Poziotinib for EGFR exon 20-mutant NSCLC: Clinical efficacy, resistance mechanisms, and impact of insertion location on drug sensitivity.

We report a phase II study of 50 advanced non-small cell lung cancer (NSCLC) patients with point mutations or insertions in EGFR exon 20 treated with poziotinib (NCT03066206). The study achieved its primary endpoint, with confirmed objective response rates (ORRs) of 32% and 31% by investigator and blinded independent review, respectively, with a median progression-free survival of 5.5 months. Using preclinical studies, in silico modeling, and molecular dynamics simulations, we found that poziotinib sensitivity was highly dependent on the insertion location, with near-loop insertions (amino acids A767 to P772) being more sensitive than far-loop insertions, an observation confirmed clinically with ORRs of 46% and 0% observed in near versus far-loop, respectively (p = 0.0015). Putative mechanisms of acquired resistance included EGFR T790M, MET amplifications, and epithelial-to-mesenchymal transition (EMT). Our data demonstrate that poziotinib is active in EGFR exon 20-mutant NSCLC, although this activity is influenced by insertion location.

Analytical Validation of the Next-Generation Sequencing Assay for a Nationwide Signal-Finding Clinical Trial: Molecular Analysis for Therapy Choice Clinical Trial.

The National Cancer Institute-Molecular Analysis for Therapy Choice (NCI-MATCH) trial is a national signal-finding precision medicine study that relies on genomic assays to screen and enroll patients with relapsed or refractory cancer after standard treatments. We report the analytical validation processes for the next-generation sequencing (NGS) assay that was tailored for regulatory compliant use in the trial. The Oncomine Cancer Panel assay and the Personal Genome Machine were used in four networked laboratories accredited for the Clinical Laboratory Improvement Amendments. Using formalin-fixed paraffin-embedded clinical specimens and cell lines, we found that the assay achieved overall sensitivity of 96.98% for 265 known mutations and 99.99% specificity. High reproducibility in detecting all reportable variants was observed, with a 99.99% mean interoperator pairwise concordance across the four laboratories. The limit of detection for each variant type was 2.8% for single-nucleotide variants, 10.5% for insertion/deletions, 6.8% for large insertion/deletions (gap ≥4 bp), and four copies for gene amplification. The assay system from biopsy collection through reporting was tested and found to be fully fit for purpose. Our results indicate that the NCI-MATCH NGS assay met the criteria for the intended clinical use and that high reproducibility of a complex NGS assay is achievable across multiple clinical laboratories. Our validation approaches can serve as a template for development and validation of other NGS assays for precision medicine.

GNAS is frequently mutated in both low-grade and high-grade disseminated appendiceal mucinous neoplasms but does not affect survival.

We analyzed a series of 55 disseminated appendiceal mucinous neoplasms treated at our institution for GNAS and KRAS mutations in an attempt to correlate mutation status with clinicopathological findings and patient survival. GNAS mutations (p.R201H, c.602G>A and p.R201C, and c.602C>T) were identified in 17 (31%) of 55 of disseminated mucinous neoplasms and were found in 8 (35%) of 23 low-grade mucinous neoplasms, 7 (37%) of 19 high-grade mucinous adenocarcinomas lacking a signet ring cell component, and 2 (15%) of 13 high-grade mucinous adenocarcinomas with a signet ring cell component. All 7 mucinous adenocarcinomas composed of pure (>95%) signet ring cells harbored wild-type GNAS. There was no significant association between GNAS mutations and sex and age (both with P > .05) or between GNAS mutations and individual adverse histologic features including cytologic grade, destructive invasion, tumor cellularity, angiolymphatic invasion, perineural invasion, and signet ring cells (all with P > .05). KRAS mutations were identified in 22 (40%) of 55 disseminated mucinous neoplasms. GNAS-mutated disseminated appendiceal mucinous neoplasms more frequently harbored concurrent KRAS mutations compared with GNAS wild-type tumors (65% versus 29%, P = .018). GNAS mutations were not significantly associated with overall survival (both with P > .05). Only overall tumor grade was an independent predictor of overall survival in the multivariate analysis (P = .01). Our results indicate that GNAS mutations are frequently identified in both low-grade and high-grade disseminated appendiceal mucinous neoplasms indicating that GNAS mutation status cannot be used to distinguish between low-grade from high-grade appendiceal mucinous neoplasms.

