William K. Funkhouser

Molecular classification of head and neck squamous cell carcinomas using patterns of gene expression.

The prognostication of head and neck squamous cell carcinoma (HNSCC) is largely based upon the tumor size and location and the presence of lymph node metastases. Here we show that gene expression patterns from 60 HNSCC samples assayed on cDNA microarrays allowed categorization of these tumors into four distinct subtypes. These subtypes showed statistically significant differences in recurrence-free survival and included a subtype with a possible EGFR-pathway signature, a mesenchymal-enriched subtype, a normal epithelium-like subtype, and a subtype with high levels of antioxidant enzymes. Supervised analyses to predict lymph node metastasis status were approximately 80% accurate when tumor subsite and pathological node status were considered simultaneously. This work represents an important step toward the identification of clinically significant biomarkers for HNSCC.

Selective activation of NF-κB subunits in human breast cancer: potential roles for NF-κB2/p52 and for Bcl-3

Members of the NF-κB/Rel transcription factor family have been shown recently to be required for cellular transformation by oncogenic Ras and by other oncoproteins and to suppress transformation-associated apoptosis. Furthermore, NF-κB has been shown to be activated by several oncoproteins including HER2/Neu, a receptor tyrosine kinase often expressed in human breast cancer. Human breast cancer cell lines, human breast tumors and normal adjacent tissue were analysed by gel mobility shift assay, immunoblotting of nuclear extracts and immunohistochemistry for activation of NF-κB. Furthermore, RNA levels for NF-κB-activated genes were analysed in order to determine if NF-κB is functionally active in human breast cancer. Our data indicate that the p65/RelA subunit of NF-κB is activated (i.e., nuclear) in breast cancer cell lines. However, breast tumors exhibit an absence or low level of nuclear p65/RelA but show activated c-Rel, p50 and p52 as compared to nontumorigenic adjacent tissue. Additionally, the IκB homolog Bcl-3, which functions to stimulate transcription with p50 or p52, was also activated in breast tumors. There was no apparent correlation between estrogen receptor status and levels of nuclear NF-κB complexes. Transcripts of NF-κB-regulated genes were found elevated in breast tumors, as compared to adjacent normal tissue, indicating functional NF-κB activity. These data suggest a potential role for a subset of NF-κB and IκB family proteins, particularly NF-κB/p52 and Bcl-3, in human breast cancer. Additionally, the activation of functional NF-κB in these tumors likely involves a signal transduction pathway distinct from that utilized by cytokines.

Lung squamous cell carcinoma mRNA expression subtypes are reproducible, clinically important, and correspond to normal cell types.

Purpose: Lung squamous cell carcinoma (SCC) is clinically and genetically heterogeneous, and current diagnostic practices do not adequately substratify this heterogeneity. A robust, biologically based SCC subclassification may describe this variability and lead to more precise patient prognosis and management. We sought to determine if SCC mRNA expression subtypes exist, are reproducible across multiple patient cohorts, and are clinically relevant. Experimental Design: Subtypes were detected by unsupervised consensus clustering in five published discovery cohorts of mRNA microarrays, totaling 382 SCC patients. An independent validation cohort of 56 SCC patients was collected and assayed by microarrays. A nearest-centroid subtype predictor was built using discovery cohorts. Validation cohort subtypes were predicted and evaluated for confirmation. Subtype survival outcome, clinical covariates, and biological processes were compared by statistical and bioinformatic methods. Results: Four lung SCC mRNA expression subtypes, named primitive, classical, secretory, and basal, were detected and independently validated (P < 0.001). The primitive subtype had the worst survival outcome (P < 0.05) and is an independent predictor of survival (P < 0.05). Tumor differentiation and patient sex were associated with subtype. The expression profiles of the subtypes contained distinct biological processes (primitive: proliferation; classical: xenobiotic metabolism; secretory: immune response; basal: cell adhesion) and suggested distinct pharmacologic interventions. Comparison with lung model systems revealed distinct subtype to cell type correspondence. Conclusions: Lung SCC consists of four mRNA expression subtypes that have different survival outcomes, patient populations, and biological processes. The subtypes stratify patients for more precise prognosis and targeted research. Clin Cancer Res; 16(19); 4864–75. ©2010 AACR.

