Harmonization and Standardization of Panel-Based Tumor Mutational Burden Measurement: Real-World Results and Recommendations of the Quality in Pathology Study

IntroductionTumor mutational burden (TMB) is a quantitative assessment of the number of somatic mutations within a tumor genome. Immunotherapy benefit has been associated with TMB assessed by whole-exome sequencing (wesTMB) and gene panel sequencing (psTMB). The initiatives of Quality in Pathology (QuIP) and Friends of Cancer Research have jointly addressed the need for harmonization among TMB testing options in tissues. This QuIP study identifies critical sources of variation in psTMB assessment.MethodsA total of 20 samples from three tumor types (lung adenocarcinoma, head and neck squamous cell carcinoma, and colon adenocarcinoma) with available WES data were analyzed for psTMB using six panels across 15 testing centers. Interlaboratory and interplatform variation, including agreement on variant calling and TMB classification, were investigated. Bridging factors to transform psTMB to wesTMB values were empirically derived. The impact of germline filtering was evaluated.ResultsSixteen samples had low interlaboratory and interpanel psTMB variation, with 87.7% of pairwise comparisons revealing a Spearman’s ρ greater than 0.6. A wesTMB cut point of 199 missense mutations projected to psTMB cut points between 7.8 and 12.6 mutations per megabase pair; the corresponding psTMB and wesTMB classifications agreed in 74.9% of cases. For three-tier classification with cut points of 100 and 300 mutations, agreement was observed in 76.7%, weak misclassification in 21.8%, and strong misclassification in 1.5% of cases. Confounders of psTMB estimation included fixation artifacts, DNA input, sequencing depth, genome coverage, and variant allele frequency cut points.ConclusionsThis study provides real-world evidence that all evaluated panels can be used to estimate TMB in a routine diagnostic setting and identifies important parameters for reliable tissue TMB assessment that require careful control. As complex or composite biomarkers beyond TMB are likely playing an increasing role in therapy prediction, the efforts by QuIP and Friends of Cancer Research also delineate a general framework and blueprint for the evaluation of such assays.     

Pulmonary Sarcomatoid Carcinomas Commonly Harbor Either Potentially Targetable Genomic Alterations or High Tumor Mutational Burden as Observed by Comprehensive Genomic Profiling

IntroductionPulmonary sarcomatoid carcinoma (PSC) is a high-grade NSCLC characterized by poor prognosis and resistance to chemotherapy. Development of targeted therapeutic strategies for PSC has been hampered because of limited and inconsistent molecular characterization.MethodsHybrid capture–based comprehensive genomic profiling was performed on DNA from formalin-fixed paraffin-embedded sections of 15,867 NSCLCs, including 125 PSCs (0.8%). Tumor mutational burden (TMB) was calculated from 1.11 megabases (Mb) of sequenced DNA.ResultsThe median age of the patients with PSC was 67 years (range 32–87), 58% were male, and 78% had stage IV disease. Tumor protein p53 gene (TP53) genomic alterations (GAs) were identified in 74% of cases, which had genomics distinct from TP53 wild-type cases, and 62% featured a GA in KRAS (34%) or one of seven genes currently recommended for testing in the National Comprehensive Cancer Network NSCLC guidelines, including the following: hepatocyte growth factor receptor gene (MET) (13.6%), EGFR (8.8%), BRAF (7.2%), erb-b2 receptor tyrosine kinase 2 gene (HER2) (1.6%), and ret proto-oncogene (RET) (0.8%). MET exon 14 alterations were enriched in PSC (12%) compared with non-PSC NSCLCs (∼3%) (p < 0.0001) and were more prevalent in PSC cases with an adenocarcinoma component. The fraction of PSC with a high TMB (>20 mutations per Mb) was notably higher than in non-PSC NSCLC (20% versus 14%, p = 0.056). Of nine patients with PSC treated with targeted or immunotherapies, three had partial responses and three had stable disease.ConclusionPotentially targetable GAs in National Comprehensive Cancer Network NSCLC genes (30%) or intermediate or high TMB (43%, >10 mutations per Mb) were identified in most of the PSC cases. Thus, the use of comprehensive genomic profiling in clinical care may provide important treatment options for a historically poorly characterized and difficult to treat disease.     

