Whole tumor section quantitative image analysis maximizes between-pathologists' reproducibility for clinical immunohistochemistry-based biomarkers.

Pathologists have had increasing responsibility for quantitating immunohistochemistry (IHC) biomarkers with the expectation of high between-reader reproducibility due to clinical decision-making especially for patient therapy. Digital imaging-based quantitation of IHC clinical slides offers a potential aid for improvement; however, its clinical adoption is limited potentially due to a conventional field-of-view annotation approach. In this study, we implemented a novel solely morphology-based whole tumor section annotation strategy to maximize image analysis quantitation results between readers. We first compare the field-of-view image analysis annotation approach to digital and manual-based modalities across multiple clinical studies (~120 cases per study) and biomarkers (ER, PR, HER2, Ki-67, and p53 IHC) and then compare a subset of the same cases (~40 cases each from the ER, PR, HER2, and Ki-67 studies) using whole tumor section annotation approach to understand incremental value of all modalities. Between-reader results for each biomarker in relation to conventional scoring modalities showed similar concordance as manual read: ER field-of-view image analysis: 95.3% (95% CI 92.0-98.2%) vs digital read: 92.0% (87.8-95.8%) vs manual read: 94.9% (91.4-97.8%); PR field-of-view image analysis: 94.1% (90.3-97.2%) vs digital read: 94.0% (90.2-97.1%) vs manual read: 94.4% (90.9-97.2%); Ki-67 field-of-view image analysis: 86.8% (82.1-91.4%) vs digital read: 76.6% (70.9-82.2%) vs manual read: 85.6% (80.4-90.4%); p53 field-of-view image analysis: 81.7% (76.4-86.8%) vs digital read: 80.6% (75.0-86.0%) vs manual read: 78.8% (72.2-83.3%); and HER2 field-of-view image analysis: 93.8% (90.0-97.2%) vs digital read: 91.0 (86.6-94.9%) vs manual read: 87.2% (82.1-91.9%). Subset implementation and analysis on the same cases using whole tumor section image analysis approach showed significant improvement between pathologists over field-of-view image analysis and manual read (HER2 100% (97-100%), P=0.013 field-of-view image analysis and 0.013 manual read; Ki-67 100% (96.9-100%), P=0.040 and 0.012; ER 98.3% (94.1-99.5%), p=0.232 and 0.181; and PR 96.6% (91.5-98.7%), p=0.012 and 0.257). Overall, whole tumor section image analysis significantly improves between-pathologist's reproducibility and is the optimal approach for clinical-based image analysis algorithms.

Impact of an alternative chromosome 17 probe and the 2013 American Society of Clinical Oncology and College of American Pathologists guidelines on fluorescence in situ hybridization for the determination of HER2 gene amplification in breast cancer.

BACKGROUND: The dual-probe fluorescence in situ hybridization (FISH) assay for human epidermal growth factor receptor 2 (HER2) gene amplification in breast cancer provides an HER2:CEP17 (centromere enumeration probe for chromosome 17) ratio. Copy number alteration (CNA) in CEP17 may skew this ratio. The authors analyzed the impact of the 2013 American Society of Oncology/College of American Pathologists (ASCO/CAP) guidelines and an alternative chromosome 17 probe on HER2 status in tumor specimens with CEP17 CNA. METHODS: Specimens with CEP17 CNA (n = 310) were selected from 3048 tumor samples that were received from January 2013 to June 2015 for testing with the alternative chromosome 17 probe D17S122. Reclassification of HER2 status was assessed using the 2007 and 2013 ASCO/CAP guidelines. RESULTS: The alternative chromosome 17 probe reclassified 82 of 310 (26.5%) and 87 of 310 (28.1%) tumors using the 2007 and 2013 guidelines, respectively. Of the 41 of 310 tumors (13.2%) that were reclassified from nonamplified to amplified according to 2007 guidelines, 28 of 41 (68.3%) had an average HER2 copy number ≥4.0 and <6.0. The 39 of 310 tumors (12.6%) that were reclassified from equivocal to amplified according to 2013 guidelines had a mean HER2 copy number between ≥4.0 and <6.0. Most of these patients had stage I, hormone receptor-positive, lymph node-negative tumors, which is an unusual clinicopathologic profile for HER2-amplified tumors, and most received HER2-targeted therapy in addition to endocrine therapy. CONCLUSIONS: Reflex testing with an alternative chromosome 17 probe using the 2013 ASCO/CAP guidelines reclassified 28.1% of tumor samples that had CEP17 CNA, converting nearly one-half from equivocal to amplified. The benefit of HER2-targeted therapy in this patient population requires further study. Cancer 2017;123:2230-2239. © 2017 American Cancer Society.