Concomitant down-regulation of BRM and BRG1 in human tumor cell lines: Differential effects on RB-mediated growth arrest vs CD44 expression

Mammalian cells express two homologs of the SWI2 subunit of the SWI/SNF chromatin-remodeling complex called BRG1 and BRM. Whether the SWI/SNF complexes formed by these two subunits perform identical or different functions remains an important question. In this report, we show concomitant down-regulation of BRG1 and BRM in six human tumor cell lines. This down-regulation occurs at the level of mRNA abundance. We tested whether BRM could affect aberrant cellular functions attributed to BRG1 in tumor cell lines. By transient transfection, we found that BRM can restore RB-mediated cell cycle arrest, induce expression of CD44 protein and suppress Cyclin A expression. Therefore, BRM may be consistently down-regulated with BRG1 during neoplastic progression because they share some redundant functions. However, assorted tissues from BRM null/BRG1-positive mice lack CD44 expression, suggesting that BRM-containing SWI/SNF complexes regulate expression of this gene under physiological conditions. Our studies further define the mechanism by which chromatin-remodeling complexes participate in RB-mediated cell cycle arrest and provide additional novel evidence that the functions of SWI/SNF complexes containing BRG1 or BRM are not completely interchangeable.

Relevance, Pathogenesis, and Testing Algorithm for Mismatch Repair–Defective Colorectal Carcinomas: A Report of the Association for Molecular Pathology

Loss-of-function defects in DNA mismatch repair (MMR), which manifest as high levels of microsatellite instability (MSI), occur in approximately 15% of all colorectal carcinomas (CRCs). This molecular subset of CRC characterizes patients with better stage-specific prognoses who experience no benefit from 5-fluorouracil chemotherapy. Most MMR-deficient (dMMR) CRCs are sporadic, but 15% to 20% are due to inherited predisposition (Lynch syndrome). High penetrance of CRCs in germline MMR gene mutation carriers emphasizes the importance of accurate diagnosis of Lynch syndrome carriers. Family-based (Amsterdam), patient/family-based (Bethesda), morphology-based, microsatellite-based, and IHC-based screening criteria do not individually detect all germline mutation carriers. These limitations support the use of multiple concurrent tests and the screening of all patients with newly diagnosed CRC. This approach is resource intensive but would increase detection of inherited and de novo germline mutations to guide family screening. Although CRC prognosis and prediction of 5-fluorouracil response are similar in both the Lynch and sporadic dMMR subgroups, these subgroups differ significantly with regard to the implications for family members. We recommend that new CRCs should be classified into sporadic MMR-proficient, sporadic dMMR, or Lynch dMMR subgroups. The concurrent use of MSI testing, MMR protein IHC, and BRAF c.1799T>A mutation analysis would detect almost all dMMR CRCs, would classify 94% of all new CRCs into these MMR subgroups, and would guide secondary molecular testing of the remainder.

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.

Phase 2 Trial of De-intensified Chemoradiation Therapy for Favorable-Risk Human Papillomavirus–Associated Oropharyngeal Squamous Cell Carcinoma

PURPOSE To perform a prospective, multi-institutional, phase 2 study of a substantial decrease in concurrent chemoradiation therapy (CRT) intensity as primary treatment for favorable-risk, human papillomavirus-associated oropharyngeal squamous cell carcinoma. METHODS AND MATERIALS The major inclusion criteria were: (1) T0 to T3, N0 to N2c, M0; (2) human papillomavirus or p16 positive; and (3) minimal/remote smoking history. Treatment was limited to 60 Gy intensity modulated radiation therapy with concurrent weekly intravenous cisplatinum (30 mg/m(2)). The primary study endpoint was pathologic complete response (pCR) rate based on required biopsy of the primary site and dissection of pretreatment positive lymph node regions, regardless of radiographic response. Power computations were performed for the null hypothesis that the pCR rate is 87% and n=40, resulting in a type 1 error of 14.2%. Secondary endpoint measures included physician-reported toxicity (Common Toxicity Terminology for Adverse Events, CTCAE), patient-reported symptoms (PRO-CTCAE), and modified barium swallow studies. RESULTS The study population was 43 patients. The pCR rate was 86% (37 of 43). The incidence of CTCAE grade 3/4 toxicity and PRO-CTCAE severe/very severe symptoms was as follows: mucositis 34%/45%, general pain 5%/48%, nausea 18%/52%, vomiting 5%/34%, dysphagia 39%/55%, and xerostomia 2%/75%. Grade 3/4 hematologic toxicities were 11%. Thirty-nine percent of patients required a feeding tube for a median of 15 weeks (range, 5-22 weeks). There were no significant differences in modified barium swallow studies before and after CRT. CONCLUSIONS The pCR rate with decreased intensity of therapy with 60 Gy of IMRT and weekly low-dose cisplatinum is very high in favorable-risk oropharyngeal squamous cell carcinoma, with evidence of decreased toxicity compared with standard therapies. ClinicalTrials.gov ID: NCT01530997.

MyD88 is required for protection from lethal infection with a mouse-adapted SARS-CoV.

