SARS-CoV-2 infection produces chronic pulmonary epithelial and immune cell dysfunction with fibrosis in mice.

A subset of individuals who recover from coronavirus disease 2019 (COVID-19) develop post-acute sequelae of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (PASC), but the mechanistic basis of PASC-associated lung abnormalities suffers from a lack of longitudinal tissue samples. The mouse-adapted SARS-CoV-2 strain MA10 produces an acute respiratory distress syndrome in mice similar to humans. To investigate PASC pathogenesis, studies of MA10-infected mice were extended from acute to clinical recovery phases. At 15 to 120 days after virus clearance, pulmonary histologic findings included subpleural lesions composed of collagen, proliferative fibroblasts, and chronic inflammation, including tertiary lymphoid structures. Longitudinal spatial transcriptional profiling identified global reparative and fibrotic pathways dysregulated in diseased regions, similar to human COVID-19. Populations of alveolar intermediate cells, coupled with focal up-regulation of profibrotic markers, were identified in persistently diseased regions. Early intervention with antiviral EIDD-2801 reduced chronic disease, and early antifibrotic agent (nintedanib) intervention modified early disease severity. This murine model provides opportunities to identify pathways associated with persistent SARS-CoV-2 pulmonary disease and test countermeasures to ameliorate PASC.

Prevalence and Mechanisms of Mucus Accumulation in COVID-19 Lung Disease.

Rationale: The incidence and sites of mucus accumulation and molecular regulation of mucin gene expression in coronavirus (COVID-19) lung disease have not been reported. Objectives: To characterize the incidence of mucus accumulation and the mechanisms mediating mucin hypersecretion in COVID-19 lung disease. Methods: Airway mucus and mucins were evaluated in COVID-19 autopsy lungs by Alcian blue and periodic acid-Schiff staining, immunohistochemical staining, RNA in situ hybridization, and spatial transcriptional profiling. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected human bronchial epithelial (HBE) cultures were used to investigate mechanisms of SARS-CoV-2-induced mucin expression and synthesis and test candidate countermeasures. Measurements and Main Results: MUC5B and variably MUC5AC RNA concentrations were increased throughout all airway regions of COVID-19 autopsy lungs, notably in the subacute/chronic disease phase after SARS-CoV-2 clearance. In the distal lung, MUC5B-dominated mucus plugging was observed in 90% of subjects with COVID-19 in both morphologically identified bronchioles and microcysts, and MUC5B accumulated in damaged alveolar spaces. SARS-CoV-2-infected HBE cultures exhibited peak titers 3 days after inoculation, whereas induction of MUC5B/MUC5AC peaked 7-14 days after inoculation. SARS-CoV-2 infection of HBE cultures induced expression of epidermal growth factor receptor (EGFR) ligands and inflammatory cytokines (e.g., IL-1α/ß) associated with mucin gene regulation. Inhibiting EGFR/IL-1R pathways or administration of dexamethasone reduced SARS-CoV-2-induced mucin expression. Conclusions: SARS-CoV-2 infection is associated with a high prevalence of distal airspace mucus accumulation and increased MUC5B expression in COVID-19 autopsy lungs. HBE culture studies identified roles for EGFR and IL-1R signaling in mucin gene regulation after SARS-CoV-2 infection. These data suggest that time-sensitive mucolytic agents, specific pathway inhibitors, or corticosteroid administration may be therapeutic for COVID-19 lung disease.

Protease-anti-protease compartmentalization in SARS-CoV-2 ARDS: Therapeutic implications.

