Establishing guidelines to harmonize tumor mutational burden (TMB): in silico assessment of variation in TMB quantification across diagnostic platforms: phase I of the Friends of Cancer Research TMB Harmonization Project.

BACKGROUND: Tumor mutational burden (TMB), defined as the number of somatic mutations per megabase of interrogated genomic sequence, demonstrates predictive biomarker potential for the identification of patients with cancer most likely to respond to immune checkpoint inhibitors. TMB is optimally calculated by whole exome sequencing (WES), but next-generation sequencing targeted panels provide TMB estimates in a time-effective and cost-effective manner. However, differences in panel size and gene coverage, in addition to the underlying bioinformatics pipelines, are known drivers of variability in TMB estimates across laboratories. By directly comparing panel-based TMB estimates from participating laboratories, this study aims to characterize the theoretical variability of panel-based TMB estimates, and provides guidelines on TMB reporting, analytic validation requirements and reference standard alignment in order to maintain consistency of TMB estimation across platforms. METHODS: Eleven laboratories used WES data from The Cancer Genome Atlas Multi-Center Mutation calling in Multiple Cancers (MC3) samples and calculated TMB from the subset of the exome restricted to the genes covered by their targeted panel using their own bioinformatics pipeline (panel TMB). A reference TMB value was calculated from the entire exome using a uniform bioinformatics pipeline all members agreed on (WES TMB). Linear regression analyses were performed to investigate the relationship between WES and panel TMB for all 32 cancer types combined and separately. Variability in panel TMB values at various WES TMB values was also quantified using 95% prediction limits. RESULTS: Study results demonstrated that variability within and between panel TMB values increases as the WES TMB values increase. For each panel, prediction limits based on linear regression analyses that modeled panel TMB as a function of WES TMB were calculated and found to approximately capture the intended 95% of observed panel TMB values. Certain cancer types, such as uterine, bladder and colon cancers exhibited greater variability in panel TMB values, compared with lung and head and neck cancers. CONCLUSIONS: Increasing uptake of TMB as a predictive biomarker in the clinic creates an urgent need to bring stakeholders together to agree on the harmonization of key aspects of panel-based TMB estimation, such as the standardization of TMB reporting, standardization of analytical validation studies and the alignment of panel-based TMB values with a reference standard. These harmonization efforts should improve consistency and reliability of panel TMB estimates and aid in clinical decision-making.

Tumor mutational burden standardization initiatives: Recommendations for consistent tumor mutational burden assessment in clinical samples to guide immunotherapy treatment decisions.

Characterization of tumors utilizing next-generation sequencing methods, including assessment of the number of somatic mutations (tumor mutational burden [TMB]), is currently at the forefront of the field of personalized medicine. Recent clinical studies have associated high TMB with improved patient response rates and survival benefit from immune checkpoint inhibitors; hence, TMB is emerging as a biomarker of response for these immunotherapy agents. However, variability in current methods for TMB estimation and reporting is evident, demonstrating a need for standardization and harmonization of TMB assessment methodology across assays and centers. Two uniquely placed organizations, Friends of Cancer Research (Friends) and the Quality Assurance Initiative Pathology (QuIP), have collaborated to coordinate efforts for international multistakeholder initiatives to address this need. Friends and QuIP, who have partnered with several academic centers, pharmaceutical organizations, and diagnostic companies, have adopted complementary, multidisciplinary approaches toward the goal of proposing evidence-based recommendations for achieving consistent TMB estimation and reporting in clinical samples across assays and centers. Many factors influence TMB assessment, including preanalytical factors, choice of assay, and methods of reporting. Preliminary analyses highlight the importance of targeted gene panel size and composition, and bioinformatic parameters for reliable TMB estimation. Herein, Friends and QuIP propose recommendations toward consistent TMB estimation and reporting methods in clinical samples across assays and centers. These recommendations should be followed to minimize variability in TMB estimation and reporting, which will ensure reliable and reproducible identification of patients who are likely to benefit from immune checkpoint inhibitors.

Treatment decisions, clinical outcomes, and pharmacoeconomics in the treatment of patients with EGFR mutated stage III/IV NSCLC in Germany: an observational study.

