Genomic profiling of solid tumors harboring BRD4-NUT and response to immune checkpoint inhibitors.

BACKGROUND: The translocation t(15:19) produces the oncogenic BRD4-NUT fusion which is pathognomonic for NUT carcinoma (NC), which is a rare, but extremely aggressive solid tumor. Comprehensive genomic profiling (CGP) by hybrid-capture based next generation sequencing of 186+ genes of a cohort of advanced cancer cases with a variety of initial diagnoses harboring BRD4-NUT may shed further insight into the biology of these tumors and possible options for targeted treatment. CASE PRESENTATION: Thirty-one solid tumor cases harboring a BRD4-NUT translocation are described, with only 16% initially diagnosed as NC and the remainder carrying other diagnoses, most commonly NSCLCNOS (22%) and lung squamous cell carcinoma (NSCLC-SCC) (16%). The cohort was all microsatellite stable and harbored a low Tumor Mutational Burden (TMB, mean 1.7 mut/mb, range 0-4). In two index cases, patients treated with immune checkpoint inhibitors (ICPI) had unexpected partial or better responses of varying duration. Notably, four cases - including the two index cases - were negative for PD-L1 expression. Neo-antigen prediction for BRD4-NUT and then affinity modeling of the peptide-MHC (pMHC) complex for an assessable index case predicted very high affinity binding, both on a ranked (99.9%) and absolute (33 nM) basis. CONCLUSIONS: CGP identifies BRD4-NUT fusions in advanced solid tumors which carry a broad range of initial diagnoses and which should be re-diagnosed as NC per guidelines. A hypothesized mechanism underlying responses to ICPI in the low TMB, PD-L1 negative index cases is the predicted high affinity of the BRD4-NUT fusion peptide to MHC complexes. Further study of pMHC affinity and response to immune checkpoint inhibitors in patients with NC harboring BRD4-NUT is needed to validate this therapeutic hypothesis.

Clinical types of checkpoint inhibitor-related pneumonitis in lung cancer patients: a multicenter experience.

BACKGROUND: Checkpoint inhibitor-related pneumonitis (CIP) is not well classified according to clinical factors. We propose different clinical sub-types of CIP based on clinical factors and investigated the corresponding clinical features, treatments, and outcomes. METHODS: We conducted a multicenter retrospective study of patients with lung cancer (including non-small cell lung cancer and small cell lung cancer) who developed CIP. The clinical characteristics, radiologic features, treatments, and outcomes of CIP were analyzed. RESULTS: A total of 55 patients developed CIP and were classified into 3 groups as follows: 21 in the pure type (PT) group, 14 in the induced type (IT) group, and 20 in the mixed type (MT) group. The incidence of severe (grade 3-5) pneumonitis was significantly higher in the IT group than in the PT and MT groups (71.4% vs. 14.3% vs. 50.0%, P=0.002). Antiviral therapy was significantly more frequent in the IT group than in the PT and MT groups. Antibiotic therapy was administered in 23.8%, 71.4%, and 80.0% of patients with the PT, IT, and MT, respectively. The improvement time in the PT group was longer than that in the IT and MT groups (0.9 vs. 0.5 vs. 0.3 months, P=0.028). Patients with the PT had a better tumor response to immune checkpoint inhibitors (ICIs) than those with the other 2 types [overall response rate (ORR), 78% vs. 31% vs. 44%, P=0.027]. CONCLUSIONS: The clinical classification of CIP may favor strategies for treatments and predict the tumor response to ICIs.

Pan-Cancer Analysis of BRCA1 and BRCA2 Genomic Alterations and Their Association With Genomic Instability as Measured by Genome-Wide Loss of Heterozygosity.