BACKGROUND: Interleukin-6 (IL-6) is elevated in SARS-CoV-2 infection. IL-6 regulates acute-phase proteins, such as alpha-1 antitrypsin (AAT), a key lung anti-protease. We investigated the protease-anti-protease balance in the circulation and pulmonary compartments in SARS-CoV-2 acute respiratory distress syndrome (ARDS) compared to non-SARS-CoV-2 ARDS (nsARDS) and the effects of tocilizumab (IL-6 receptor antagonist) on anti-protease defence in SARS-CoV-2 infection. METHODS: Levels and activity of AAT and neutrophil elastase (NE) were measured in plasma, airway tissue and tracheal secretions (TA) of people with SARS-CoV-2 ARDS or nsARDS. AAT and IL-6 levels were evaluated in people with moderate SARS-CoV-2 infection who received standard of care +/- tocilizumab. FINDINGS: AAT plasma levels doubled in SARS-CoV-2 ARDS. In lung parenchyma AAT levels were increased, as was the percentage of neutrophils involved in NET formation. A protease-anti-protease imbalance was detected in TA with active NE and no active AAT. The airway anti-protease, secretory leukoprotease inhibitor was decreased in SARS-CoV-2-infected lungs and cleaved in TA. In nsARDS, plasma AAT levels were elevated but TA samples had less AAT cleavage, with no detectable active NE in most samples. Induction of AAT in ARDS occurred mainly through IL-6. Tocilizumab down-regulated AAT during SARS-CoV-2 infection. INTERPRETATION: There is a protease-anti-protease imbalance in the airways of SARS-CoV-2-ARDS patients. This imbalance is a target for anti-protease therapy. FUNDING: NIH Serological Sciences Network, National Heart, Lung, and Blood Institute and National Institute of Diabetes and Digestive and Kidney Diseases.

A model of persistent post SARS-CoV-2 induced lung disease for target identification and testing of therapeutic strategies.

COVID-19 survivors develop post-acute sequelae of SARS-CoV-2 (PASC), but the mechanistic basis of PASC-associated lung abnormalities suffers from a lack of longitudinal samples. Mouse-adapted SARS-CoV-2 MA10 produces an acute respiratory distress syndrome (ARDS) in mice similar to humans. To investigate PASC pathogenesis, studies of MA10-infected mice were extended from acute disease through clinical recovery. At 15-120 days post-virus clearance, histologic evaluation identified subpleural lesions containing collagen, proliferative fibroblasts, and chronic inflammation with tertiary lymphoid structures. Longitudinal spatial transcriptional profiling identified global reparative and fibrotic pathways dysregulated in diseased regions, similar to human COVID-19. Populations of alveolar intermediate cells, coupled with focal upregulation of pro-fibrotic markers, were identified in persistently diseased regions. Early intervention with antiviral EIDD-2801 reduced chronic disease, and early anti-fibrotic agent (nintedanib) intervention modified early disease severity. This murine model provides opportunities to identify pathways associated with persistent SARS-CoV-2 pulmonary disease and test countermeasures to ameliorate PASC.

Rapid idiosyncratic mechanisms of clinical resistance to KRAS G12C inhibition.

BACKGROUNDThe KRAS proto-oncogene is among the most frequently mutated genes in cancer, yet for 40 years it remained an elusive therapeutic target. Recently, allosteric inhibitors that covalently bind to KRAS G12C mutations have been approved for use in lung adenocarcinomas. Although responses are observed, they are often short-lived, thus making in-depth characterization of the mechanisms of resistance of paramount importance.METHODSHere, we present a rapid-autopsy case of a patient who had a KRASG12C-mutant lung adenocarcinoma who initially responded to a KRAS G12C inhibitor but then rapidly developed resistance. Using deep-RNA and whole-exome sequencing comparing pretreatment, posttreatment, and matched normal tissues, we uncover numerous mechanisms of resistance to direct KRAS inhibition.RESULTSIn addition to decreased KRAS G12C-mutant allele frequency in refractory tumors, we also found reactivation of the MAPK pathway despite no new mutations in KRAS or its downstream mediators. Tumor cell-intrinsic and non-cell autonomous mechanisms included increased complement activation, coagulation, and tumor angiogenesis, and several lines of evidence of immunologic evasion.CONCLUSIONTogether, our findings reveal numerous mechanisms of resistance to current KRAS G12C inhibitors through enrichment of clonal populations, KRAS-independent downstream signaling, and diverse remodeling of the tumor microenvironment.FUNDINGRichard and Fran Duley, Jimmy and Kay Mann, the NIH, and the North Carolina Biotechnology Center.

Correction: Molecular Subtypes in Head and Neck Cancer Exhibit Distinct Patterns of Chromosomal Gain and Loss of Canonical Cancer Genes.

[This corrects the article DOI: 10.1371/journal.pone.0056823.].

Integrated RNA and DNA sequencing reveals early drivers of metastatic breast cancer.

