HNSCC Specimens Research Articles

Deglycosylation of pathological specimens alters performance of diagnostic PDL1 antibodies.

Immunohistochemical (IHC) predictive quantitation of PDL1 expression is obligatory in many cancer entities with improved response to immune checkpoint inhibition in PDL1-positive subgroups. With recent demonstration of increased positivity rates after enzymatic deglycosylation in breast cancer specimens, a comparative analysis with two different antibodies and extended controls was performed in a cohort of head and neck squamous cell cancer samples (HNSCC).Formalin-fixed paraffin-embedded tissue from HNSCC specimens was used for initial on-slide method optimization based on the PNGase F assay. SDS-PAGE and immunoblotting with the PDL1 antibody 28-8 was performed to evaluate deglycosylation efficiency. A tissue micro array of n = 527 tissue cores of 181 patients with HNSCC was used to determine the effects of deglycosylation on staining pattern and intensity with PDL1 antibodies 28-8 and E1L3N.Successful on-slide deglycosylation with PNGase F was confirmed by immunoblot but varied across replicates. Using E1L3N (intracellular binding domain, most probably not glycosylated), mean signal intensity as well as the fraction of PDL1 positive cells was increased by deglycosylation. Opposite effects were observed with 28-8 (extracellular binding domain, glycosylated).Deglycosylation reduces diagnostic performance of the PDL1 antibody 28-8. In contrast, effects for E1L3N are complex and probably involve reduction of off-target binding leading to specifically improved signal intensity. However, enzymatic deglycosylation adds further variance to IHC.

Immunological profiling of tumor-infiltrating CD8+ T lymphocytes in non-small cell lung cancer, head and neck squamous cell carcinoma, breast cancer, and renal cell cancer

Abstract To explore whether the variation in clinical response to immune checkpoint blockade (ICB) reflects intrinsic characteristics of tumor-infiltrating lymphocytes (TIL), TILs from multiple tumor types were analyzed by multiparameter flow cytometry. Recent work identified CD39+CD103+ double positive (DP) CD8 TIL as tumor-reactive, therefore we assessed this phenotype in primary NSCLC (n=28), BCa (n=23), HNSCC (n=23), and RCC (n=23) specimens. TILs from NSCLC and HNSCC had significantly higher frequencies of DP T cells than BCa and RCC (NSCLC: median=34.2, IQR=15.0–53.8%; HNSCC: median=28.6%, IQR=13.5–44.5%), while TILs from BCa and RCC had low frequencies of DP T cells (BCa: median=2.4%, IQR=1.1–3.9%; RCC: median=4.2%, IQR=1.5–16.6%). Additionally, DP cells in NSCLC and HNSCC were co-expressed immune checkpoint markers PD-1, TIM-3, and LAG-3 at a higher frequency than RCC or BCa (P<0.05). DP CD8 T cells in NSCLC and HNSCC also exhibited increased effector potential (granzyme B+) as compared to BCa and RCC, suggesting functional differences. Expression of the transcription factor Eomesodermin, associated with T cell exhaustion, was higher in effector CD8 T cells from RCC (median=84.5%, IQR=44.9–97.4%) as compared to NSCLC (median=28.7%, IQR=20.7–61.4%), BCa (median=30.1%, IQR=0–50%), and HNSCC (median=40.3%, IQR=20.1–61.2%). Additionally, 4-1BB, an indicator of TCR engagement, trended higher in RCC (mean=8.6%, IQR=1.4–46.7%) as compared to BCa (median=1.9%, IQR=0.4–8.6%). These data highlight the heterogeneity of human TILs isolated from distinct tumor types and provide insight into the basal expression of actionable therapeutic targets. Supported by GlaxoSmithKline and the Providence Portland Medical Foundation

Fewer tumour-specific PD-1+CD8+ TILs in high-risk \u201cInfiltrating\u201d HPV\u2212 HNSCC