Comparison of Three Sequencing Panels Used for the Assessment of Tumor Mutational Burden in NSCLC Reveals Low Comparability

IntroductionTumor mutational burden (TMB) has been proposed as a novel predictive biomarker for the stratification of patients with NSCLC undergoing immune checkpoint inhibitor (ICI) treatment. The assessment of TMB has recently been established using large targeted sequencing panels, and numerous studies are ongoing to harmonize TMB assessment. “Correlation” or the coefficient of determination has generally been used to evaluate the association between different panels. We hypothesized that these metrics might overestimate the comparability, especially for lower TMB values.MethodsA total of 30 samples from patients with NSCLC undergoing ICI treatment were consecutively sequenced using the following three large, targeted sequencing panels: FoundationOne, Oncomine TML, and QiaSeq TMB. The TMB values were compared in the whole patient population and in a subset of patients in which the TMB assessed by FoundationOne was between 5 and 25 mutations/Mb. Prediction of durable clinical benefit (>6 mo with no progression) was assessed using receiver operator characteristics, and optimal cutoff values were calculated using the Youden J statistic.ResultsCorrelation between the three targeted sequencing panels was strong in the whole patient population (R² > 0.79) but was dramatically reduced in the subset of patients with TMB of 5 to 25 mutations/Mb. The agreement assessed using the Bland-Altman method was also very low. All panels were able to predict durable clinical benefit in the TMB-high population.ConclusionsAssessment of TMB using the three targeted sequencing panels was possible and predictive of response to ICI treatment, but correlation was an inappropriate measurement to assess the association between the respective panels.     

Complete and Durable Response to Nivolumab in Recurrent Poorly Differentiated Pancreatic Neuroendocrine Carcinoma with High Tumor Mutational Burden

Poorly differentiated pancreatic neuroendocrine carcinomas (NECs) are rare and aggressive malignancies with rapid disease progression and early widespread metastasis. Given histology similarity, they are commonly treated with platinum-based chemotherapy as small cell lung cancer (SCLC). However, no standard treatment has been established for recurrent or progressive disease. We present an Asian patient with recurrent poorly differentiated pancreatic NEC after curative surgery and adjuvant chemotherapy with cisplatin and etoposide. The tumor mutational burden (TMB) was high. The patient received chemotherapy combined with maintenance immunotherapy with nivolumab and achieved promising and durable response, suggesting TMB could be a biomarker to identify NEC patients for immune checkpoint inhibitor (ICI) treatment.     

Deep Targeted Sequencing and Its Potential Implication for Cancer Therapy in Chinese Patients with Gastric Adenocarcinoma

IntroductionGastric cancer (GC) has a high incidence and mortality rate, especially in East Asians, and about 90% of GCs are adenocarcinomas. Histological and etiological heterogeneity and ethnic diversity make molecular subtyping of GC complicated, thus making it difficult to determine molecular division systems and standard treatment modalities. Limited cohorts from South Korea, Singapore, Australia, and Japan have been studied; however, the mutational landscape of gastric adenocarcinomas in Chinese patients is still unknown.MethodsWe performed a targeted sequencing panel focusing on cancer‐related genes and tumor‐associated microorganisms of 529 gastric adenocarcinoma samples with matched blood controls. We identified 449 clinically relevant gene mutations.ResultsApproximately 47.1% of Chinese patients with GC harbored at least one actionable mutation. The top somatic mutations were TP53, ARID1A, LRP1B, PIK3CA, ERBB2, CDH1, KRAS, FAT4, CCNE1, and KMT2D. Truncation mutations of ARID1A, KMT2D, RNF43, TGFBR2, and CIC occurred in patients with high tumor mutational burden. Gene amplifications of ERBB2, CCNE1, CDK12, and CCND1 were detected in patients with low tumor mutational burden. Pathway analysis revealed common gene alterations in the Wnt and PI3K/Akt signaling pathways. The ratio of patients with high microsatellite instability was significantly lower than other cohorts, and high microsatellite instability and Epstein‐Barr virus (EBV)–positive features seemed mutually inclusive in Chinese patients with GC. In 44 (8.3%) patients, 45 germline mutations were identified, among which SPINK1 mutations, all SPINK1 c.194 + 2T > C, were present in 15.9% (7/44) of patients. Microorganisms found in Chinese patients with GC included Helicobacter pylori, EBV, hepatitis B virus, and human papillomavirus types 16 and 18.ConclusionIdentification of varied molecular features by targeted next‐generation sequencing provides more insight into patient stratification and offers more possibilities for both targeted therapies and immunotherapies of Chinese patients with GC.Implications for PracticeThis study investigated the genomic alteration profile of 529 Chinese patients with gastric adenocarcinoma by deep targeting sequencing, which might be the largest Chinese cohort on the genomic research of gastric adenocarcinoma up to now.     