BACKGROUND: We evaluated treatment decisions and outcomes in a cohort of predominately Caucasian patients with EGFR mutation-positive (EGFR Mut+) non-small-cell lung cancer (NSCLC). METHODS: REASON (NCT00997230) was a non-interventional study in German patients with stage IIIB/IV NSCLC. Secondary endpoints for EGFR Mut + NSCLC included progression-free survival (PFS), overall survival (OS), adverse event (AE) management, and pharmacoeconomic outcomes. RESULTS: Among 334 patients with EGFR Mut + NSCLC, tyrosine kinase inhibitors (TKIs) were the most common first-line therapy (56.6%, 53.0% gefitinib). Among patients who received TKIs/gefitinib before first disease progression, PFS was longer compared with those who did not receive a TKI (median 10.1/10.0 vs. 7.0 months; HR 0.67/0.69; log-rank p = 0.012/p = 0.022). OS was longer for those patients who ever received a TKI/gefitinib during their complete therapy course compared with those who never received a TKI (median 18.4/18.1 vs. 13.6 months; HR 0.53/0.55; p = 0.003/p = 0.005). Total mean first-line treatment healthcare costs per person were higher for those receiving TKIs (€46,443) compared with those who received chemotherapy (€27,182). Mean outpatient and inpatient costs were highest with chemotherapy. Rash, diarrhea, and dry skin were the most commonly reported AEs for patients receiving gefitinib. CONCLUSIONS: In REASON, TKI therapy was the most common first- and second-line treatment for EGFR Mut + NSCLC, associated with increased drug costs compared with chemotherapy. Patients who received gefitinib or a TKI ever during their complete therapy course had prolonged PFS and OS compared with patients who did not receive a TKI. TRIAL REGISTRATION: The trial was registered on October, 2009 with ClinicalTrials.gov : https://clinicaltrials.gov/ct2/show/NCT00997230?term=NCT00997230&rank=1.

Ioncopy: a novel method for calling copy number alterations in amplicon sequencing data including significance assessment.

Recently, it has been demonstrated that calling of copy number alterations (CNAs) from amplicon sequencing (AS) data is feasible. Most approaches, however, require non-tumor (germline) DNA for data normalization. Here, we present the method Ioncopy for CNA detection which requires no normal controls and includes a significance assessment for each detected alteration.Ioncopy was evaluated in a cohort of 184 clinically annotated breast carcinomas. A total number of 252 amplifications were detected, of which 183 (72.6%) could be validated by a call of an additional amplicon interrogating the same gene. Moreover, a total number of 33 deletions were found, whereof 27 (81.8%) could be validated. Analyzing the 16 most frequently amplified genes, validation rates of over 89% could be achieved for 11 of these genes. 11 of the top 16 genes showed significant overexpression in the amplified tumors. 89.5% of the HER2-amplified tumors were GRB7 and STARD3 co-amplified, whereas 68.4% of the HER2-amplified tumors had additional MED1 amplifications. Correlations between CNAs measured by amplicons in HER2 exons 19, 20 and 21 were strong (all R > 0.93). AS based detection of HER2 amplifications had a sensitivity of 90.0% and a specificity of 98.8% compared to the gold standard of HER2 immunohistochemistry combined with in situ hybridization.In summary, we developed and validated a novel method for detection and significance assessment of CNAs in amplicon sequencing data. Using Ioncopy, AS offers a straightforward and efficient approach to simultaneously analyze gene amplifications and gene deletions together with simple somatic mutations in a single assay.

Assessment of the PD-L1 status by immunohistochemistry: challenges and perspectives for therapeutic strategies in lung cancer patients.

Immunotherapy targeting the PD-L1/PD-1 axis has recently shown spectacular efficacy and promise for the future of patients with metastatic lung cancer. In the setting of second-line treatment of metastatic disease, this therapy has increased overall survival of patients by several months when compared to chemotherapy, both for squamous cell carcinoma (SCC) and adenocarcinoma (ADC) of the lung. Clinical trials targeting the PD-1/PD-L1 axis have shown a tendency towards higher efficacy if expression of PD-L1 is relatively high, as evaluated by immunohistochemistry (IHC) of tumour samples. Targeting the PD-1/PD-L1 axis is of crucial importance not only for metastatic non-small cell lung cancer (NSCLC) but probably also for patients with small cell lung cancer. Nivolumab, an antibody targeting PD-1, has recently received FDA and EMA approval for NSCLC, regardless of the PDL1 expression status (for both tumour types in the USA and for only SCC in EU). However, the need for a biomarker that allows better selection of patients is essential, to improve treatment efficacy and to manage cost of these therapies. Assessment of PD-L1 expression through immunohistochemical staining is advocated by many as one such potential biomarker. This prospect raises several questions, in particular how to define a threshold for positive PD-L1 labelling on biopsy tissue samples, taking into account that certain patients respond to treatment targeting PD-L1/PD-1, despite low or absent immunoreactivity of this biomarker. This review discusses major challenges related to detection of PD-L1 by immunohistochemistry as a companion diagnostic test, along with immune checkpoint blockade treatment of lung cancer.

Quantitative analysis of diagnostic guidelines for HER2-status assessment.