Preoperative GNAS and KRAS testing in the diagnosis of pancreatic mucinous cysts.

PURPOSE: Management guidelines for pancreatic intraductal papillary mucinous neoplasms (IPMN) and mucinous cystic neoplasms (MCN) are based on the assumption that mucinous cysts can be accurately distinguished from other pancreatic cystic lesions. Previous studies using surgical material have identified recurrent mutations in GNAS and KRAS in pancreatic mucinous neoplasms. Yet, the diagnostic utility of testing for both genes in pancreatic cyst fluid obtained by endoscopic ultrasound-fine-needle aspiration (EUS-FNA) remains unclear. EXPERIMENTAL DESIGN: GNAS and KRAS testing was performed on EUS-FNA pancreatic cyst fluid from 91 pancreatic cysts: 41 IPMNs, 9 IPMNs with adenocarcinoma, 16 MCNs, 10 cystic pancreatic neuroendocrine tumors (PanNET), 9 serous cystadenomas (SCA), 3 retention cysts, 2 pseudocysts, and 1 lymphoepithelial cyst. RESULTS: Mutations in GNAS were detected in 16 (39%) IPMNs and 2 (22%) IPMNs with adenocarcinoma. KRAS mutations were identified in 28 (68%) IPMNs, 7 (78%) IPMNs with adenocarcinoma, and 1 (6%) MCN. Mutations in either gene were present in 34 (83%) IPMNs, 8 (89%) IPMNs with adenocarcinoma, and 1 (6%) MCN. No mutations were found in cystic PanNETs, SCAs, retention cysts, pseudocysts, and a lymphoepithelial cyst. GNAS and KRAS mutations had 100% specificity [95% confidence interval (CI), 0.83-1.00] but 65% sensitivity (95% CI, 0.52-0.76) for mucinous differentiation. Among IPMNs, mutations in either gene had 98% specificity (95% CI, 0.86-1.00) and 84% sensitivity (95% CI, 0.70-0.92). CONCLUSIONS: The combination of GNAS and KRAS testing was highly specific and sensitive for IPMNs; however, the lack of sensitivity for MCNs highlights the need for additional markers to improve the detection of pancreatic mucinous neoplasms.

Integration of KRAS testing in the diagnosis of pancreatic cystic lesions: a clinical experience of 618 pancreatic cysts.

With improvements in abdominal imaging, detection of incidental pancreatic cysts are becoming increasingly common. Analysis of pancreatic cyst fluid from fine-needle aspiration is particularly important in identifying intraductal papillary mucinous neoplasms (IPMNs) and mucinous cystic neoplasms (MCNs), which have significant implications in clinical intervention and follow-up. Previous controlled studies have shown that KRAS mutations in cyst fluid are highly specific for mucinous differentiation in pancreatic cysts; however, this has not been examined in the clinical setting. Over a 6-year study period, 618 pancreatic cyst fluids obtained by fine-needle aspiration at the time of endoscopic ultrasound were tested for KRAS mutations as part of routine evaluation for a cystic neoplasm. Of the 618 specimens, 603 (98%) from 546 patients were satisfactory for molecular analysis. Patients ranged in age from 17 to 90 years (mean, 63.9 years) and were predominantly female (68%). Pancreatic cysts were relatively evenly distributed throughout the pancreas and ranged in size from 0.6 to 11.0 cm (mean, 2.3 cm). Mutations in KRAS were detected in 232 of 603 (38%) aspirates. Although sufficient for molecular analysis, 320 of 603 (53%) specimens were either less than optimal (38%) or unsatisfactory (15%) for cytopathologic diagnosis. Surgical follow-up information was available for 142 (26%) patients and consisted of 53 KRAS-mutated and 89 KRAS-wild-type cysts. Overall, KRAS mutations had a specificity of 100%, but a sensitivity of 54% for mucinous differentiation. When stratified by cyst type, KRAS had a sensitivity of 67% and 14% for IPMNs and MCNs, respectively. In summary, KRAS mutations were highly specific for mucinous differentiation, but were inadequate in identifying MCNs. Future molecular studies and the combination of other fluid markers are required to improve the detection and classification of pancreatic mucinous neoplasms by endoscopic ultrasound fine-needle aspiration.