HER2 intratumoral heterogeneity is independently associated with incomplete response to anti-HER2 neoadjuvant chemotherapy in HER2-positive breast carcinoma.

PURPOSE: Anti-HER2 neoadjuvant chemotherapy has been widely used in HER2-positive breast cancer patients; however, pathologic complete response (pCR) is achieved in only 40-50% of patients. The aim of this study was to investigate the association of HER2 intratumoral heterogeneity (ITH) with response to anti-HER2 neoadjuvant chemotherapy. METHODS: Assessment of HER2 ITH was performed on whole tissue sections of pre-treatment samples from a cohort of 64 invasive breast carcinoma cases originally considered positive for HER2 and treated with anti-HER2 neoadjuvant chemotherapy. Both HER2 gene signal and protein expression were simultaneously evaluated by means of a single-slide dual assay, designated as a HER2 gene-protein assay (GPA). HER2 GPA was carried out as well on surgical resection tissues from 25 cases with incomplete therapeutic response. RESULTS: Nineteen of 64 cases (30%) showed HER2 ITH. Significantly more cases with HER2 ITH were found in the incomplete response group (56%, 14/25) than in the pCR group (13%, 5/39). Patients without ITH detectable by GPA had a 76% pCR outcome (34/45), as compared to 26% (5/19) for those with detectable ITH. Multivariate analysis demonstrated HER2 ITH, progesterone receptor positivity, and relatively low HER2/chromosome 17 centromere ratio to be significantly associated with incomplete response. CONCLUSIONS: HER2 ITH analyses conducted with GPA method revealed that HER2 ITH is an independent factor predicting incomplete response to anti-HER2 neoadjuvant chemotherapy.

MET and PTEN gene copy numbers and Ki-67 protein expression associate with pathologic complete response in ERBB2-positive breast carcinoma patients treated with neoadjuvant trastuzumab-based therapy.

BACKGROUND: Pathologic complete response (pCR) after neoadjuvant chemotherapy for breast cancer is associated with improved prognosis in aggressive tumor subtypes, including ERBB2- positive tumors. Recent adoption of pCR as a surrogate endpoint for clinical trials in early stage breast cancer in the neoadjuvant setting highlights the need for biomarkers that, alone or in combination, help predict the likelihood of response to treatment. METHODS: Biopsy specimens from 29 patients with invasive ductal carcinoma treated with trastuzumab-based therapy prior to definitive resection and pathologic staging were evaluated by dual color bright field in situ hybridization (dual ISH) using probes for MET, TOP2A, PTEN, and PIK3CA genes, each paired with centromeric probes to their respective chromosomes (chromosomes 7, 17, 10, and 3). Ki-67 expression was assessed by immunohistochemistry (IHC). Various parameters describing copy number alterations were evaluated for each gene and centromere probe to identify the optimal parameters for clinical relevance. Combinations of ISH parameters and IHC expression for Ki-67 were also evaluated. RESULTS: Of the four genes and their respective chromosomes evaluated by ISH, two gene copy number parameters provided statistically significant associations with pCR: MET gain or loss relative to chromosome 7 (AUC = 0.791, sensitivity = 92 % and specificity = 67 % at optimal cutoff, p = 0.0032) and gain of PTEN (AUC = 0.674, sensitivity = 38 % and specificity = 100 % at optimal cutoff, p = 0.039). Ki-67 expression was also found to associate significantly with pCR (AUC = 0.726, sensitivity = 100 % and specificity = 42 % at optimal cutoff, p = 0.0098). Combining gain or loss of MET relative to chromosome 7 with Ki-67 expression further improved the association with pCR (AUC = 0.847, sensitivity = 92 % and specificity = 83 % at optimal cutoffs, p = 0.0006). CONCLUSIONS: An immunogenotypic signature of low complexity comprising MET relative copy number and Ki-67 expression generated by dual ISH and IHC may help predict pCR in ERBB2-positive breast cancer treated with neoadjuvant chemotherapy and trastuzumab. These findings require validation in additional patient cohorts.