PURPOSE: BRCA1 or BRCA2 loss of function results in homologous recombination deficiency (HRD), which is targetable by poly (ADP-ribose) polymerase (PARP) inhibitors and other DNA-damaging agents. In cancers associated with germline BRCA1/2 alterations (BRCA1/2-associated cancers: breast, ovarian, pancreatic, prostate), BRCA1/2 alterations result in HRD and are biomarkers for PARP inhibitor use. In other (non-BRCA1/2-associated) cancer types, the association between BRCA1/2 alteration and HRD is less clear. METHODS: A total of 234,154 tumor samples were sequenced by hybrid capture-based comprehensive genomic profiling. Somatic, germline, and zygosity status was determined computationally. BRCA1/2 alterations were classified as predicted germline/somatic and biallelic/monoallelic. Genome-wide loss of heterozygosity (gLOH) was evaluated as a marker of HRD. RESULTS: BRCA1/2 alterations were observed at a 4.7% frequency. BRCA1/2 mutations were predicted germline in 57.4% of BRCA1/2-associated and 37.2% of non-BRCA1/2-associated cancers. The fraction of BRCA1/2-altered cases that were biallelic was 68.7%, with a higher biallelic fraction in BRCA1/2-associated (89.9%) versus non-BRCA1/2-associated cancers (43.6%). Differences in tissue distribution of biallelic BRCA1 versus BRCA2 alterations were noted, including a higher rate of biallelic BRCA2 alteration in prostate cancer. Biallelic BRCA1/2 alteration was observed at a 3.2% frequency (BRCA1/2-associated cancers, 8.9%; non-BRCA1/2-associated cancers, 1.3%) and > 1% frequency in at least 13 cancer types. Across cancer types, biallelic BRCA1/2 alteration was associated with increased gLOH versus monoallelic or wild-type BRCA1/2; predicted germline or somatic mutations were both associated with elevated gLOH. CONCLUSION: Biallelic BRCA1/2 alterations were associated with elevated gLOH in diverse cancer types, including those not traditionally associated with BRCA1/2 cancer syndromes. Biomarker development for PARP inhibitors should integrate methods to distinguish biallelic from monoallelic BRCA1/2 status, and biallelic BRCA1/2 alteration should be broadly evaluated across cancer types as a biomarker for underlying HRD and PARP inhibitor sensitivity.

Retrospective analysis of real-world data to determine clinical outcomes of patients with advanced non-small cell lung cancer following cell-free circulating tumor DNA genomic profiling.

OBJECTIVES: Liquid biopsy and comprehensive genomic profiling (CGP) of circulating tumor DNA (ctDNA) are increasingly used for detection of targetable genomic alterations (GA) in non-small cell lung cancer (NSCLC). To examine the clinical outcomes for patients following CGP using liquid biopsy versus tissue biopsy, receipt of matched targeted therapy post-CGP and associated outcomes were evaluated in the real-world setting. METHODS: 6491 patients with NSCLC and liquid biopsy (N = 937 tests) and/or tissue (N = 5582 tests) CGP were included in a de-identified commercial clinico-genomic database. Targetable GAs included National Comprehensive Cancer Network NSCLC guideline biomarkers. Clinical characteristics, real-world progression, and real-world response (rwR) were obtained via technology-enabled abstraction of clinician notes and radiology/pathology reports. RESULTS: At the time of liquid biopsy CGP, 53% (496/937) of patients were documented to have received ≥1 line of prior therapy (tissue CGP: 13%, 735/5582). 90% (832/928) of liquid biopsy cases had evidence of ctDNA. A targetable GA was detected in 20% (188/937) of liquid biopsy and 22% (1215/5582) of tissue CGP cases. Use of matched targeted therapy overall was similar post-liquid biopsy or post-tissue CGP but varied considerably across emerging (25%, 79/317) versus standard of care (SOC) (74%, 475/640) GA. Real-world-progression free survival for patients receiving SOC first line matched targeted therapy administered following liquid biopsy (n = 33) and tissue (n = 229) CGP were similar (13.8 vs 10.6 months; aHR = 0.68 [0.36-1.26]). Among patients evaluated for rwR, overall response rate (partial/complete response) to matched targeted therapy post-liquid biopsy CGP was 75% (39/52) versus 66% post-tissue CGP (254/385, P = 0.51). CONCLUSION: Retrospective analysis of real-world clinico-genomic data demonstrated that clinical outcomes on matched targeted therapy were similar following liquid biopsy and tissue CGP in NSCLC, which suggests routine clinical use of liquid biopsy CGP can reliably guide therapy selection.

Germline and somatic DNA repair gene alterations in prostate cancer.