The prognostic value of Ki-67, p53, epidermal growth factor receptor, 1p36, 9p21, 10q23, and 17p13 in skull base chordomas.

CONTEXT: Skull base chordomas are rare, locally aggressive, notochord-derived neoplasms for which prognostically relevant biomarkers are not well established. OBJECTIVE: To evaluate whether newly discovered molecular alterations in chordomas have prognostic significance similar to what has been described regarding Ki-67 proliferation index. DESIGN: We conducted a retrospective study of 28 cases of primary clival chordomas. RESULTS: Ki-67 proliferation index 5% or more, p53 accumulation, and epidermal growth factor receptor expression were seen in 32%, 44%, and 8% of chordomas, respectively. 1p loss of heterozygosity (LOH) and/or 1p36 hemizygous deletion was seen in 30% of tumors, while 9p LOH and/or 9p21 homozygous deletion was seen in 21% of cases. Loss of heterozygosity at 10q23 and 17p13 were identified in 57% and 52% of cases, respectively. Ki-67 proliferation index 5% or more and 9p LOH were significantly associated with a shorter overall survival, while homozygous deletion at 9p21 via fluorescence in situ hybridization approached significance. No correlation with survival was found for p53 or epidermal growth factor receptor expression, 1p36 hemizygous deletion, or LOH at 1p, 10q23, or 17p13. CONCLUSIONS: Chordomas with elevated Ki-67 proliferation index or deletion at 9p21 may be at risk for a more aggressive clinical course and shorter survival. These biomarkers may thus be used to improve therapeutic stratification.

Molecular alterations in atypical adenomatous hyperplasia occurring in benign and cancer-bearing lungs.

Atypical adenomatous hyperplasia (AAH) is considered to be a precursor lesion of the lung adenocarcinoma. Several genetic abnormalities have been reported in AAH associated with adenocarcinoma, but little is known about AAH associated with benign lung lesions. To address this we compared the molecular characteristics of AAH present in benign conditions to those coexisting with carcinoma. Seven cases of AAH from resected non-neoplastic lungs (AAH-B) and 12 cases from lungs resected for primary lung carcinoma (AAH-M) were analyzed for loss of heterozygosity (LOH) using 21 polymorphic microsatellite markers situated in proximity to known tumor suppressor genes on chromosomes 3p, 5q, 7p, 9p, 10q, and 17p. Direct DNA sequencing for K-ras mutation was also performed. There was a broad range of LOH in both groups. No LOH was identified in 3 cases (25%) of AAH-M, but all cases of AAH-B showed LOH (P=0.26). Six cases (50%) of AAH-M and 3 cases (43%) of AAH-B showed loss at 1 marker (P=0.99). LOH at 2 or more markers was identified in 3 (25%) cases of AAH-M and 4 (57%) cases of AAH-B (P=0.32). LOH was most frequently detected on chromosomes 3p and 10q in both groups. The difference in overall fractional allelic loss between the 2 groups did not reach statistical significance. K-ras mutations were not identified in either group. Our results showed a significant overlap in LOH patterns between AAH with or without coexistent lung malignancy. Therefore, AAH may represent a smoking induced low-grade neoplastic lesion that may be a precursor lesion of only a subset of invasive lung adenocarcinoma.

Overexpression of Dicer in precursor lesions of lung adenocarcinoma.