Emerging Biomarkers in Personalized Therapy of Lung Cancer.

The two clinically validated and Food and Drug Administration approved lung cancer predictive biomarkers (epidermal growth factor receptor mutations and anaplastic lymphoma kinase (ALK) translocations) occur in only about 20 % of lung adenocarcinomas and acquired resistance develops to first generation drugs. Several other oncogenic drivers for lung adenocarcinoma have emerged as potentially druggable targets with new predictive biomarkers. Oncologists are requesting testing for ROS1 translocations which predict susceptibility to crizotinib, already approved for ALK positive lung cancers. Other potential biomarkers which are currently undergoing clinical trials are RET, MET, HER2 and BRAF. Detection of these biomarkers includes fluorescent in situ hybridization and/or reverse transcriptase polymerase chain reaction (ROS1, RET, HER2), mutation analysis (BRAF) and immunohistochemistry (MET). Screening by immunohistochemistry may be useful for some biomarkers (ROS1, BRAF). Targeted next generation sequencing techniques may be useful as well. These five biomarkers are under consideration for inclusion in revised lung cancer biomarker guidelines by the College of American Pathologists, International Association for the Study of Lung Cancer and Association for Molecular Pathology.

Next-generation sequencing-based multi-gene mutation profiling of solid tumors using fine needle aspiration samples: promises and challenges for routine clinical diagnostics.

Increasing use of fine needle aspiration for oncological diagnosis, while minimally invasive, poses a challenge for molecular testing by traditional sequencing platforms due to high sample requirements. The advent of affordable benchtop next-generation sequencing platforms such as the semiconductor-based Ion Personal Genome Machine (PGM) Sequencer has facilitated multi-gene mutational profiling using only nanograms of DNA. We describe successful next-generation sequencing-based testing of fine needle aspiration cytological specimens in a clinical laboratory setting. We selected 61 tumor specimens, obtained by fine needle aspiration, with known mutational status for clinically relevant genes; of these, 31 specimens yielded sufficient DNA for next-generation sequencing testing. Ten nanograms of DNA from each sample was tested for mutations in the hotspot regions of 46 cancer-related genes using a 318-chip on Ion PGM Sequencer. All tested samples underwent successful targeted sequencing of 46 genes. We showed 100% concordance of results between next-generation sequencing and conventional test platforms for all previously known point mutations that included BRAF, EGFR, KRAS, MET, NRAS, PIK3CA, RET and TP53, deletions of EGFR and wild-type calls. Furthermore, next-generation sequencing detected variants in 19 of the 31 (61%) patient samples that were not detected by traditional platforms, thus increasing the utility of mutation analysis; these variants involved the APC, ATM, CDKN2A, CTNNB1, FGFR2, FLT3, KDR, KIT, KRAS, MLH1, NRAS, PIK3CA, SMAD4, STK11 and TP53 genes. The results of this study show that next-generation sequencing-based mutational profiling can be performed on fine needle aspiration cytological smears and cell blocks. Next-generation sequencing can be performed with only nanograms of DNA and has better sensitivity than traditional sequencing platforms. Use of next-generation sequencing also enhances the power of fine needle aspiration by providing gene mutation results that can direct personalized cancer therapy.

Delay to formalin fixation 'cold ischemia time': effect on ERBB2 detection by in-situ hybridization and immunohistochemistry.