Breast cancer metastasis remains a clinical challenge, even within a single patient across multiple sites of the disease. Genome-wide comparisons of both the DNA and gene expression of primary tumors and metastases in multiple patients could help elucidate the underlying mechanisms that cause breast cancer metastasis. To address this issue, we performed DNA exome and RNA sequencing of matched primary tumors and multiple metastases from 16 patients, totaling 83 distinct specimens. We identified tumor-specific drivers by integrating known protein-protein network information with RNA expression and somatic DNA alterations and found that genetic drivers were predominantly established in the primary tumor and maintained through metastatic spreading. In addition, our analyses revealed that most genetic drivers were DNA copy number changes, the TP53 mutation was a recurrent founding mutation regardless of subtype, and that multiclonal seeding of metastases was frequent and occurred in multiple subtypes. Genetic drivers unique to metastasis were identified as somatic mutations in the estrogen and androgen receptor genes. These results highlight the complexity of metastatic spreading, be it monoclonal or multiclonal, and suggest that most metastatic drivers are established in the primary tumor, despite the substantial heterogeneity seen in the metastases.

Frequency of breast cancer subtypes among African American women in the AMBER consortium.

BACKGROUND: Breast cancer subtype can be classified using standard clinical markers (estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2)), supplemented with additional markers. However, automated biomarker scoring and classification schemes have not been standardized. The aim of this study was to optimize tumor classification using automated methods in order to describe subtype frequency in the African American Breast Cancer Epidemiology and Risk (AMBER) consortium. METHODS: Using immunohistochemistry (IHC), we quantified the expression of ER, PR, HER2, the proliferation marker Ki67, and two basal-like biomarkers, epidermal growth factor receptor (EGFR) and cytokeratin (CK)5/6, in 1381 invasive breast tumors from African American women. RNA-based (prediction analysis of microarray 50 (PAM50)) subtype, available for 574 (42%) cases, was used to optimize classification. Subtype frequency was calculated, and associations between subtype and tumor characteristics were estimated using logistic regression. RESULTS: Relative to ER, PR and HER2 from medical records, central IHC staining and the addition of Ki67 or combined tumor grade improved accuracy for classifying PAM50-based luminal subtypes. Few triple negative cases (< 2%) lacked EGFR and CK5/6 expression, thereby providing little improvement in accuracy for identifying basal-like tumors. Relative to luminal A subtype, all other subtypes had higher combined grade and were larger, and ER-/HER2+ tumors were more often lymph node positive and late stage tumors. The frequency of basal-like tumors was 31%, exceeded only slightly by luminal A tumors (37%). CONCLUSIONS: Our findings indicate that automated IHC-based classification produces tumor subtype frequencies approximating those from PAM50-based classification and highlight high frequency of basal-like and low frequency of luminal A breast cancer in a large study of African American women.

Racial Differences in PAM50 Subtypes in the Carolina Breast Cancer Study.

Background: African American breast cancer patients have lower frequency of hormone receptor-positive (HR+)/human epidermal growth factor receptor 2 (HER2)-negative disease and higher subtype-specific mortality. Racial differences in molecular subtype within clinically defined subgroups are not well understood. Methods: Using data and biospecimens from the population-based Carolina Breast Cancer Study (CBCS) Phase 3 (2008-2013), we classified 980 invasive breast cancers using RNA expression-based PAM50 subtype and recurrence (ROR) score that reflects proliferation and tumor size. Molecular subtypes (Luminal A, Luminal B, HER2-enriched, and Basal-like) and ROR scores (high vs low/medium) were compared by race (blacks vs whites) and age (≤50 years vs > 50 years) using chi-square tests and analysis of variance tests. Results: Black women of all ages had a statistically significantly lower frequency of Luminal A breast cancer (25.4% and 33.6% in blacks vs 42.8% and 52.1% in whites; younger and older, respectively). All other subtype frequencies were higher in black women (case-only odds ratio [OR] = 3.11, 95% confidence interval [CI] = 2.22 to 4.37, for Basal-like; OR = 1.45, 95% CI = 1.02 to 2.06, for Luminal B; OR = 2.04, 95% CI = 1.33 to 3.13, for HER2-enriched). Among clinically HR+/HER2- cases, Luminal A subtype was less common and ROR scores were statistically significantly higher among black women. Conclusions: Multigene assays highlight racial disparities in tumor subtype distribution that persist even in clinically defined subgroups. Differences in tumor biology (eg, HER2-enriched status) may be targetable to reduce disparities among clinically ER+/HER2- cases.

Two fatal herpesvirus cases: Treatable but easily missed diagnoses.