The Underlying Tumor Genomics of Predominant Histologic Subtypes in Lung Adenocarcinoma

IntroductionThe purpose of the study is to genomically characterize the biology and related therapeutic opportunities of prognostically important predominant histologic subtypes in lung adenocarcinoma (LUAD).MethodsWe identified 604 patients with stage I to III LUAD who underwent complete resection and targeted next-generation sequencing using the Memorial Sloan Kettering–Integrated Mutation Profiling of Actionable Cancer Targets platform. Tumors were classified according to predominant histologic subtype and grouped by architectural grade (lepidic [LEP], acinar or papillary [ACI/PAP], and micropapillary or solid [MIP/SOL]). Associations among clinicopathologic factors, genomic features, mutational signatures, and recurrence were evaluated within subtypes and, when appropriate, quantified using competing-risks regression, with adjustment for pathologic stage and extent of resection.ResultsMIP/SOL tumors had higher tumor mutational burden (p < 0.001), fraction of genome altered (p = 0.001), copy number amplifications (p = 0.021), rate of whole-genome doubling (p = 0.008), and number of oncogenic pathways altered ( p < 0.001) as compared with LEP and ACI/PAP tumors. Across all tumors, mutational signatures attributed to APOBEC activity were associated with the highest risk of postresection recurrence: SBS2 (p = 0.021) and SBS13 (p = 0.005). Three oncogenic pathways (p53, Wnt, Myc) were altered with statistical significance in MIP/SOL tumors. Compared with LEP and ACI/PAP tumors, MIP/SOL tumors had a higher frequency of targetable BRAF-V600E mutations (p = 0.046). Among ACI/PAP tumors, alterations in the cell cycle (p < 0.001) and PI3K (p = 0.002) pathways were associated with recurrence; among MIP/SOL tumors, only PI3K alterations were associated with recurrence (p = 0.049).ConclusionsThese results provide the first in-depth assessment of tumor genomic profiling of predominant LUAD histologic subtypes, their associations with recurrence, and their correlation with targetable driver alterations in patients with surgically resected LUAD.     

EGFR-TKI治疗肺腺癌的疗效与血清肿瘤标志物的相关性

目的探讨表皮生长因子受体酪氨酸激酶抑制剂(epidermal growth factor receptor tyrosine kinaseinhibitor,EGFR-TKI)对肺腺癌的疗效和血清肿瘤标志物间的关系。方法 回顾分析48例应用EGFR-TKI治疗的晚期肺腺癌患者的临床特征、生存时间和治疗前血清肿瘤标志物水平的相关性。结果 EGFR-TKI治疗后有效率为58.3%,控制率为65.6%;中位生存时间为13.2月。统计学分析显示:吸烟史、血清CEA和CA19-9水平与EGFR-TKI的疗效相关(P<0.05);治疗前血清CEA,CA19-9水平高者有着更高的治疗有效率、控制率和更长的生存期(P<0.05)。结论 EGFR-TKI治疗前血清CA19-9和CEA的水平对预测EGFR-TRI对肺腺癌患者的疗效有参考价值。     