The American Society of Clinical Oncology/College of American Pathologists ERBB2 testing guidelines address several pre-analytical variables known to affect ERBB2 testing accuracy. According to 2010 updated guidelines, the pre-analytical variable of time to tissue fixation (cold ischemia time) should be kept to <1 h, however, little has been published about cold ischemia time and its significance in ERBB2 testing. To that end, this study evaluated ERBB2 status using two different FDA-approved in-situ hybridization methods and an FDA-approved immunohistochemistry (IHC) assay in the largest cohort to date (n=84) of invasive breast carcinomas with tracked cold ischemia time. Cold ischemia time was stratified into four groups (<1 h (n=45), 1-2 h (n=27), 2-3 h (n=6), and >3 h (n=6)) and ERBB2 status was evaluated in each group by IHC (4B5) and by in-situ hybridization methodologies (PathVysion(®) fluorescence in situ hybridization and the INFORM HER2(®) dual in situ DNA probe assay). Both in-situ hybridization methods were evaluated using three ERBB2 scoring criteria (dual-probe guidelines, single-probe guidelines, and the FDA package insert scoring instructions). Fluorescence in-situ hybridization (FISH) and INFORM HER2(®) demonstrated 100% concordance in the detection of ERBB2 amplification by all three scoring guidelines at all cold ischemia time points. Agreement between in-situ hybridization methodologies and IHC was superior using single-probe guidelines compared with dual probe or FDA scoring instructions. In addition, Inform HER2(®) in-situ hybridization signals were significantly more intense than FISH at all cold ischemia time points, however, no significant loss of either chromosome 17 or ERBB2 signal was detected by FISH or Inform HER2(®) in-situ hybridization in cold ischemia times up to 3 h. On the basis of our findings, cold ischemia time up to 3 h has no deleterious effect on the detection of ERBB2 via in-situ hybridization or IHC.

Comparison of HercepTest™ mAb pharmDx (Dako Omnis, GE001) with Ventana PATHWAY anti-HER-2/neu (4B5) in breast cancer: correlation with HER2 amplification and HER2 low status.

Performance of the new CE-IVD-marked HercepTest™ mAb pharmDx (Dako Omnis) assay (HercepTest (mAb)) was compared against the PATHWAY® anti-HER-2/neu (4B5) (PATHWAY 4B5) assay using 119 pre-selected breast cancer samples covering the entire range of HER2 immunohistochemistry (IHC) expression scores (0, 1 + , 2 + , 3 +). The sensitivity and specificity of both assays were assessed based on consensus IHC scores and amplification status, as determined by fluorescence in situ hybridization (FISH) according to 2018 ASCO/CAP testing guidelines. There was a high concordance between results from the HercepTest (mAb) and PATHWAY 4B5 assays for HER2-negative (IHC 0, 1 + , 2 + and FISH negative) and HER2-positive (IHC 3 + , 2 + and FISH positive) breast carcinomas (98.2%). Regarding individual IHC scores, complete agreement was achieved in 69.7% (83/119) of cases, and all but one of the discordant cases were due to higher HER2-status scoring using the HercepTest (mAb). Thus, more tumors were overscored as IHC 2 + by HercepTest (mAb) (27 versus 15) as evidenced by their lower FISH positivity rate (48.1% versus 80%). However, two amplified tumors identified as IHC 2 + by HercepTest (mAb) were missed by PATHWAY 4B5 (IHC 1 +). Four additional cases identified as IHC 2 + by HercepTest (mAb), with FISH ratio < 2 but elevated gene counts (≥ 4 to < 6), were recorded negative by PATHWAY 4B5. The HercepTest (mAb) detects HER2 expression with higher sensitivity in tumors with gene amplification (ISH group 1) and increased gene counts (ISH group 4) as well as in HER2-low tumors (HER2 IHC2 + /FISH negative or IHC 1 +). Future studies will demonstrate whether this translates into improved patient selection especially for new HER2-directed therapies.

Neoplastic Cellularity Assessment in Molecular Testing.

CONTEXT.­: Neoplastic cellularity assessment has become an essential component of molecular oncology testing; however, there are currently no best practice recommendations or guidelines for this potentially variable step in the testing process. OBJECTIVE.­: To describe the domestic and international practices of neoplastic cellularity assessment and to determine how variations in laboratory practices affect neoplastic cellularity assessment accuracy. DESIGN.­: Data were derived from 57 US and international laboratories that participated in the 2019 College of American Pathologists Neoplastic Cellularity Proficiency Testing Survey (NEO-B 2019). NEO-B 2019 included 29 laboratory practice questions and 5 images exhibiting challenging histologic features. Participants assessed the neoplastic cellularity of hematoxylin-eosin-stained digital images, and results were compared to a criterion standard derived from a manual cell count. RESULTS.­: The survey responses showed variations in the laboratory practices for the assessment of neoplastic cellularity, including the definition of neoplastic cellularity, assessment methodology, counting practices, and quality assurance practices. In some instances, variation in laboratory practice affected neoplastic cellularity assessment performance. CONCLUSIONS.­: The results highlight the need for a consensus definition and improved standardization of the assessment of neoplastic cellularity. We put forth an initial set of best practice recommendations to begin the process of standardizing neoplastic cellularity assessment.