Ill or immunosuppressed hospital patients are at increased risk for herpes simplex and herpes zoster virus infections, with high potential morbidity and mortality. Here, we present two cases of reactivation of herpes virus infections with delay in diagnosis, with ultimately fatal results. Since these infections are treatable, it is important to keep a high index of suspicion to facilitate early diagnosis and treatment.

Performance of Three-Biomarker Immunohistochemistry for Intrinsic Breast Cancer Subtyping in the AMBER Consortium.

BACKGROUND: Classification of breast cancer into intrinsic subtypes has clinical and epidemiologic importance. To examine accuracy of IHC-based methods for identifying intrinsic subtypes, a three-biomarker IHC panel was compared with the clinical record and RNA-based intrinsic (PAM50) subtypes. METHODS: Automated scoring of estrogen receptor (ER), progesterone receptor (PR), and HER2 was performed on IHC-stained tissue microarrays comprising 1,920 cases from the African American Breast Cancer Epidemiology and Risk (AMBER) consortium. Multiple cores (1-6/case) were collapsed to classify cases, and automated scoring was compared with the clinical record and to RNA-based subtyping. RESULTS: Automated analysis of the three-biomarker IHC panel produced high agreement with the clinical record (93% for ER and HER2, and 88% for PR). Cases with low tumor cellularity and smaller core size had reduced agreement with the clinical record. IHC-based definitions had high agreement with the clinical record regardless of hormone receptor positivity threshold (1% vs. 10%), but a 10% threshold produced highest agreement with RNA-based intrinsic subtypes. Using a 10% threshold, IHC-based definitions identified the basal-like intrinsic subtype with high sensitivity (86%), although sensitivity was lower for luminal A, luminal B, and HER2-enriched subtypes (76%, 40%, and 37%, respectively). CONCLUSION: Three-biomarker IHC-based subtyping has reasonable accuracy for distinguishing basal-like from nonbasal-like, although additional biomarkers are required for accurate classification of luminal A, luminal B, and HER2-enriched cancers. IMPACT: Epidemiologic studies relying on three-biomarker IHC status for subtype classification should use caution when distinguishing luminal A from luminal B and when interpreting findings for HER2-enriched cancers.

Alterations of LKB1 and KRAS and risk of brain metastasis: comprehensive characterization by mutation analysis, copy number, and gene expression in non-small-cell lung carcinoma.

BACKGROUND: Brain metastases are one of the most malignant complications of lung cancer and constitute a significant cause of cancer related morbidity and mortality worldwide. Recent years of investigation suggested a role of LKB1 in NSCLC development and progression, in synergy with KRAS alteration. In this study, we systematically analyzed how LKB1 and KRAS alteration, measured by mutation, gene expression (GE) and copy number (CN), are associated with brain metastasis in NSCLC. MATERIALS AND METHODS: Patients treated at University of North Carolina Hospital from 1990 to 2009 with NSCLC provided frozen, surgically extracted tumors for analysis. GE was measured using Agilent 44,000 custom-designed arrays, CN was assessed by Affymetrix GeneChip Human Mapping 250K Sty Array or the Genome-Wide Human SNP Array 6.0 and gene mutation was detected using ABI sequencing. Integrated analysis was conducted to assess the relationship between these genetic markers and brain metastasis. A model was proposed for brain metastasis prediction using these genetic measurements. RESULTS: 17 of the 174 patients developed brain metastasis. LKB1 wild type tumors had significantly higher LKB1 CN (p<0.001) and GE (p=0.002) than the LKB1 mutant group. KRAS wild type tumors had significantly lower KRAS GE (p<0.001) and lower CN, although the latter failed to be significant (p=0.295). Lower LKB1 CN (p=0.039) and KRAS mutation (p=0.007) were significantly associated with more brain metastasis. The predictive model based on nodal (N) stage, patient age, LKB1 CN and KRAS mutation had a good prediction accuracy, with area under the ROC curve of 0.832 (p<0.001). CONCLUSION: LKB1 CN in combination with KRAS mutation predicted brain metastasis in NSCLC.

Prediction of lung cancer histological types by RT-qPCR gene expression in FFPE specimens.