61例肺实性型腺癌的临床特征及预后分析

探讨肺实性型腺癌的临床特征及影响预后的因素。方法：回顾性分析1975年7月至2010年4月收治的61例肺实性型腺癌患者的临床资料,其中行手术为主的综合治疗54例、非手术治疗7例。分别对性别、年龄、吸烟史、肿瘤部位、肿瘤大小、淋巴结转移、pTNM分期等因素进行预后分析。Kaplan-Meier法计算生存率,Log-rank法进行生存率显著性检验,Cox比例风险回归模型进行单因素和多因素分析,评价各因素对预后的影响。结果：全部患者的1、3和5年生存率分别为77.3%、44.8%和25.2%。单因素分析显示：肿瘤大小（P<0.001）、有无淋巴结转移（P=0.014）、M分期（P=0.013）、pTNM分期（P=0.006）、治疗方式（P<0.001）和手术方式（P=0.006）是影响预后的因素。多因素分析显示：肿瘤大小（P=0.015）、有无淋巴结转移（P<0.001）、M分期（P=0.013）和治疗方式是影响预后的独立因素。结论：肺实性型腺癌发生率低,缺乏特异性临床表征,较易发生淋巴结转移,预后差。影响其预后的因素主要为肿瘤大小、淋巴结转移、M分期和治疗方式。根治性手术是其主要治疗手段。争取早期正确诊断、选择正确的治疗方法,是提高生存率的方法。     

多项肺系统肿瘤标志物异常在晚期肺腺癌治疗中的作用

背景与目的 肺癌的常用肿瘤标志物中,癌胚抗原(carcinoembryonic antigen, CEA)与糖类抗原125(carbohydrate antigen 125, CA125)、细胞角蛋白19片段(cytokeratin 19, CYFRA21-1)与鳞状细胞癌抗原(squamous carcinoma antigen, SCC)、神经元特异性烯醇化酶(neuron specific enolase, NSE)与胃泌素释放肽前体(pro-gastrin-releasing peptide, ProGRP)分别在肺腺癌、肺鳞状细胞癌和小细胞肺癌中有较高表达.本研究旨在通过对比多项肿瘤标志物异常(A组)和仅CEA和/或CA125异常(B组)的两组晚期肺腺癌患者,探讨多项肿瘤标志物异常在疗效评价和预测复发方面的价值.方法 纳入中国医学科学院肿瘤医院的IV期肺腺癌初治病例,回顾性分析其临床数据,包括临床特征、治疗前的血清肿瘤标志物水平、疗效及无进展生存期.结果 除CEA和CA125外,A组异常比率最高的肿瘤标志物是CYFRA21-1(93%),其次是NSE(36%)、SCC(13%)和ProGRP (12%).多项肿瘤标志物异常的患者更易出现远处多部位转移(P<0.001),治疗后的无进展生存期更短(中位时间5.3个月 vs 7.3个月,P=0.016).两组中进行维持治疗的患者均比未行维持治疗的患者复发风险低(P均<0.001).结论 多项肿瘤标志物异常患者复发风险高,维持治疗可降低复发风险.     

Relapsed and De Novo Metastatic HER2-positive Breast Cancer Treated With Trastuzumab: Tumor Genotypes and Clinical Measures Associated With Patient Outcome

Background

We examined tumor genotype characteristics of human epidermal growth factor receptor 2 (HER2)-positive relapsed (R-) and de novo (dn-) metastatic breast cancer (MBC) in trastuzumab-treated patients who were previously not exposed to this agent.

Correlation Between EGFR Mutations and Serum TumorMarkers in Lung Adenocarcinoma Patients

Background: Mutations affecting the epidermal growth factor receptor (EGFR) are good predictors ofclinical efficacy of EGFR tyrosine kinase inhibitors (TKI) in patients with non-small cell lung cancer. Serumcarcinoembryonic antigen (CEA) levels are also regarded as predictive for the efficacy of EGFR-TKI andEGFR gene mutations. This study analyzed the association between EGFR gene mutations and clinical features,including serum tumor marker levels in lung adenocarcinomas patients. Patients and Methods: A total of70 lung adenocarcinoma patients with complete clinical data and pathological specimens were investigated.EGFR gene mutations at exons 19 and 21 were assessed. Serum tumor markers were detected by protein chipchemiluminescenceat the corresponding time, and correlations were analyzed. Results: Mutations of the EGFRgene were detected in 27 of the 70 patients and the serum CEA and CA242 concentrations were found to besignificantly associated with the incidence of EGFR gene mutations (P<0.05). The AUCs for CEA and CA242 were0.724 (95% CI: 0.598~0.850, P<0.05) and 0.769 (95% CI: 0.523~0.800, P<0.05) respectively. Conclusions: SerumCEA and CA242 levels are associated with mutations of the EGFR gene in patients with lung adenocarcinomas.     