Immunohistochemical Validation of Rare Tissues and Antigens With Low Frequency of Occurrence: Recommendations From The Anatomic Pathology Patient Interest Association (APPIA).

Laboratories worldwide find it challenging to identify enough tissues and cases for verification and validation studies of low-incidence, rare antigens. These antigens have a low frequency of occurrence in the population, or have little or no expression in normal tissues. Validation studies are essential to assure testing standardization before introducing a new instrument, product, or test into the clinical laboratory. The College of American Pathologists has published comprehensive guidelines for the verification and validation of new immunohistochemical tests introduced into the laboratory menu. Within the guidelines, varied numbers of cases are required for nonpredictive versus predictive markers. However, regarding low-incidence antigens, the laboratory medical director determines the extent of validation required. Recommended practical solutions available to clinical laboratories for low-incidence validation include developing internal resources using the laboratory information system with retrospective and prospective search(s) of archival material and purchase of tissue microarray blocks, slides, or cell lines from external resources. Utilization of homemade multitissue blocks has proved to be extremely valuable in biomarker research and demonstrated great utility in clinical immunohistochemistry laboratories. Participation in External Quality Assessment program(s) may provide insufficient numbers or the ability to calculate concordance rates. However, supplementation with in-house tissues can allow a laboratory to reach the optimal number of cases needed for verification and/or validation schemes. An alternative approach is conducting a thorough literature search and correlating staining patterns of the new test to the expected results. These solutions may be used uniquely or together to assure consistent standardized testing.

Proficiency Testing of Standardized Samples Shows Very High Interlaboratory Agreement for Clinical Next-Generation Sequencing-Based Oncology Assays.

CONTEXT.­: Next-generation sequencing-based assays are being increasingly used in the clinical setting for the detection of somatic variants in solid tumors, but limited data are available regarding the interlaboratory performance of these assays. OBJECTIVE.­: To examine proficiency testing data from the initial College of American Pathologists (CAP) Next-Generation Sequencing Solid Tumor survey to report on laboratory performance. DESIGN.­: CAP proficiency testing results from 111 laboratories were analyzed for accuracy and associated assay performance characteristics. RESULTS.­: The overall accuracy observed for all variants was 98.3%. Rare false-negative results could not be attributed to sequencing platform, selection method, or other assay characteristics. The median and average of the variant allele fractions reported by the laboratories were within 10% of those orthogonally determined by digital polymerase chain reaction for each variant. The median coverage reported at the variant sites ranged from 1922 to 3297. CONCLUSIONS.­: Laboratories demonstrated an overall accuracy of greater than 98% with high specificity when examining 10 clinically relevant somatic single-nucleotide variants with a variant allele fraction of 15% or greater. These initial data suggest excellent performance, but further ongoing studies are needed to evaluate the performance of lower variant allele fractions and additional variant types.

Rapid assay for quantitative measurement of apoptosis in cultured cells and brain tissue.

Analysis of apoptosis in brain tissue following ischemia, hypoxia, or oxidative stress has technical limitations. The use of counting cells displaying apoptotic morphology is time intensive, vulnerable to sampling errors, and suffers from low numbers of total recorded events. Other cell death assays such as agarose gel analysis of DNA fragmentation, TUNEL, or ELISA are time intensive, limited to a single endpoint measure, and can be technically difficult to perform or reproduce. To overcome these limitations, we set out to develop a technique using flow cytometry to measure apoptosis based on the physical properties of light scatter produced from isolated nuclei. This dye/marker free approach would bypass many of the inherent encumbrances and reproducibility problems found in other apoptosis assays. Here we demonstrate that this new technique, using flow cytometry performed on isolated nuclei, allows rapid quantitation of apoptosis in a variety of brain tissues without the need for intercalating dyes or fluorescent markers. We conclude that this technique significantly improves currently available protocols to quantify apoptosis from tissue and offers the possibility to perform additional analysis on the same population of nuclei via downstream assays.

Clinical Validation of a Novel Commercial Reverse Transcription-Quantitative Polymerase Chain Reaction Screening Assay for Detection of ALK Translocations and Amplifications in Non-Small Cell Lung Carcinomas.