Lung cancer histologic diagnosis is clinically relevant because there are histology-specific treatment indications and contraindications. Histologic diagnosis can be challenging owing to tumor characteristics, and it has been shown to have less-than-ideal agreement among pathologists reviewing the same specimens. Microarray profiling studies using frozen specimens have shown that histologies exhibit different gene expression trends; however, frozen specimens are not amenable to routine clinical application. Herein, we developed a gene expression-based predictor of lung cancer histology for FFPE specimens, which are routinely available in clinical settings. Genes predictive of lung cancer histologies were derived from published cohorts that had been profiled by microarrays. Expression of these genes was measured by quantitative RT-PCR (RT-qPCR) in a cohort of patients with FFPE lung cancer. A histology expression predictor (HEP) was developed using RT-qPCR expression data for adenocarcinoma, carcinoid, small cell carcinoma, and squamous cell carcinoma. In cross-validation, the HEP exhibited mean accuracy of 84% and κ = 0.77. In separate independent validation sets, the HEP was compared with pathologist diagnoses on the same tumor block specimens, and the HEP yielded similar accuracy and precision as the pathologists. The HEP also exhibited good performance in specimens with low tumor cellularity. Therefore, RT-qPCR gene expression from FFPE specimens can be effectively used to predict lung cancer histology.

Molecular subtypes in head and neck cancer exhibit distinct patterns of chromosomal gain and loss of canonical cancer genes.

Head and neck squamous cell carcinoma (HNSCC) is a frequently fatal heterogeneous disease. Beyond the role of human papilloma virus (HPV), no validated molecular characterization of the disease has been established. Using an integrated genomic analysis and validation methodology we confirm four molecular classes of HNSCC (basal, mesenchymal, atypical, and classical) consistent with signatures established for squamous carcinoma of the lung, including deregulation of the KEAP1/NFE2L2 oxidative stress pathway, differential utilization of the lineage markers SOX2 and TP63, and preference for the oncogenes PIK3CA and EGFR. For potential clinical use the signatures are complimentary to classification by HPV infection status as well as the putative high risk marker CCND1 copy number gain. A molecular etiology for the subtypes is suggested by statistically significant chromosomal gains and losses and differential cell of origin expression patterns. Model systems representative of each of the four subtypes are also presented.

Validation of interobserver agreement in lung cancer assessment: hematoxylin-eosin diagnostic reproducibility for non-small cell lung cancer: the 2004 World Health Organization classification and therapeutically relevant subsets.

CONTEXT: Precise subtype diagnosis of non-small cell lung carcinoma is increasingly relevant, based on the availability of subtype-specific therapies, such as bevacizumab and pemetrexed, and based on the subtype-specific prevalence of activating epidermal growth factor receptor mutations. OBJECTIVES: To establish a baseline measure of interobserver reproducibility for non-small cell lung carcinoma diagnoses with hematoxylin-eosin for the current 2004 World Health Organization classification, to estimate interobserver reproducibility for the therapeutically relevant squamous/nonsquamous subsets, and to examine characteristics that improve interobserver reproducibility. DESIGN: Primary, resected lung cancer specimens were converted to digital (virtual) slides. Based on a single hematoxylin-eosin virtual slide, pathologists were asked to assign a diagnosis using the 2004 World Health Organization classification. Kappa statistics were calculated for each pathologist-pair for each slide and were summarized by classification scheme, pulmonary pathology expertise, diagnostic confidence, and neoplastic grade. RESULTS: The 12 pulmonary pathology experts and the 12 community pathologists each independently diagnosed 48 to 96 single hematoxylin-eosin digital slides derived from 96 cases of non-small cell lung carcinoma resection. Overall agreement improved with simplification from the comprehensive 44 World Health Organization diagnoses (κ  =  0.25) to their 10 major header subtypes (κ  =  0.48) and improved again with simplification into the therapeutically relevant squamous/nonsquamous dichotomy (κ  =  0.55). Multivariate analysis showed that higher diagnostic agreement was associated with better differentiation, better slide quality, higher diagnostic confidence, similar years of pathology experience, and pulmonary pathology expertise. CONCLUSIONS: These data define the baseline diagnostic agreement for hematoxylin-eosin diagnosis of non-small cell lung carcinoma, allowing future studies to test for improved diagnostic agreement with reflex ancillary tests.

Differential pathogenesis of lung adenocarcinoma subtypes involving sequence mutations, copy number, chromosomal instability, and methylation.