The Implication of Anthracosis in the Development of Pulmonary Adenocarcinoma

The relationship between anthracosis, which is the deposition of black dust matter in the lung parenchyma, and the development of pulmonary adenocarcinoma has not been fully characterized. In order to clarify whether background black dust matter deposition could be implicated in the development of pulmonary adenocarcinoma, we measured the level of anthracosis at autopsy in 47 patients who had died of pulmonary adenocarcinoma. Both lungs of all 47 cadavers were examined. Twenty-micrometer sections were cut from formalin-fixed, paraffin-embedded blocks of the largest cut surface of each lung. Black dust matter was extracted from the sections and blotted onto a nitrocellulose membrane. The density of the blotted black dust matter was then analyzed using an imaging densitometer. There were no significant differences in the density of black dust matter deposition between lungs affected by pulmonary adenocarcinoma and control lungs. However, well differentiated adenocarcinomas tended to develop more frequently than poorly differentiated ones in lungs showing less deposition. We found a very strong correlation between the degree of black dust matter deposition and smoking history. Patients with severe anthracosis tended to have a poorer prognosis than those with mild anthracosis.     

A Grading System for Invasive Pulmonary Adenocarcinoma: A Proposal From the International Association for the Study of Lung Cancer Pathology Committee

IntroductionA grading system for pulmonary adenocarcinoma has not been established. The International Association for the Study of Lung Cancer pathology panel evaluated a set of histologic criteria associated with prognosis aimed at establishing a grading system for invasive pulmonary adenocarcinoma.MethodsA multi-institutional study involving multiple cohorts of invasive pulmonary adenocarcinomas was conducted. A cohort of 284 stage I pulmonary adenocarcinomas was used as a training set to identify histologic features associated with patient outcomes (recurrence-free survival [RFS] and overall survival [OS]). Receiver operating characteristic curve analysis was used to select the best model, which was validated (n = 212) and tested (n = 300, including stage I–III) in independent cohorts. Reproducibility of the model was assessed using kappa statistics.ResultsThe best model (area under the receiver operating characteristic curve [AUC] = 0.749 for RFS and 0.787 for OS) was composed of a combination of predominant plus high-grade histologic pattern with a cutoff of 20% for the latter. The model consists of the following: grade 1, lepidic predominant tumor; grade 2, acinar or papillary predominant tumor, both with no or less than 20% of high-grade patterns; and grade 3, any tumor with 20% or more of high-grade patterns (solid, micropapillary, or complex gland). Similar results were seen in the validation (AUC = 0.732 for RFS and 0.787 for OS) and test cohorts (AUC = 0.690 for RFS and 0.743 for OS), confirming the predictive value of the model. Interobserver reproducibility revealed good agreement (k = 0.617).ConclusionsA grading system based on the predominant and high-grade patterns is practical and prognostic for invasive pulmonary adenocarcinoma.     

Association between Smoking and Tumor Progression in Japanese Women with Adenocarcinoma of the Lung

We studied the effect of smoking on tumor progression in 3312 patients with lung cancer registered at the National Matsudo Hospital and National Cancer Center Hospital East between 1977 and 1996. The odds ratios of the following variables for tumor extent (localized versus advanced disease) and hazard ratios for survival were calculated in both sexes separately using the logistic regression and Cox proportional hazard models, respectively: smoking history, number of cigarettes smoked per day, pack-years smoked, age, histological type, and the year of admission. Of the 943 women, 367 (38.9%) were smokers and 694 (73.6%) had adenocarcinoma, whereas of the 2369 men, 2255 (95.2%) were smokers and 1010 (42.6%) had adenocarcinoma. In female adenocarcinoma patients, the odds ratio (95% confidence interval) for advanced disease and the hazard ratio (95% confidence interval) for survival with an increase of 30 cigarettes smoked per day were 2.86 (1.49-5.49) and 1.52 (1.13-2.04), respectively, but in those with non-adenocarcinoma, the odds ratio and hazard ratio were 0.96 (0.41-2.23) and 1.13(0.75-1.70), respectively. In male patients, smoking history influenced tumor progression regardless of histological type, but the odds ratios and hazard ratios were lower than those for women with adenocarcinoma. In conclusion, smoking habit was closelycorrelated with progression of adenocarcinoma in women. This association was not observed in women with non-adenocarcinoma and was weaker in men, suggesting various effects of smoking on lung cancer development depending on gender and the histological typeof the tumor