resistance to tyrosine-kinase inhibitors (TKIs) used for treatment of lung adenocarcinoma (LADC).

Experimental design.

Thirty-five LADC patients who received epidermal growth factor receptor (EGFR)-TKI therapy, including ten who received tumor rebiopsy after development of resistance, were subjected to picoliter-ddPCR-cfDNA analysis to determine the fraction of cfDNA with TKI-sensitive (L858R and inflame exon 19 deletions) and -resistant (i.e., T790M) mutations, as well as their concordance with mutation status in rebiopsied tumor tissues.

Results.

cfDNA samples from 15 (94%) of 16 patients who acquired resistance were positive for TKI-sensitive mutations. Also, 7 (44%) were positive for the T790M mutation, with fractions of T790M (+) cfDNA ranging from 7.4% to 97%. T790M positivity in cfDNA was consistent in eight of ten patients for whom rebiopsied tumor tissues were analyzed, whereas the remaining cases were negative in cfDNA and positive in rebiopsied tumors. Prior to EGFR-TKI therapy, cfDNAs from 9 (38%) and 0 of 24 patients were positive for TKI-sensitive and T790M mutations, respectively. Next-generation sequencing of cfDNA from one patient who exhibited innate resistance to TKI despite a high fraction of TKI-sensitive mutations and the absence of the T790M mutation in his cfDNA revealed the presence of the L747P mutation, a known driver of TKI resistance.

Conclusion.

Picoliter-ddPCR examination of cfDNA, supported by next-generation sequencing analysis, enables noninvasive assessment of EGFR mutations that confer resistance to TKIs.

Implications for Practice:

Noninvasive monitoring of the predominance of tumors harboring the secondary T790M mutation in the activating mutation in EGFR gene is necessary for precise and effective treatment of lung adenocarcinoma. Because cells harboring the T790M mutation are resistant to epidermal growth factor receptor-tyrosine-kinase inhibitors (TKIs), the predominance of tumor cells harboring the T790M mutations influences the choice of whether to use conventional or next-generation TKIs. Digital polymerase chain reaction-based examination of cfDNA is a promising method; however, its feasibility, including its consistency with examination of rebiopsied tumor tissue, has not been fully proven. Here, picoliter-droplet digital polymerase chain reaction technology is presented as a candidate method for testing cfDNA and assessing the predominance of T790M-mutant tumors.     

Comprehensive Genomic Profiling of Advanced Esophageal Squamous Cell Carcinomas and Esophageal Adenocarcinomas Reveals Similarities and Differences

Background.

Esophageal squamous cell carcinomas (ESCCs) and esophageal adenocarcinomas (EACs) account for >95% of esophageal malignancies and represent a major global health burden. ESCC is the dominant histology globally but represents a minority of U.S. cases, with EAC accounting for the majority of U.S. cases. The patient outcomes for advanced ESCC and EAC are poor, and new therapeutic options are needed. Using a sensitive sequencing assay, we compared the genomic profiles of ESCC and EAC with attention to identification of therapeutically relevant genomic alterations.

Methods.

Next-generation sequencing-based comprehensive genomic profiling was performed on hybridization-captured, adaptor ligation-based libraries to a median coverage depth of >650× for all coding exons of 315 cancer-related genes plus selected introns from 28 genes frequently rearranged in cancer. Results from a single sample were evaluated for all classes of genomic alterations (GAs) including point mutations, short insertions and deletions, gene amplifications, homozygous deletions, and fusions/rearrangements. Clinically relevant genomic alterations (CRGAs) were defined as alterations linked to approved drugs and those under evaluation in mechanism-driven clinical trials.

Results.

There were no significant differences by sex for either tumor type, and the median age for all patients was 63 years. All ESCCs and EACs were at an advanced stage at the time of sequencing. All 71 ESCCs and 231 EACs featured GAs on profiling, with 522 GAs in ESCC (7.4 per sample) and 1,303 GAs in EAC (5.6 per sample). The frequency of clinically relevant GAs in ESCC was 94% (2.6 per sample) and 93% in EAC (2.7 per sample). CRGAs occurring more frequently in EAC included KRAS (23% EAC vs. 6% ESCC) and ERBB2 (23% EAC vs. 3% ESCC). ESCC samples were enriched for CRGA in PIK3CA (24% ESCC vs. 10% EAC), PTEN (11% ESCC vs. 4% EAC), and NOTCH1 (17% ESCC vs. 3% EAC). Other GAs that differed significantly between histologic tumor types included SMAD4 (14% EAC vs. 1% ESCC), RB1 (14% ESCC vs. 2% EAC), SOX2 (18% ESCC vs. 1% EAC), and NFE2L2 (24% ESCC vs. 1% EAC).

Conclusion.

ESCC and EAC share similarly high frequencies of overall and clinically relevant genomic alterations; however, the profiles of genomic alterations in the two diseases differ widely, with KRAS and ERBB2 far more frequently altered in EAC compared with ESCC and with mammalian target of rapamycin (MTOR) pathway genes (PIK3CA and PTEN) and NOTCH1 more frequently altered in ESCC compared with EAC. Comprehensive genomic profiling highlights the promise of identifying clinically relevant genomic alterations in both ESCC and EAC and suggests new avenues for molecularly directed therapies in esophageal cancer.

Implications for Practice:

Both esophageal squamous cell carcinoma and esophageal adenocarcinoma are aggressive cancers with poor patient response to conventional chemotherapy and radiation treatment. In this study, comprehensive genomic profiling was performed for 302 advanced esophageal cancers, and it was found that the frequently altered genes and biological pathways differed between the two subtypes. Also, a high frequency of clinically relevant genomic alterations was noted for both types of esophageal cancer as a means of finding a potential targeted therapy to be used in addition to or as an alternative to conventional treatment.     

The spectrum of BRCA mutations and characteristics of BRCA‐associated breast cancers in China: Screening of 2,991 patients and 1,043 controls by next‐generation sequencing

To characterize the prevalence of BRCA mutations and characteristics of BRCA carriers in China and to update the clinical recommendations for BRCA testing, we conducted a wide screen for BRCA mutations using next‐generation sequencing (NGS). A total of 4,034 Chinese subjects were screened for germline BRCA1/2 mutations, including 2,991 breast cancer patients and 1,043 healthy individuals from the community enrolled as controls. We developed an NGS‐based approach to perform BRCA1/2 screening. BRCA mutations were identified in 9.1% (232/2,560) of cases with at least one risk factor, in 3.5% (15/431) of sporadic patients and in 0.38% (4/1,043) of healthy controls. The mutation frequency ranged from 8.9 to 15.2% in cohorts with a single risk factor to 16.6–100% in groups with multiple risk factors. We identified 70 novel BRCA mutations. A high frequency of BRCA1 c.5470_5477del was detected, accounting for 13.9% (16/115) of the BRCA1 mutations detected in our study. Clinical characteristics such as family history, invasive carcinoma, negative human epidermal growth factor receptor 2 (HER2), high Ki67 index, lymph node status, and high tumour grade were closely related to BRCA mutations. BRCA2 carriers had poorer disease‐free survival among HER2‐ or hormone receptor‐positive patients (hazard ratio = 1.892; 95% confidence interval: 1.132–3.161; p = 0.013). This study shows that BRCA mutation carriers could be frequently identified among breast cancer patients with multiple risk factors. Importantly, we established an NGS‐based pipeline for BRCA1/2 testing in clinical practice and strongly suggest that breast cancer patients of premier‐ and moderate‐grade risks receive BRCA1/2 mutations testing in China.