BACKGROUND: Lung adenocarcinoma (LAD) has extreme genetic variation among patients, which is currently not well understood, limiting progress in therapy development and research. LAD intrinsic molecular subtypes are a validated stratification of naturally-occurring gene expression patterns and encompass different functional pathways and patient outcomes. Patients may have incurred different mutations and alterations that led to the different subtypes. We hypothesized that the LAD molecular subtypes co-occur with distinct mutations and alterations in patient tumors. METHODOLOGY/PRINCIPAL FINDINGS: The LAD molecular subtypes (Bronchioid, Magnoid, and Squamoid) were tested for association with gene mutations and DNA copy number alterations using statistical methods and published cohorts (n = 504). A novel validation (n = 116) cohort was assayed and interrogated to confirm subtype-alteration associations. Gene mutation rates (EGFR, KRAS, STK11, TP53), chromosomal instability, regional copy number, and genomewide DNA methylation were significantly different among tumors of the molecular subtypes. Secondary analyses compared subtypes by integrated alterations and patient outcomes. Tumors having integrated alterations in the same gene associated with the subtypes, e.g. mutation, deletion and underexpression of STK11 with Magnoid, and mutation, amplification, and overexpression of EGFR with Bronchioid. The subtypes also associated with tumors having concurrent mutant genes, such as KRAS-STK11 with Magnoid. Patient overall survival, cisplatin plus vinorelbine therapy response and predicted gefitinib sensitivity were significantly different among the subtypes. CONCLUSIONS/ SIGNIFICANCE: The lung adenocarcinoma intrinsic molecular subtypes co-occur with grossly distinct genomic alterations and with patient therapy response. These results advance the understanding of lung adenocarcinoma etiology and nominate patient subgroups for future evaluation of treatment response.

Epstein-Barr virus infection is common in inflamed gastrointestinal mucosa.

BACKGROUND AND AIMS: Epstein-Barr virus (EBV) is present in the malignant epithelial cells of 10% of all gastric adenocarcinomas; however, localization of the virus in normal gastrointestinal mucosa is largely unexplored. In the present study, we measured EBV DNA and localized viral gene products in gastritis specimens (n = 89), normal gastric and colonic mucosa (n = 14), Crohn's disease (n = 9), and ulcerative colitis (n = 11) tissues. METHODS: A battery of sensitive and specific quantitative polymerase chain reactions targeted six disparate regions of the EBV genome: BamH1 W, EBNA1, LMP1, LMP2, BZLF1, and EBER1. EBV infection was localized by EBV-encoded RNA (EBER) in situ hybridization and by immunohistochemical stains for viral latent proteins LMP1 and LMP2 and for viral lytic proteins BMRF1 and BZLF1. B lymphocytes were identified using CD20 immunostains. RESULTS: EBV DNA was essentially undetectable in normal gastric mucosa but was present in 46% of gastritis lesions, 44% of normal colonic mucosa, 55% of Crohn's disease, and 64% of ulcerative colitis samples. Levels of EBV DNA exceeded what would be expected based on the numbers of B lymphocytes in inflamed tissues, suggesting that EBV is preferentially localized to inflammatory gastrointestinal lesions. Histochemical staining revealed EBER expression in lymphoid cells of some PCR-positive lesions. The viral lytic viral proteins, BMRF1 and BZLF1, were expressed in lymphoid cells of two ulcerative colitis tissues, both of which had relatively high viral loads by quantitative PCR. CONCLUSION: EBV-infected lymphocytes are frequently present in inflamed gastric and colonic mucosa. Active viral replication in some lesions raises the possibility of virus-related perpetuation of gastrointestinal inflammation.

Association of p16(INK4a) overexpression with improved outcomes in young patients with squamous cell cancers of the oral tongue.

BACKGROUND: The aim of this study was to examine biomolecular profiles in a cohort of young adults with squamous cell cancers (SCCs) of the oral tongue. METHODS: We identified all patients aged 18 to 39 years diagnosed with SCC of the oral tongue at our institution. Immunohistochemical (IHC) staining was performed for p16(INK4a) , epidermal growth factor receptor (EGFR), phosphorylated-EGFR (pEGFR), p53, and ERCC1. Human papillomavirus (HPV) testing was performed using in situ hybridization (ISH) and polymerase chain reaction (PCR). Biomarker expression and HPV status were correlated with outcomes. RESULTS: We identified 25 patients with sufficient tumor samples. Median age at diagnosis was 30 years (range, 20-39 years). p16(INK4a) overexpression was observed in 11 of 25 patients, whereas HPV-16 positivity was observed in none of the tumor samples by ISH and 2 of the tumor samples by PCR. p16(INK4a) positivity was correlated with improved relapse-free survival (hazard ratio [HR] = 0.23, p = .01) and overall survival (HR = 0.28, p = .05). Neither EGFR, pEGFR, p53, nor excision repair cross-complementing rodent repair deficiency, complementation group 1 (ERCC1) expression correlated with outcome on univariate analysis. CONCLUSIONS: p16(INK4a) overexpression was common and was a marker of favorable prognosis. p16(INK4a) overexpression was not a reliable predictor of HPV positivity in our cohort.