BACKGROUND: Emerging evidence has suggested that DNA repair gene alterations may be important in prostate cancer pathogenesis. In the current study, the authors sought to characterize alterations in DNA repair pathway genes in both primary and metastatic prostate tumors with attention to tissue distribution as well as specific genomic alterations. METHODS: The authors studied the distribution and type of alterations in 24 genes that are considered important for DNA repair in 944 prostate cancers harvested from localized and metastatic tumors. Tumor DNA underwent hybrid capture for all coding exons of 287 or 395 cancer-related genes plus select introns from 19 or 31 genes frequently rearranged in cancer. Captured libraries were sequenced to a median exon coverage depth of >×500. Specific genomic alterations were characterized and the frequencies of mutations by tissue site (prostate vs metastases) were compared using logistic regression. RESULTS: A total of 152 patients from the cohort of 944 men (16%) harbored a germline or somatic mutation in ≥1 DNA repair genes. The most frequently mutated genes were BRCA2 (11.4%) and ATM (5.8%), followed by MSH6 (2.5%) and MSH2 (2.1%). Mutations were identified in approximately 20.1% of primary prostate tumors compared with 18.8% of bone metastases. When stratified by tissue site, the highest rates of DNA repair mutations were found in solid organ metastases, including brain and visceral metastases, compared with prostate. CONCLUSIONS: DNA repair gene mutations are more common in metastatic than localized prostate tumors. Visceral and other solid organ metastases appear enriched for these mutations compared with localized tumors or bone and lymph node metastases.

Genomic profiling of cell-free circulating tumor DNA in patients with colorectal cancer and its fidelity to the genomics of the tumor biopsy.

BACKGROUND: Liquid biopsy offers the ability to non-invasively analyze the genome of a tumor through circulating tumor DNA (ctDNA) to identify targetable and prognostic genomic alterations. Few studies have rigorously analyzed ctDNA results and determined the fidelity with which they recapitulate the genomics of a sequenced tissue sample obtained from the same tumor. The clinical utility study (CUS) for the FoundationACT™ ctDNA assay (Foundation Medicine, Cambridge, MA, USA; NCT02620527) is a multi-center prospective clinical study for multiple solid tumor types to compare genomic profiling of paired tissue and blood samples from the same patient. In this subset of the study, paired specimens from 96 patients with colorectal cancer (CRC) were analyzed with comprehensive genomic profiling (CGP) of the tumor tissue sample (FoundationOne®) and blood sample (FoundationACT™). METHODS: Both samples underwent CGP using the hybrid capture-based Illumina Hi-Seq technology. Maximum somatic allele frequency (MSAF) was used to estimate the fraction of ctDNA in the sample. The set of genes and targeted regions common to both tumor and liquid were compared for each subject. RESULTS: Among these patients, 61% were male; 74% had clinical stage IV disease, 19% had clinical stage III disease, and 7% had clinical stage II disease. Time between the tissue biopsy and liquid biopsy (range, 0-709 days) had a significant impact on the positive percent agreement (PPA) between the two assays. Eighty percent of cases had evidence of ctDNA in the blood (MSAF >0). For all cases with MSAF >0, 171 base substitutions and insertions/deletions (indels) were identified in the tumor, and 79% (PPA) of these identical alterations were also identified in matched ctDNA samples; PPA increased to 87% for cases <270 days between the tissue and liquid biopsy, 95% for <90 days, and 100% PPA for <30 days. All known and likely short variants in KRAS, NRAS, and BRAF were analyzed independently as testing of these genes is recommended by the National Comprehensive Cancer Network (NCCN) for patients with CRC and have therapeutic implications. For NCCN genes, PPA was 80% for all time points for short variants; PPA increased to 90% for cases <270 days between the tissue and liquid biopsy. There was high concordance for KRAS G12X between tissue and liquid: overall percent agreement (97%), PPA (93%), negative percent agreement (NPA) (100%), positive predictive value (PPV) (100%), and negative predictive value (NPV) (96%) for the <270 day cohort. CONCLUSIONS: In cases where tumor tissue profiling is not possible, these results provide compelling evidence that genomic profiling of ctDNA in late stage CRC shows a high concordance with tumor tissue sequencing results and can be used to identify most clinically relevant alterations capable of guiding therapy for these patients.

Molecular testing strategies in non-small cell lung cancer: optimizing the diagnostic journey.