A novel human coronavirus, SARS-CoV, emerged suddenly in 2003, causing approximately 8000 human cases and more than 700 deaths worldwide. Since most animal models fail to faithfully recapitulate the clinical course of SARS-CoV in humans, the virus and host factors that mediate disease pathogenesis remain unclear. Recently, our laboratory and others developed a recombinant mouse-adapted SARS-CoV (rMA15) that was lethal in BALB/c mice. In contrast, intranasal infection of young 10-week-old C57BL/6 mice with rMA15 results in a nonlethal infection characterized by high titer replication within the lungs, lung inflammation, destruction of lung tissue, and loss of body weight, thus providing a useful model to identify host mediators of protection. Here, we report that mice deficient in MyD88 (MyD88−/−), an adapter protein that mediates Toll-like receptor (TLR), IL-1R, and IL-18R signaling, are far more susceptible to rMA15 infection. The genetic absence of MyD88 resulted in enhanced pulmonary pathology and greater than 90% mortality by day 6 post-infection. MyD88−/− mice had significantly higher viral loads in lung tissue throughout the course of infection. Despite increased viral loads, the expression of multiple proinflammatory cytokines and chemokines within lung tissue and recruitment of inflammatory monocytes/macrophages to the lung was severely impaired in MyD88−/− mice compared to wild-type mice. Furthermore, mice deficient in chemokine receptors that contribute to monocyte recruitment to the lung were more susceptible to rMA15-induced disease and exhibited severe lung pathology similar to that seen in MyD88−/−mice. These data suggest that MyD88-mediated innate immune signaling and inflammatory cell recruitment to the lung are required for protection from lethal rMA15 infection.

SARS-CoV pathogenesis is regulated by a STAT1 dependent but a type I, II and III interferon receptor independent mechanism.

Severe acute respiratory syndrome coronavirus (SARS-CoV) infection often caused severe end stage lung disease and organizing phase diffuse alveolar damage, especially in the elderly. The virus-host interactions that governed development of these acute end stage lung diseases and death are unknown. To address this question, we evaluated the role of innate immune signaling in protection from human (Urbani) and a recombinant mouse adapted SARS-CoV, designated rMA15. In contrast to most models of viral pathogenesis, infection of type I, type II or type III interferon knockout mice (129 background) with either Urbani or MA15 viruses resulted in clinical disease outcomes, including transient weight loss, denuding bronchiolitis and alveolar inflammation and recovery, identical to that seen in infection of wildtype mice. This suggests that type I, II and III interferon signaling play minor roles in regulating SARS pathogenesis in mouse models. In contrast, infection of STAT1−/− mice resulted in severe disease, high virus titer, extensive pulmonary lesions and 100% mortality by day 9 and 30 post-infection with rMA15 or Urbani viruses, respectively. Non-lethal in BALB/c mice, Urbani SARS-CoV infection in STAT1−/− mice caused disseminated infection involving the liver, spleen and other tissues after day 9. These findings demonstrated that SARS-CoV pathogenesis is regulated by a STAT1 dependent but type I, II and III interferon receptor independent, mechanism. In contrast to a well documented role in innate immunity, we propose that STAT1 also protects mice via its role as an antagonist of unrestrained cell proliferation.

Novel critical role of Toll-like receptor 4 in lung ischemia-reperfusion injury and edema

Toll-like receptors (TLRs) of the innate immune system contribute to noninfectious inflammatory processes. We employed a murine model of hilar clamping (1 h) with reperfusion times between 15 min and 3 h in TLR4-sufficient (C3H/OuJ) and TLR4-deficient (C3H/HeJ) anesthetized mice with additional studies in chimeric and myeloid differentiation factor 88 (MyD88)- and TLR4-deficient mice to determine the role of TLR4 in lung ischemia-reperfusion injury. Human pulmonary microvascular endothelial monolayers were subjected to simulated warm ischemia and reperfusion with and without CRX-526, a competitive TLR4 inhibitor. Functional TLR4 solely on pulmonary parenchymal cells, not bone marrow-derived cells, mediates early lung edema following ischemia-reperfusion independent of MyD88. Activation of MAPKs and NF-kappaB was significantly blunted and/or delayed in lungs of TLR4-deficient mice as a consequence of ischemia-reperfusion injury, but edema development appeared to be independent of activation of these signaling pathways. Pretreatment with a competitive TLR4 inhibitor prevented edema in vivo and reduced actin cytoskeletal rearrangement and gap formation in pulmonary microvascular endothelial monolayers subjected to simulated warm ischemia and reperfusion. In addition to its well-accepted role to alter gene transcription, functioning TLR4 on pulmonary parenchymal cells plays a key role in very early and profound pulmonary edema in murine lung ischemia-reperfusion injury. This may be due to a novel mechanism: regulation of endothelial cell cytoskeleton affecting microvascular endothelial cell permeability.