BackgroundThe prognosis of HPV- HNSCC was worse than that of HPV+ HNSCC. Analysis of tumours and tumour-infiltrating lymphocytes (TILs) may provide insight into the progression of HPV− HNSCC.MethodsThe tumour and TIL phenotypic characteristics of 134 HNSCC specimens (HPV− tumours were classified into “Infiltrating” and “Pushing” subtypes based on their different tumour nest configuration and prognosis) were retrospectively analysed. HNSCC data from the Cancer Genome Atlas (n = 263) were analysed for CD8α, HPV and overall survival (OS). A murine HNSCC model was used to verify the antitumour role of PD-1+CD8+ TILs.ResultsThe “Infiltrating” HPV− subtype showed shorter OS than the “Pushing” subtype. Moreover, there is a tendency from “Pushing” to “Infiltrating” subtype from the primary to the recurrent lesion. Different from total CD8+ TILs, tumour-specific PD-1+CD8+ TILs were fewer in invasive margin (IM) of “Infiltrating” HPV− tumours. PD-1+CD8+ TILs recognised autologous HNSCC cells and showed stronger inhibition of tumour growth in a murine HNSCC model resistant to PD-1 blockade.ConclusionsCoevolution of HPV− HNSCC and TILs is characterised by an “Infiltrating” phenotype and less tumour-specific PD-1+CD8+ TILs, which may provide a framework for further translational studies and patient stratification.

Head and Neck Cancers Promote an Inflammatory Transcriptome through Coactivation of Classic and Alternative NF-κB Pathways.

Head and neck squamous cell carcinomas (HNSCC) promote inflammation in the tumor microenvironment through aberrant NF-κB activation, but the genomic alterations and pathway networks that modulate NF-κB signaling have not been fully dissected. Here, we analyzed genome and transcriptome alterations of 279 HNSCC specimens from The Cancer Genome Atlas (TCGA) cohort and identified 61 genes involved in NF-κB and inflammatory pathways. The top 30 altered genes were distributed across 96% of HNSCC samples, and their expression was often correlated with genomic copy-number alterations (CNA). Ten of the amplified genes were associated with human papilloma virus (HPV) status. We sequenced 15 HPV- and 11 HPV+ human HNSCC cell lines, and three oral mucosa keratinocyte lines, and supervised clustering revealed that 28 of 61 genes exhibit altered expression patterns concordant with HNSCC tissues and distinct signatures related to their HPV status. RNAi screening using an NF-κB reporter line identified 16 genes that are induced by TNFα or Lymphotoxin-β (LTβ) and implicated in the classic and/or alternative NF-κB pathways. Knockdown of TNFR, LTBR, or selected downstream signaling components established cross-talk between the classic and alternative NF-κB pathways. TNFα and LTβ induced differential gene expression involving the NF-κB, IFNγ, and STAT pathways, inflammatory cytokines, and metastasis-related genes. Improved survival was observed in HNSCC patients with elevated gene expression in T-cell activation, immune checkpoints, and IFNγ and STAT pathways. These gene signatures of NF-κB activation, which modulate inflammation and responses to the immune therapy, could serve as potential biomarkers in future clinical trials.

The Nedd8-activating enzyme inhibitor MLN4924 (TAK-924/Pevonedistat) induces apoptosis via c-Myc-Noxa axis in head and neck squamous cell carcinoma.

ObjectivesThe present study aimed to reveal expression status of the neddylation enzymes in HNSCC and to elucidate the anticancer efficacy and the underlying mechanisms of inhibiting neddylation pathway.Materials and methodsThe expression levels of neddylation enzymes were estimated by Western blotting in human HNSCC specimens and bioinformatics analysis of the cancer genome atlas (TCGA) database. Cell apoptosis was evaluated by Annexin V fluorescein isothiocyanate/propidium iodide (Annexin V‐FITC/PI) stain and fluorescence‐activated cell sorting (FACS). Small interfering RNA (siRNA) and the CRISPR‐Cas9 system were used to elucidate the underlying molecular mechanism of MLN4924‐induced HNSCC apoptosis.ResultsExpression levels of NAE1 and UBC12 were prominently higher in HNSCC tissues than that in normal tissues. Inactivation of the neddylation pathway significantly inhibited malignant phenotypes of HNSCC cells. Mechanistic studies revealed that MLN4924 induced the accumulation of CRL ligase substrate c‐Myc that transcriptionally activated pro‐apoptotic protein Noxa, which triggered apoptosis in HNSCC.ConclusionsThese findings determined the over‐expression levels of neddylation enzymes in HNSCC and revealed novel mechanisms underlying neddylation inhibition induced growth suppression in HNSCC cells, which provided preclinical evidence for further clinical evaluation of neddylation inhibitors (eg, MLN4924) for the treatment of HNSCC.