Molecular testing identifies patients with advanced non-small cell lung cancer (NSCLC) who may benefit from targeted therapy or immunotherapy (i.e., immune checkpoint inhibitor treatment for patients with high tumor mutational burden (TMB), microsatellite instability-high or mismatch repair-deficient tumors). Current guidelines state that molecular testing should be conducted at the time of initial diagnosis and tumor progression on targeted therapy. In real-world clinical practice in the United States (US), molecular testing is often not conducted or happens late in the diagnostic journey, resulting in delayed or inappropriate treatment. Herein, we review the rationale for molecular testing in advanced NSCLC, along with best-practice guidelines based on published recommendations and our own clinical experience, including a case study. We propose three strategies to optimize molecular testing in newly diagnosed patients with advanced NSCLC: (I) pulmonologists, interventional radiologists, or thoracic surgeons order molecular tests as soon as advanced NSCLC with an adenocarcinoma component is suspected; (II) liquid biopsies conducted early in the diagnostic pathway; and (III) pathologist-directed reflex testing, as conducted in other areas of oncology. To help facilitate these strategies, we outline our recommendations for optimal sample collection techniques and stewardship. In summary, we believe that implementation of these individual strategies will allow clinicians to effectively leverage available treatment options for advanced NSCLC, reducing the time to optimal treatment and improving patient outcomes.

MET Genomic Alterations in Head and Neck Squamous Cell Carcinoma (HNSCC): Rapid Response to Crizotinib in a Patient with HNSCC with a Novel MET R1004G Mutation.

Identification of effective targeted therapies for recurrent/metastatic head and neck squamous cell carcinoma (HNSCC) remains an unmet medical need. A patient with platinum-refractory recurrent oral cavity HNSCC underwent comprehensive genomic profiling (CGP) that identified an activating MET mutation (R1004). The patient was treated with the oral MET tyrosine kinase inhibitor crizotinib with rapid response to treatment.Based on this index case, we determined the frequency of MET alterations in 1,637 HNSCC samples, which had been analyzed with hybrid capture-based CGP performed in the routine course of clinical care. The specimens were sequenced to a median depth of >500× for all coding exons from 182 (version 1, n = 24), 236 (version 2, n = 326), or 315 (version 3, n = 1,287) cancer-related genes, plus select introns from 14 (version 1), 19 (version 2), or 28 (version 3) genes frequently rearranged in cancer. We identified 13 HNSCC cases (0.79%) with MET alterations (4 point mutation events and 9 focal amplification events). MET-mutant or amplified tumors represent a small but potentially actionable molecular subset of HNSCC. KEY POINTS: This case report is believed to be the first reported pan-cancer case of a patient harboring a MET mutation at R1004 demonstrating a clinical response to crizotinib, in addition to the first documented case of head and neck squamous cell carcinoma (HNSCC) with any MET alteration responding to crizotinib.The positive response to MET inhibition in this patient highlights the significance of comprehensive genomic profiling in advanced metastatic HNSCC to identify actionable targetable molecular alterations as current treatment options are limited.

A Novel Next-Generation Sequencing Approach to Detecting Microsatellite Instability and Pan-Tumor Characterization of 1000 Microsatellite Instability-High Cases in 67,000 Patient Samples.

Microsatellite instability (MSI) is an important biomarker for predicting response to immune checkpoint inhibitor therapy, as emphasized by the recent checkpoint inhibitor approval for MSI-high (MSI-H) solid tumors. Herein, we describe and validate a novel method for determining MSI status from a next-generation sequencing comprehensive genomic profiling assay using formalin-fixed, paraffin-embedded samples. This method is 97% (65/67) concordant with current standards, PCR and immunohistochemistry. We further apply this method to >67,000 patient tumor samples to identify genes and pathways that are enriched in MSI-stable or MSI-H tumor groups. Data show that although rare in tumors other than colorectal and endometrial carcinomas, MSI-H samples are present in many tumor types. Furthermore, the large sample set revealed that MSI-H tumors selectively share alterations in genes across multiple common pathways, including WNT, phosphatidylinositol 3-kinase, and NOTCH. Last, MSI is sufficient, but not necessary, for a tumor to have elevated tumor mutation burden. Therefore, MSI can be determined from comprehensive genomic profiling with high accuracy, allowing for efficient MSI-H detection across all tumor types, especially those in which routine use of immunohistochemistry or PCR-based assays would be impractical because of a rare incidence of MSI. MSI-H tumors are enriched in alterations in specific signaling pathways, providing a rationale for investigating directed immune checkpoint inhibitor therapies in combination with pathway-targeted therapies.