Human epidermal growth factor receptor 2 (HER2, alias ERBB2)-targeted therapy in breast and gastric cancers depends on the reliable assessment of HER2 protein expression and (in equivocal cases) the quantitative evaluation of HER2 gene amplification. Typically, HER2 and centromere 17 gene copy numbers are evaluated using in situ hybridization (ISH) to calculate ratios for which cutoff values dividing nonamplified and amplified cases have been proposed. Although several studies have investigated how laboratory procedures affect diagnostics, a rigorous quantitative assessment of the diagnostic guidelines for data analysis is still missing. Here, we analyze the dependence of the diagnosed HER2/chromosome 17 ratios on i) sample size (evaluated cells), ii) gene/chromosome signal distributions, and iii) the approach used for quotient calculation using Monte Carlo simulations. Our data show that the current recommendation may lead to statistical HER2/CHR17 ratio variations of up to 0.94 and may therefore lead to incorrect HER2 status diagnoses, given the ratio threshold of 2.0 defined by the Food and Drug Administration. Moreover, borderline cases may receive different amplification diagnoses, depending on the ratio calculation approach: Brightfield-silver ISH with aggregated signal counts may underestimate the HER2/CHR17 ratio compared with two-color fluorescence ISH. Our results provide a basis for quantitative rationales behind HER2 diagnostic guidelines that call for increased numbers of evaluated cells and emphasize the importance of well-designed data analysis methods in diagnostic pathology, especially for predictive clinical application.

Quantitative determination of estrogen receptor, progesterone receptor, and HER2 mRNA in formalin-fixed paraffin-embedded tissue--a new option for predictive biomarker assessment in breast cancer.

The development of optimized therapy strategies against malignant tumors is critically dependent on the assessment of tissue-based biomarkers in routine diagnostic tissue samples. We investigated a novel, fully automated, and xylene-free method for RNA isolation and biomarker determination using formalin-fixed paraffin-embedded (FFPE) tissue. The aim was to show that this approach is feasible and gives results that are comparable to the current gold standards. Expression of the breast cancer biomarkers ESR1, PGR, and HER2 was measured in a total of 501 FFPE tissue samples from 167 breast carcinomas, which had been stored for up to 21 years. Total RNA was extracted from tissue sections and biomarker expression was measured by kinetic RT-PCR (RT-kPCR). The results of the new method were compared with immunohistochemistry as the current gold standard.RNA was successfully isolated from all samples, with a mean yield of 1.4 µg/sample and fragment lengths of at least 150 bp in 99% of samples. RT-kPCR analysis of ESR1, PGR, and HER2 was possible in all samples. Comparing RT-kPCR results with standard IHC, we found a good concordance for ESR1 (agreement: 98.4%), PGR (84.4%), and HER2 (89.8%). We observed a low section-to-section variability of kPCR results for all 3 biomarkers (root of mean squared errors: 0.2 to 0.5 Ct values). The new approach is a reliable high-throughput instrument for standardized testing of biomarkers in clinical routine and for research studies on archived FFPE material up to 21 years old. For the assessment of ESR1, PGR, and HER2 the results are comparable to the current gold-standard.

EGFR mutation detection in NSCLC--assessment of diagnostic application and recommendations of the German Panel for Mutation Testing in NSCLC.

EGFR mutation testing in non-small cell lung cancer (NSCLC) is a novel and important molecular pathological diagnostic assay that is predictive of response to anti-epidermal growth factor receptor (EGFR) therapy. A comprehensive compilation of a large number of EGFR mutation analyses of the German Panel for Mutation Analyses in NSCLC demonstrates (a) a higher than previously reported mutation frequency outside the conventionally tested exons 19 and 21 and (b) an overall superiority of sequencing based assays over mutation-specific PCR. The implications for future diagnostic EGFR mutation testing are discussed.

Silver in situ hybridization (SISH) for determination of HER2 gene status in breast carcinoma: comparison with FISH and assessment of interobserver reproducibility.

The importance of HER2 status in breast cancer management has focused attention on the ability of clinical assays to correctly assign HER2 amplification status. There is no consensus as to the best method for assessing HER2 status. Disadvantages of fluorescence in situ hybridization (FISH) testing include longer time required for staining and scoring slides, requirements for specialized training and fluorescence microscopy, and loss of the signal due to quenching of the fluorescent dye. Silver-enhanced in situ hybridization (SISH) is a rapid fully automated assay providing permanently stained slides that are interpreted by conventional bright field microscopy which enables pathologists to evaluate slides within the context of tissue morphology. This study evaluates the concordance between SISH and FISH assays in determining the status of HER2 gene amplification in a cohort of 298 primary invasive breast carcinomas. Furthermore, we assessed in detail the variables contributing to interobserver interpretive reproducibility of HER2 SISH among 10 pathologists. HER2 was quantified using the ratio of HER2 to CHR17 signals using the conventional historical interpretation scale and also by the American Society of Clinical Oncology/College of American Pathologists reporting scheme. For SISH status determined by consensus among 10 pathologists, overall concordance between SISH and FISH was identified in 288 of 298 cases (96.6%) using the conventional Food and Drug Administration approved criteria. Overall agreement was observed in 282 of 285 cases (98.9%) using the American Society of Clinical Oncology/College of American Pathologists result reporting scheme (with equivocal cases removed). In conclusion, SISH represents a novel approach for the determination of HER2 status in breast cancer. The overall concordance between SISH and FISH is excellent, and the interpretation of SISH results by pathologists is most reproducible using the HER2/CHR17 ratio.