High XRCC1 protein expression is associated with poorer survival in patients with head and neck squamous cell carcinoma.

PURPOSE: We evaluated X-ray repair complementing defective repair in Chinese hamster cells 1 (XRCC1) protein in head and neck squamous cell carcinoma (HNSCC) patients in association with outcome. EXPERIMENTAL DESIGN: XRCC1 protein expression was assessed by immunohistochemical (IHC) staining of pretreatment tissue samples in 138 consecutive HNSCC patients treated with surgery (n = 31), radiation (15), surgery and radiation (23), surgery and adjuvant chemoradiation (17), primary chemoradiation (51), and palliative measures (1). RESULTS: Patients with high XRCC1 expression by IHC (n = 77) compared with patients with low XRCC1 expression (n = 60) had poorer median overall survival (OS; 41.0 months vs. OS not reached, P = 0.009) and poorer progression-free survival (28.0 months vs. 73.0 months, P = 0.031). This association was primarily due to patients who received chemoradiation (median OS of high- and low-XRCC1 expression patients, 35.5 months and not reached respectively, HR 3.48; 95% CI: 1.44-8.38; P = 0.006). In patients treated with nonchemoradiation modalities, there was no survival difference by XRCC1 expression. In multivariable analysis, high XRCC1 expression and p16(INK4a)-positive status were independently associated with survival in the overall study population (HR = 2.62; 95% CI: 1.52-4.52; P < 0.001 and HR = 0.21; 95% CI: 0.06-0.71; P = 0.012, respectively) and among chemoradiation patients (HR = 6.02; 95% CI: 2.36-15.37; P < 0.001 and HR = 0.26; 95% CI: 0.08-0.92, respectively; P = 0.037). CONCLUSIONS: In HNSCC, high XRCC1 protein expression is associated with poorer survival, particularly in patients receiving chemoradiation. Future validation of these findings may enable identification of HNSCC expressing patients who benefit from chemoradiation treatment.

Amplification of thymidylate synthetase in metastatic colorectal cancer patients pretreated with 5-fluorouracil-based chemotherapy.

Resistance to 5-fluorouracil (5-FU) represents a major contributor to cancer-related mortality in advanced colorectal cancer patients. Genetic variations and expression alterations in genes involved in 5-FU metabolism and effect have been shown to modulate 5-FU sensitivity in vitro, however these alterations do not fully explain clinical resistance to 5-FU-based chemotherapy. To determine if alterations of DNA copy number in genes involved in 5-FU metabolism-impacted clinical resistance to 5-FU-based chemotherapy, we assessed thymidylate synthetase (TYMS) and thymidine phosphorylase (TYMP) copy number in colorectal liver metastases. DNA copy number of TYMS and TYMP was evaluated using real time quantitative PCR in frozen colorectal liver metastases procured from 62 patients who were pretreated with 5-FU-based chemotherapy prior to surgical resection (5-FU exposed) and from 51 patients who received no pretreatment (unexposed). Gain of TYMS DNA copy number was observed in 18% of the 5-FU exposed metastases, while only 4% of the unexposed metastases exhibited TYMS copy gain (p = 0.036). No significant differences were noted in TYMP copy number alterations between 5-FU-exposed and -unexposed metastases. Median survival time was similar in 5-FU-exposed patients with metastases containing TYMS amplification and those with no amplification. However, TYMS amplification was associated with shorter median survival in patients receiving post-resection chemotherapy (hazard ratio = 2.7, 95% confidence interval = 1.1-6.6; p = 0.027). These results suggest amplification of TYMS amplification as a putative mechanism for clinical resistance to 5-FU-based chemotherapy and may have important ramifications for the post-resection chemotherapy choices for metastatic colorectal cancer.