Prospective Comprehensive Genomic Profiling of Primary and Metastatic Prostate Tumors.

PURPOSE: Comprehensive genomic profiling (CGP) is increasingly used for routine clinical management of prostate cancer. To inform targeted treatment strategies, 3,476 clinically advanced prostate tumors were analyzed by CGP for genomic alterations (GAs) and signatures of genomic instability. METHODS: Prostate cancer samples (1,660 primary site and 1,816 metastatic site tumors from unmatched patients) were prospectively analyzed by CGP (FoundationOne Assay; Foundation Medicine, Cambridge, MA) for GAs and genomic signatures (genome-wide loss of heterozygosity [gLOH], microsatellite instability [MSI] status, tumor mutational burden [TMB]). RESULTS: Frequently altered genes were TP53 (44%), PTEN (32%), TMPRSS2-ERG (31%), and AR (23%). Potentially targetable GAs were frequently identified in DNA repair, phosphatidylinositol 3-kinase, and RAS/RAF/MEK pathways. DNA repair pathway GAs included homologous recombination repair (23%), Fanconi anemia (5%), CDK12 (6%), and mismatch repair (4%) GAs. BRCA1/2, ATR, and FANCA GAs were associated with high gLOH, whereas CDK12-altered tumors were infrequently gLOH high. Median TMB was low (2.6 mutations/Mb). A subset of cases (3%) had high TMB, of which 71% also had high MSI. Metastatic site tumors were enriched for the 11q13 amplicon (CCND1/FGF19/FGF4/FGF3) and GAs in AR, LYN, MYC, NCOR1, PIK3CB, and RB1 compared with primary tumors. CONCLUSION: Routine clinical CGP in the real-world setting identified GAs that are investigational biomarkers for targeted therapies in 57% of cases. gLOH and MSI/TMB signatures could further inform selection of poly (ADP-ribose) polymerase inhibitors and immunotherapies, respectively. Correlation of DNA repair GAs with gLOH identified genes associated with homologous recombination repair deficiency. GAs enriched in metastatic site tumors suggest therapeutic strategies for metastatic prostate cancer. Lack of clinical outcome correlation was a limitation of this study.

Squamous Cell Transformation of Primary Lung Adenocarcinoma in a Patient With EML4-ALK Fusion Variant 5 Refractory to ALK Inhibitors.

Histologic transformation from adenocarcinoma to squamous cell carcinoma in lung cancer has not been reported as a mechanism of resistance to ALK inhibition. This report describes the clinical course of a female former light smoker with metastatic lung adenocarcinoma whose tumor underwent histologic transformation from a well-differentiated lung adenocarcinoma to a well-differentiated lung squamous cell carcinoma in the same location at the left mainstem bronchus while maintaining the ALK fusion oncogene without any resistance mutations. After experiencing disease progression while on crizotinib, the patient participated in clinical trials that provided early access to the novel ALK inhibitors ceritinib and alectinib before they were commercially available. Tumor recurrence occurred at the primary and metastatic central nervous system sites (ie, brain and spine). At tumor progression, liquid biopsy and tumor genomic profiling of plasma cell-free DNA next-generation sequencing (NGS) provided an accurate diagnosis with a short turnaround time compared with the tissue-based targeted capture NGS. The patient received several courses of radiation primarily to the brain and spine during her disease course. Her disease did not respond to the immune checkpoint inhibitor nivolumab, and she died on home hospice approximately 4 years after diagnosis. This case supports the importance of both histopathologic assessment and comprehensive genomic profiling in selecting appropriate treatment for patients with refractory, metastatic, ALK oncogene-driven non-small cell lung cancer. Use of symptom-directed radiation in tandem with ALK inhibitors contributed to the disease and symptomatic control and prolonged survival in this patient.

Clinical utility of tumor genomic profiling in patients with high plasma circulating tumor DNA burden or metabolically active tumors.