KRAS genotyping of paraffin-embedded colorectal cancer tissue in routine diagnostics: comparison of methods and impact of histology.

KRAS mutation testing before anti-epidermal growth factor receptor therapy of metastatic colorectal cancer has become mandatory in Europe. However, considerable uncertainty exists as to which methods for detection can be applied in a reproducible and economically sound manner in the routine diagnostic setting. To answer this question, we examined 263 consecutive routine paraffin slide specimens. Genomic DNA was extracted from microdissected tumor tissue. The DNA was analyzed prospectively by Sanger sequencing and array analysis as well as retrospectively by melting curve analysis and pyrosequencing; the results were correlated to tissue characteristics. The methods were then compared regarding the reported results, costs, and working times. Approximately 40% of specimens contained KRAS mutations, and the different methods reported concordant results (kappa values >0.9). Specimens harboring fewer than 10% tumor cells showed lower mutation rates regardless of the method used, and histoanatomical variables had no influence on the frequency of the mutations. Costs per assay were higher for array analysis and melting curve analysis when compared with the direct sequencing methods. However, for sequencing methods equipment costs were much higher. In conclusion, Sanger sequencing, array analysis, melting curve analysis, and pyrosequencing were equally effective for routine diagnostic KRAS mutation analysis; however, interpretation of mutation results in conjunction with histomorphologic tissue review and on slide tumor tissue dissection is required for accurate diagnosis.

Cut-resistant protective gloves in pathology--effective and cost-effective.

Cutting injuries and needle-stitch injuries constitute a potentially fatal danger to both pathologists and autopsy personnel. We evaluated such injuries in a large German institute of pathology from 2002 to 2007 and analysed the effect of the introduction of cut-resistant gloves on the incidence of these injuries. In the observation period, 64 injuries (48 cutting injuries and 16 needle-stitch injuries) were noted in the injury report books. Most injuries were located at the non-dominant hand, preferentially at the index finger and the thumb. Around one fifths of the injuries were at the side of handedness. The average number of injuries per month was 1.22 for the 50 months prior to the introduction of cut-resistant gloves, more than seven times higher than after their introduction (0.158; 19 months; p < 0.001). Considering the medical and administrational costs of such injuries, cut-resistant protective gloves are an effective and cost-effective completion of personal occupational safety measures in surgical pathology and autopsy. We strongly recommend the use of such gloves, especially for autopsy personnel.

Immunohistochemical double-staining of renal allograft tissue: critical assessment of three different protocols.

Quantitative analyses of renal allograft tissue using immunohistochemical double-staining could be a useful tool to extend the existing knowledge on renal allograft immunopathology. Due to technical reasons, this method has been only rarely applied in the past. The use of indirect immunohistochemistry for double-staining bears the risk of nonspecific cross reactions between the two staining sequences. To date, various procedures have been refined to avoid such cross reactions. Here we assessed the validity of three different protocols for indirect immunohistochemical double-staining on frozen sections of renal transplant biopsies ( n=12). Both colocalized antigens and antigens with a non-overlapping distribution were stained according to each of the three protocols. Differentiation between the two staining sequences was achieved by employing different colored substrates of alkaline phosphatase (protocol 1), different enzymes (peroxidase and alkaline phosphatase) together with the use of 3,3'-diaminobenzidine-tetrahydrochloride substrate in the first staining sequence (protocol 2), or primary antibodies from different species (protocol 3). Sensitivity and specificity of each protocol were determined by quantitative comparison with control single-stainings of adjacent sections. Sensitivity of the first staining sequence was about 100% with each of the three protocols investigated. In the second staining sequence, sensitivities of protocols 1 (50%) and 2 (54-66%) were much lower than of protocol 3 (100%). Specificity of the second staining sequence was only 44% with protocol 1 compared with 98% with protocol 2 and 100% with protocol 3. In conclusion, protocols 1 and 2 are not recommended for quantitative double-staining analyses. In contrast, protocol 3 provided maximum sensitivity and specificity, even for antigens that are colocalized on the same cell type. Thus, the use of primary antibodies from different species is by far the most reliable technique for quantitative double-staining analyses in renal allograft tissue.