CONTEXT: -EGFR mutations and anaplastic lymphoma kinase (ALK) translocations have significant biologic and therapeutic implications in lung cancers, particularly lung adenocarcinomas. ALK translocations are less frequent compared with EGFR mutations; interestingly, these two abnormalities are most commonly mutually exclusive. The 2013 College of American Pathologists/Association for Molecular Pathology/International Association for the Study of Lung Cancer molecular testing guideline for lung cancers recommend a testing algorithm in which detection of ALK translocations using fluorescence in situ hybridization (FISH) is to be performed following testing for EGFR mutations. Such an algorithm is cost-effective but potentially slows down turnaround time; and as a secondary test, ALK FISH assay may not be completed because it requires the use of additional tissue, and the small biopsies or cytology specimens may have been exhausted in the extraction of nucleic acid for EGFR mutation screening. OBJECTIVE: -To provide efficient testing of both EGFR and ALK genetic alterations in small biopsies and cytology specimens. DESIGN: -We validated a highly sensitive ALK reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay as a screening tool for ALK translocations and amplifications. RESULTS: -We performed a retrospective review of cases previously tested by FISH and found that all FISH ALK translocation-positive specimens were RT-qPCR positive, and all FISH ALK translocation-negative cases were RT-qPCR negative (the sensitivity and specificity of the ALK RT-qPCR assay were 100%). CONCLUSION: -This assay allows rapid identification of ALK alterations, can be performed in conjunction with EGFR testing, and does not require use of valuable additional tumor tissue.

Next-Generation Sequencing of Matched Primary and Metastatic Rectal Adenocarcinomas Demonstrates Minimal Mutation Gain and Concordance to Colonic Adenocarcinomas.

CONTEXT: -Colorectal carcinoma is the third most common cause of cancer death in males and females in the United States. Rectal adenocarcinoma can have distinct therapeutic and surgical management from colonic adenocarcinoma owing to its location and anatomic considerations. OBJECTIVE: -To determine the oncologic driver mutations and better understand the molecular pathogenesis of rectal adenocarcinoma in relation to colon adenocarcinoma. DESIGN: -Next-generation sequencing was performed on 20 cases of primary rectal adenocarcinoma with a paired lymph node or solid organ metastasis by using an amplicon-based assay of more than 2800 Catalogue of Somatic Mutations in Cancer (COSMIC)-identified somatic mutations. RESULTS: -Next-generation sequencing data were obtained on both the primary tumor and metastasis from 16 patients. Most rectal adenocarcinoma cases demonstrated identical mutations in the primary tumor and metastasis (13 of 16, 81%). The mutations identified, listed in order of frequency, included TP53, KRAS, APC, FBXW7, GNAS, FGFR3, BRAF, NRAS, PIK3CA, and SMAD4. CONCLUSIONS: -The somatic mutations identified in our rectal adenocarcinoma cohort showed a strong correlation to those previously characterized in colonic adenocarcinoma. In addition, most rectal adenocarcinomas harbored identical somatic mutations in both the primary tumor and metastasis. These findings demonstrate evidence that rectal adenocarcinoma follows a similar molecular pathogenesis as colonic adenocarcinoma and that sampling either the primary or metastatic lesion is valid for initial evaluation of somatic mutations and selection of possible targeted therapy.

Next-Generation Sequencing of a Cohort of Pulmonary Large Cell Carcinomas Reclassified by World Health Organization 2015 Criteria.