BACKGROUND: This retrospective study was undertaken to determine if the plasma circulating tumor DNA (ctDNA) level and tumor biological features in patients with advanced solid tumors affected the detection of genomic alterations (GAs) by a plasma ctDNA assay. METHOD: Cell-free DNA (cfDNA) extracted from frozen plasma (N = 35) or fresh whole blood (N = 90) samples were subjected to a 62-gene hybrid capture-based next-generation sequencing assay FoundationACT. Concordance was analyzed for 51 matched FoundationACT and FoundationOne (tissue) cases. The maximum somatic allele frequency (MSAF) was used to estimate the amount of tumor fraction of cfDNA in each sample. The detection of GAs was correlated with the amount of cfDNA, MSAF, total tumor anatomic burden (dimensional sum), and total tumor metabolic burden (SUVmax sum) of the largest ten tumor lesions on PET/CT scans. RESULTS: FoundationACT detected GAs in 69 of 81 (85%) cases with MSAF > 0. Forty-two of 51 (82%) cases had ≥ 1 concordance GAs matched with FoundationOne, and 22 (52%) matched to the National Comprehensive Cancer Network (NCCN)-recommended molecular targets. FoundationACT also detected 8 unique molecular targets, which changed the therapy in 7 (88%) patients who did not have tumor rebiopsy or sufficient tumor DNA for genomic profiling assay. In all samples (N = 81), GAs were detected in plasma cfDNA from cancer patients with high MSAF quantity (P = 0.0006) or high tumor metabolic burden (P = 0.0006) regardless of cfDNA quantity (P = 0.2362). CONCLUSION: This study supports the utility of using plasma-based genomic assays in cancer patients with high plasma MSAF level or high tumor metabolic burden.

Multiple configurations of EGFR exon 20 resistance mutations after first- and third-generation EGFR TKI treatment affect treatment options in NSCLC.

After sequential treatment with first- and third-generation EGFR tyrosine kinase inhibitors (TKIs), EGFR-mutant non-small cell lung cancers frequently harbor multiple resistance mutations in exon 20 of EGFR including T790M, mediating resistance to first-generation TKIs, and at codons 792, 796, or 797 mediating resistance to third-generation TKIs. However, whether these resistance mutations are in cis or trans has therapeutic implications for patients. We analyzed a cohort of 29 patients with NSCLC harboring EGFR mutations at codons 792, 796, or 797 to establish the configuration of these mutations. We performed hybrid capture-based, next-generation sequencing on formalin-fixed paraffin-embedded biopsy tissue or liquid biopsy. 27 samples had both a T790M mutation and a mutation at codons 792, 796, or 797. In all of these cases, the mutations were found in the cis configuration; the trans configuration was not observed. Two patients' samples harbored a mutation at codon 797 but no T790M mutation. In these two cases, longitudinal analysis showed earlier biopsies harbored EGFR T790M, which was undetectable following osimertinib treatment. Treatment of one these patients with both first- and third-generation EGFR TKIs resulted in a mixed response. Here we describe multiple configurations of EGFR T790M and third-generation TKI resistance mutations at codons 792, 796, and 797. These mutations are most commonly found in cis, which confers resistance to all current EGFR TKIs. We also describe two patients that exhibited T790M loss with acquisition of a mutation at codon 797. In addition, one of these patients, with an EGFR C797S in a lung biopsy was subsequently found to have EGFR C797N in a later biopsy of pleural fluid, highlighting the dynamic multiclonal nature of advanced NSCLC.

Analytical Validation of a Hybrid Capture-Based Next-Generation Sequencing Clinical Assay for Genomic Profiling of Cell-Free Circulating Tumor DNA.

Genomic profiling of circulating tumor DNA derived from cell-free DNA (cfDNA) in blood can provide a noninvasive method for detecting genomic biomarkers to guide clinical decision making for cancer patients. We developed a hybrid capture-based next-generation sequencing assay for genomic profiling of circulating tumor DNA from blood (FoundationACT). High-sequencing coverage and molecular barcode-based error detection enabled accurate detection of genomic alterations, including short variants (base substitutions, short insertions/deletions) and genomic re-arrangements at low allele frequencies (AFs), and copy number amplifications. Analytical validation was performed on 2666 reference alterations. The assay achieved >99% overall sensitivity (95% CI, 99.1%-99.4%) for short variants at AF >0.5%, >95% sensitivity (95% CI, 94.2%-95.7%) for AF 0.25% to 0.5%, and 70% sensitivity (95% CI, 68.2%-71.5%) for AF 0.125% to 0.25%. No false positives were detected in 62 samples from healthy volunteers. Genomic alterations detected by FoundationACT demonstrated high concordance with orthogonal assays run on the same clinical cfDNA samples. In 860 routine clinical FoundationACT cases, genomic alterations were detected in cfDNA at comparable frequencies to tissue; for the subset of cases with temporally matched tissue and blood samples, 75% of genomic alterations and 83% of short variant mutations detected in tissue were also detected in cfDNA. On the basis of analytical validation results, FoundationACT has been approved for use in our Clinical Laboratory Improvement Amendments-certified/College of American Pathologists-accredited/New York State-approved laboratory.