Differential microRNA (miR) expression is described in non-small cell lung carcinoma. miR biogenesis requires a set of proteins collectively referred to as the miR machinery. In the proposed multistep carcinogenesis model, peripheral adenocarcinoma of the lung develops from noninvasive precursor lesions known as atypical adenomatous hyperplasia (AAH) and bronchioloalveolar carcinoma (BAC). The gene array analysis of BAC and adenocarcinoma showed a transient up-regulation of Dicer (a key effector protein for small interfering RNA and miR function) and PACT along with down-regulation of most genes encoding miR machinery proteins. Immunohistochemically, Dicer was up-regulated in AAH and BAC and down-regulated in areas of invasion and in advanced adenocarcinoma. A fraction of adenocarcinomas lose Dicer as a result of deletions at the Dicer locus. Expanded immunohistochemical and Western blot analysis showed higher Dicer level in squamous cell carcinoma (SCC) of the lung when compared with adenocarcinoma. Other proteins of the RNA-induced silencing complex (RISC; SND1, PACT, and FXR1) were also present at higher levels in a SCC cell line when compared with an adenocarcinoma cell line. In conclusion, the stoichiometry of miR machinery and RISC depends on histologic subtype of lung carcinoma, varies along the AAH-BAC-adenocarcinoma sequence, and might explain the observed abnormal miR profile in lung cancer. The status of the endogenous miR machinery in various histologic subtypes and stages of lung cancer may help to predict the toxicity of and susceptibility to future RNA interference-based therapy.

Analysis of loss of heterozygosity in single-system and multisystem Langerhans' cell histiocytosis.

The contribution of molecular mutations to the progression of Langerhans' cell histiocytosis (LCH) is not well understood. This study analyzes fractional allelic loss (FAL) across a series of tumor suppressor genes (TSGs) comparing LCH of various clinical stages and LCH involving organs of various degrees of prognostic risk. Polymerase chain reaction (PCR) amplification, using fluorescent-labeled primers targeting 6 TSGs, was performed on extracted DNA. The PCR products were analyzed using a capillary electrophoresis genetic analyzer. Ratios of the peak heights in informative cases were compared between unaffected and lesional tissue to identify loss of heterozygosity (LOH). Fisher's exact testing was used to analyze the results. Fourteen children with single-system involvement (SS-LCH) and 10 with multisystem involvement (MS-LCH) were included. High-risk or special-site organ involvement was seen in 13 children and low-risk organ involvement in 10. The mean FAL in MS-LCH (62.7%) was statistically significantly higher than in SS-LCH (40.3%). The FAL in children with risk or special-site LCH (53.2%) was also significantly higher than in children with low-risk LCH (39.6%). Markers on 5q had significantly higher FAL in MS-LCH (76.3%) and children with risk or special-site organ involvement (72.7%) compared with SS-LCH (46.2%) and children with low-risk organ involvement (37.5%). These data suggest that more extensive and higher-risk forms of LCH have evidence of more mutational events at TSGs. Further investigation of the potential use of LOH at 5q23 will be necessary to establish utility for this assay to predict disease progression and poor outcome.

BRAF mutation is unusual in chronic lymphocytic thyroiditis-associated papillary thyroid carcinomas and absent in non-neoplastic nuclear atypia of thyroiditis.

Chronic lymphocytic thyroiditis (CLT) has an epidemiological relationship to papillary thyroid carcinoma (PTC). The follicular epithelium in CLT can be markedly atypical, with cytologic changes ranging from oncocytic morphology to clearing and overlapping. At the molecular level, the association between CLT and PTC is more controversial. In order to further characterize the molecular changes in CLT, this study examined the BRAF gene in 27 patient samples with CLT and without carcinoma and 28 samples with CLT and carcinoma (12 conventional papillary carcinomas, 13 follicular variants, and 3 tall cell variants). Microdissection, PCR, and sequencing of exon 15 of the BRAF gene were performed. BRAF mutations were uncommon in the cases studied with only two microscopic and two clinically sized PTCs had BRAF mutations (14%). There was no evidence of BRAF mutation in any of the areas with atypical follicular epithelium in CLT. These data suggest that BRAF is a less frequent mechanism of tumorigenesis in a background of CLT and that BRAF mutation is not present in the atypical follicular epithelium of CLT.