CONTEXT: The classification of pulmonary large cell carcinoma has undergone a major revision with the recent World Health Organization (WHO) 2015 Classification. Many large cell carcinomas are now reassigned to either adenocarcinoma with solid pattern or nonkeratinizing squamous cell carcinoma based on immunopositivity for adenocarcinoma markers or squamous cell carcinoma markers, respectively. Large cell carcinomas that are negative for adenocarcinoma and squamous cell carcinoma immunomarkers are now classified as large cell carcinoma with null immunohistochemical features (LCC-N). Although a few studies investigated the mutation profile of large cell carcinomas grouped by immunostain profile before the publication of the new WHO classification, investigation of tumors previously diagnosed as large cell carcinoma and reclassified according to the 2015 WHO classification has not, to our knowledge, been reported. OBJECTIVE: To determine the mutation profiles of pulmonary large cell carcinomas reclassified by WHO 2015 criteria. DESIGN: Archival cases of non-small cell lung carcinoma with large cell carcinoma morphology (n = 17) were reclassified according to 2015 WHO criteria. To determine mutation profile, we employed Ion Torrent (Life Technologies, Carlsbad, California)-based next-generation sequencing (50 genes; more than 2800 mutations) in addition to real-time quantitative reverse transcription polymerase chain reaction for ALK translocation detection. RESULTS: Two of 17 cases (12%) were reclassified as LCC-N, and both had mutations-BRAF D594N in one case and KRAS G12C in the other case. Seven of 17 cases (41%) were reclassified in the adenocarcinoma with solid pattern group, which showed one KRAS G12C and one EGFR E709K + G719C double mutation in addition to mutations in TP53. Eight of 17 cases (47%) were reclassified in the nonkeratinizing squamous cell carcinoma group, which showed mutations in PIK3CA, CDKN2A, and TP53. No ALK translocations or amplifications were detected. CONCLUSIONS: The adenocarcinoma with solid pattern group showed mutations typical of adenocarcinoma, whereas the nonkeratinizing squamous cell carcinoma group showed mutations typical of squamous cell carcinoma. Both LCC-N cases had mutations associated with adenocarcinoma, supporting the hypothesis that LCC-N is related to adenocarcinoma.

BET protein antagonist JQ1 is synergistically lethal with FLT3 tyrosine kinase inhibitor (TKI) and overcomes resistance to FLT3-TKI in AML cells expressing FLT-ITD.

Recently, treatment with bromodomain and extraterminal protein antagonist (BA) such as JQ1 has been shown to inhibit growth and induce apoptosis of human acute myelogenous leukemia (AML) cells, including those expressing FLT3-ITD. Here, we demonstrate that cotreatment with JQ1 and the FLT3 tyrosine kinase inhibitor (TKI) ponatinib or AC220 synergistically induce apoptosis of cultured and primary CD34(+) human AML blast progenitor cells (BPC) expressing FLT3-ITD. Concomitantly, as compared with each agent alone, cotreatment with JQ1 and the FLT3-TKI caused greater attenuation of c-MYC, BCL2, and CDK4/6. Simultaneously, cotreatment with JQ1 and the FLT3-TKI increased the levels of p21, BIM, and cleaved PARP, as well as mediated marked attenuation of p-STAT5, p-AKT, and p-ERK1/2 levels in AML BPCs. Conversely, cotreatment with JQ1 and FLT3-TKI was significantly less active against CD34(+) normal bone marrow progenitor cells. Knockdown of BRD4 by short hairpin RNA also sensitized AML cells to FLT3-TKI. JQ1 treatment induced apoptosis of mouse Ba/F3 cells ectopically expressing FLT3-ITD with or without FLT3-TKI-resistant mutations F691L and D835V. Compared with the parental human AML FLT3-ITD-expressing MOLM13, MOLM13-TKIR cells resistant to AC220 were markedly more sensitive to JQ1-induced apoptosis. Furthermore, cotreatment with JQ1 and the pan-histone deacetylase inhibitor (HDI) panobinostat synergistically induced apoptosis of FLT3-TKI-resistant MOLM13-TKIR and MV4-11-TKIR cells. Collectively, these findings support the rationale for determining the in vivo activity of combined therapy with BA and FLT3-TKI against human AML cells expressing FLT3-ITD or with BA and HDI against AML cells resistant to FLT3-TKI.

Quantitative assessment of mutant allele burden in solid tumors by semiconductor-based next-generation sequencing.

OBJECTIVES: Identification of tumor-specific somatic mutations has had a significant impact on both disease diagnosis and therapy selection. The ability of next-generation sequencing (NGS) to provide a quantitative assessment of mutant allele burden, in numerous target genes in a single assay, provides a significant advantage over conventional qualitative genotyping platforms. METHODS: We assessed the quantitative capability of NGS and a primer extension-based matrix-assisted laser desorption ionization-time-of-flight (PE-MALDI) assay and directly correlated NGS mutant allele burden determination to morphologic assessment of tumor percentage in H&E-stained slides. RESULTS: Our results show a 100% concordance between NGS and PE-MALDI in mutant allele detection and a significant correlation between NGS and PE-MALDI for determining mutant allele burden when mutant allele burden is 10% or more. CONCLUSIONS: NGS-based mutation screening provides a quantitative assessment comparable to that of PE-MALDI. In addition, NGS also allows for a high degree of multiplexing and uses nanogram quantities of DNA, thereby preserving precious material for future analysis. Furthermore, this study provides evidence that H&E-based morphologic assessment of tumor burden does not correlate to actual tumor mutant allele burden frequency.