KIBRA (WWC1) Is a Metastasis Suppressor Gene Affected by Chromosome 5q Loss in Triple-Negative Breast Cancer.

Triple-negative breast cancers (TNBCs) display a complex spectrum of mutations and chromosomal aberrations. Chromosome 5q (5q) loss is detected in up to 70% of TNBCs, but little is known regarding the genetic drivers associated with this event. Here, we show somatic deletion of a region syntenic with human 5q33.2-35.3 in a mouse model of TNBC. Mechanistically, we identify KIBRA as a major factor contributing to the effects of 5q loss on tumor growth and metastatic progression. Re-expression of KIBRA impairs metastasis in vivo and inhibits tumorsphere formation by TNBC cells in vitro. KIBRA functions co-operatively with the protein tyrosine phosphatase PTPN14 to trigger mechanotransduction-regulated signals that inhibit the nuclear localization of oncogenic transcriptional co-activators YAP/TAZ. Our results argue that the selective advantage produced by 5q loss involves reduced dosage of KIBRA, promoting oncogenic functioning of YAP/TAZ in TNBC.

High frequency of the PNPLA3 rs738409 [G] single-nucleotide polymorphism in Hmong individuals as a potential basis for a predisposition to chronic liver disease.

BACKGROUND: An exploratory study was performed to determine the prevalence of the patatin-like phospholipase domain-containing protein 3 (PNPLA3) rs78409 [G] allele among the Hmong as a risk factor for nonalcoholic fatty liver disease (NAFLD). NAFLD/nonalcoholic steatohepatitis is the world's most common chronic liver disease and is expected to replace viral hepatitis as the leading cause of cirrhosis and potential precursor to hepatocellular carcinoma (HCC). Of all populations in California, the Hmong experience the highest risk of death from HCC and the highest prevalence of metabolic syndrome risk factors among Asians that predispose them to NAFLD. Here a genetic explanation was sought for the high rates of chronic liver disease among the Hmong. The literature pointed to the PNPLA3 rs738409 [G] allele as a potential genetic culprit. METHODS: Cell-free DNA was isolated from 26 serum samples previously collected in community settings. Quantitative polymerase chain reaction-based single-nucleotide polymorphism (SNP) genotyping was performed with a validated TaqMan SNP genotyping assay, and results were analyzed with TaqMan Genotyper software. RESULTS: The PNPLA3 rs738409 [C>G] variant occurred at a frequency of 0.46 (12 of 26; 95% confidence interval, 0.27-0.67). This carrier rate would rank the Hmong as the third highest population in the 1000 Genomes Project. CONCLUSIONS: Although this small sample size limits the generalizability, the high frequency rates of this allele along with the presence of metabolic syndrome risk factors warrant further studies into the etiology of NAFLD among the Hmong. Cancer 2018;124:1583-9. © 2018 American Cancer Society.

Optimal Molecular Methods in Detecting p190 (BCR-ABL) Fusion Variants in Hematologic Malignancies: A Case Report and Review of the Literature.

Patients with BCR-ABL1 positive hematologic malignancies and Philadelphia-like B-lymphoblastic leukemia (B-ALL) are potential candidates for targeted therapy with tyrosine kinase inhibitors (TKI). Before TKIs, patients with B-ALL had a much worse prognosis and current treatments with targeted TKI therapy have improved outcomes. Thus, the detection of BCR-ABL1 is crucial and a false negative BCR-ABL1 result may adversely affect patient care. We report a case of a 76-year-old male with a new diagnosis of B-ALL who was initially found to be BCR-ABL1 negative by quantitative polymerase chain reaction (PCR). A concurrent qualitative PCR was performed which detected a positive BCR-ABL1 result that was confirmed by a next generation sequencing (NGS) based assay and identified as the rare fusion variant e1a3 of p190(BCR-ABL). Based on this result, the patient was placed on dasatinib as a targeted therapy. In the era of molecular diagnostic medicine and targeted therapy, it is essential to have an understanding of the limitations of molecular assays and to follow a comprehensive diagnostic approach in order to detect common abnormalities and rare variants. Incorporating NGS methods in an algorithmic manner into the standard diagnostic PCR-based approach for BCR-ABL1 will aid in minimizing false negative results.

Transgenic mammary epithelial osteopontin (spp1) expression induces proliferation and alveologenesis.

Osteopontin (OPN) Spp1 is involved in differentiation of the mammary gland. We engineered mice to overexpress OPN in mammary epithelium and describe an altered mammary phenotype. Three transgenic (Tg) founder lines FVB/N Tg(MMTV-Opn)((1-3BOR)) were propagated after FVB/NJ pronuclear injections. Mammary glands from Tg-OPN mice compared to littermate controls showed, at 4 weeks of age, exaggerated terminal end buds; at 8 and 12 weeks, more numerous and complex ducts with increased luminal protein; and at 16 weeks, increased lobulogenesis. Lactational Tg-OPN mammary glands showed more rapid lobulogenesis and lactational changes with slower gland involution and regression following weaning. Ex vivo lobulogenesis was noticeably increased from organoids of Tg-OPN mice. Immunohistochemistry revealed cytoplasmic OPN accumulation and increased Ki-67 positive mammary epithelial cells in Tg-OPN mammary glands. OPN appears to convey a proliferative stimulus for mammary epithelial cells and alters development and differentiation. These OPN mammary overexpressing mice provide a means to study the role of OPN in cancer progression.

Modulation of blood cell gene expression by DHA supplementation in hypertriglyceridemic men.

Our previous study with docosahexaenoic acid (DHA) supplementation to hypertriglyceridemic men showed that DHA reduced several risk factors for cardiovascular disease, including the plasma concentration of inflammatory markers. To determine the effect of DHA supplementation on the global gene expression pattern, we performed Affymetrix GeneChip microarray analysis of blood cells [treated with lipopolysaccharide (LPS) or vehicle] drawn before and after the supplementation of DHA from the hypertriglyceridemic men who participated in that study. Genes that were significantly differentially regulated by the LPS treatment and DHA supplementation were identified. Differential regulation of 18 genes was then verified by quantitative real-time polymerase chain reaction (qRT-PCR). Both microarray and qRT-PCR data showed that DHA supplementation significantly suppressed the expression of low-density lipoprotein (LDL) receptor and cathepsin L1, both of which were also up-regulated by LPS. DHA supplementation also suppressed oxidized LDL (lectin-like) receptor 1 (OLR1). However, LPS did not induce OLR1 mRNA expression. Enrichment with Gene Ontology categories demonstrated that the genes related to transcription factor activity, immunity, host defense and inflammatory responses were inversely regulated by LPS and DHA. These results provide supporting evidence for the anti-inflammatory effects of DHA supplementation, and reveal previously unrecognized genes that are regulated by DHA and are associated with risk factors of cardiovascular diseases.

Correlations between gene expression and mercury levels in blood of boys with and without autism.

Gene expression in blood was correlated with mercury levels in blood of 2- to 5-year-old boys with autism (AU) compared to age-matched typically developing (TD) control boys. This was done to address the possibility that the two groups might metabolize toxicants, such as mercury, differently. RNA was isolated from blood and gene expression assessed on whole genome Affymetrix Human U133 expression microarrays. Mercury levels were measured using an inductively coupled plasma mass spectrometer. Analysis of covariance (ANCOVA) was performed and partial correlations between gene expression and mercury levels were calculated, after correcting for age and batch effects. To reduce false positives, only genes shared by the ANCOVA models were analyzed. Of the 26 genes that correlated with mercury levels in both AU and TD boys, 11 were significantly different between the groups (P(Diagnosis*Mercury) ≤ 0.05). The expression of a large number of genes (n = 316) correlated with mercury levels in TD but not in AU boys (P ≤ 0.05), the most represented biological functions being cell death and cell morphology. Expression of 189 genes correlated with mercury levels in AU but not in TD boys (P ≤ 0.05), the most represented biological functions being cell morphology, amino acid metabolism, and antigen presentation. These data and those in our companion study on correlation of gene expression and lead levels show that AU and TD children display different correlations between transcript levels and low levels of mercury and lead. These findings might suggest different genetic transcriptional programs associated with mercury in AU compared to TD children.