Genomic microarray analysis on formalin-fixed paraffin-embedded material for uveal melanoma prognostication.

Cytogenetic alterations are strong outcome prognosticators in uveal melanoma (UVM). Monosomy 3 (-3) and MYC amplification at 8q24 are commonly tested by fluorescence in situ hybridization (FISH). Alternatively, microarray analysis provides whole genome data, detecting partial chromosome loss, loss of heterozygosity (LOH), or abnormalities unrepresented by FISH probes. Nonfixed frozen tissue is conventionally used for microarray analysis but may not always be available. We assessed the feasibility of genomic microarray analysis for high resolution interrogation of UVM using formalin-fixed paraffin-embedded tissue (FFPET) as an alternative to frozen tissue (FZT). Enucleations from 44 patients (clinical trial NCT00952939) yielded sufficient DNA from FFPET (n = 34) and/or frozen tissue (n = 41) for comparative genomic hybridization and select single nucleotide polymorphism analysis (CGH/SNP) on Roche-NimbleGen OncoChip arrays. CEP3 FISH analysis was performed on matched cytology ThinPrep material. CGH/SNP analysis was successful in 30 of 34 FFPET and 41 of 41 FZT samples. Of 27 paired FFPET/FZT samples, 26 (96.3%) were concordant for at least four of six major recurrent abnormalities (-3, +8q, -1p, +6p, -6q, -8p), and 25 of 27 (92.6%) were concordant for -3. Results of CGH/SNP were concordant with the CEP3 FISH results in 27 of 30 (90%) FFPET and 38 of 41 (92.6%) FZT cases; partial -3q was detected in two CEP3 FISH-negative cases and whole chromosome 3, 4, and 6 SNP-LOH in one case. CGH detection of -3, +8q, -8p on FFPET and FZT showed significant correlation with the clinical outcome measures (metastasis development, time to progression, survival). Results of the UVM genotyping by CGH/SNP on FFPET are highly concordant with those of the FZT analysis and with those of the CEP3 FISH analysis, and therefore CGH/SNP is a practical method for UVM prognostication. Genome-wide coverage provides additional data with potential relevance to UVM biology, diagnosis, and prognosis.

Highly active combination of BRD4 antagonist and histone deacetylase inhibitor against human acute myelogenous leukemia cells.

The bromodomain and extra-terminal (BET) protein family members, including BRD4, bind to acetylated lysines on histones and regulate the expression of important oncogenes, for example, c-MYC and BCL2. Here, we demonstrate the sensitizing effects of the histone hyperacetylation-inducing pan-histone deacetylase (HDAC) inhibitor panobinostat on human acute myelogenous leukemia (AML) blast progenitor cells (BPC) to the BET protein antagonist JQ1. Treatment with JQ1, but not its inactive enantiomer (R-JQ1), was highly lethal against AML BPCs expressing mutant NPM1c+ with or without coexpression of FLT3-ITD or AML expressing mixed lineage leukemia fusion oncoprotein. JQ1 treatment reduced binding of BRD4 and RNA polymerase II to the DNA of c-MYC and BCL2 and reduced their levels in the AML cells. Cotreatment with JQ1 and the HDAC inhibitor panobinostat synergistically induced apoptosis of the AML BPCs, but not of normal CD34(+) hematopoietic progenitor cells. This was associated with greater attenuation of c-MYC and BCL2, while increasing p21, BIM, and cleaved PARP levels in the AML BPCs. Cotreatment with JQ1 and panobinostat significantly improved the survival of the NOD/SCID mice engrafted with OCI-AML3 or MOLM13 cells (P < 0.01). These findings highlight cotreatment with a BRD4 antagonist and an HDAC inhibitor as a potentially efficacious therapy of AML.