The expression and biological information analysis of miR-375-3p in head and neck squamous cell carcinoma based on 1825 samples from GEO, TCGA, and peer-reviewed publications

BackgroundThe specific expression level and clinical significance of miR-375-3p in HNSCC had not been fully stated, as well as the overall biological function and molecular mechanisms. Therefore, we purpose to carry out a comprehensive meta-analysis to further explore the clinical significance and potential function mechanism of miR-375-3p in HNSCC. MethodsHNSCC-related data was gained from Gene Expression Omnibus (GEO), The Cancer Genome Atlas (TCGA), and peer-reviewed journals. A meta-analysis was carried out to comprehensively explore the relationship between miR-375-3p expression level and clinicopathological features of HNSCC. And summary receiver operating characteristic (SROC) curve analysis was applied for evaluating disease diagnosis value of miR-375-3p. In addition, a biological pathway analysis was also performed to assess the possible molecular mechanism of miR-375-3p in HNSCC. ResultsA total of 24 available records and references were added into analysis. The overall pooled meta-analysis outcome revealed a relatively lower expression level of miR-375-3p in HNSCC specimens than that in non-cancerous controls (P < 0.001). And SROC curve analysis showed that the pooled area under the SROC curve (AUC) was 0.90 (95%CI: 0.88–0.93). In addition, biological pathway analysis indicated that LAMC1, EDIL3, FN1, VEGFA, IGF2BP2, and IGF2BP3 maybe the latent target genes of miR-375-3p, which were greatly enriched in the pathways of beta3 integrin cell surface interactions and the binding of RNA via the insulin-like growth factor-2 mRNA-binding protein (IGF2BPs/IMPs/VICKZs). ConclusionMiR-375-3p expression clearly decreased in HNSCC samples compared with non-cancerous controls. Meanwhile, miR-375-3p may serve as a tumor suppressor via regulating tumor-related genes LAMC1, EDIL3, FN1, VEGFA, IGF2BP2, and IGF2BP3 in HNSCC.

Interaction between N-cadherin and decoy receptor-2 regulates apoptosis in head and neck cancer.

N-cadherin is a neural cell adhesion molecule that aberrantly occurs in head and neck cancers to promote cancer cell growth. However, the underlying mechanisms remain unclear. Here we report that N-cadherin increases cancer cell growth by inhibiting apoptosis. Apoptosis eliminates old, unnecessary, and unhealthy cells. However, tumor cells have the ability of avoiding apoptosis that increases cancer cell growth. Recent studies have found that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in tumor cells by reacting with four distinct cell surface receptors: TRAIL-R1 (DR-4), TRAIL-R2 (DR-5), TRAIL-R3 (DcR-1), and TRAIL-R4 (DcR-2). Among these TRAIL receptors, the death receptors DR-4 and DR-5 transmit apoptotic signals owing to the death domain in the intracellular portion. Conversely, the decoy receptors DcR-1 and DcR-2 lack a complete intracellular portion, so neither can transmit apoptotic signals. DcR-1 or DcR-2 overexpression suppresses TRAIL-induced apoptosis.In this study, N-cadherin overexpression increased DcR-2 expression and decreased DR-5 expression. In contrast, knockdown of N-cadherin expression upregulated DR-5 expression and downregulated DcR-2 expression. A significantly positive relationship between N-cadherin and DcR-2 expression was also found in HNSCC specimens. Those specimens with a lower apoptotic index showed a higher expression of N-cadherin and/or DcR-2. In addition, we demonstrated that N-cadherin interacts directly with DcR-2. Notably, DcR-2 induces cancer cell survival through the cleavage of caspases and PARP by activating MAPK/ERK pathway and suppressing NF-kB/ p65 phosphorylation, which has a very important role in resistance to chemotherapy.

Stimulation of the toll-like receptor 3 promotes metabolic reprogramming in head and neck carcinoma cells.

In this study, a possible link between the innate immune recognition receptor TLR3 and metabolic reprogramming in Head and Neck carcinoma (HNC) cells was investigated. The effects of TLR3 stimulation/knock-down were assessed under several culture conditions in 4 HNC cell-lines by cell growth assays, targeted metabolomics, and glycolysis assays based on time-resolved analysis of proton release (Seahorse analyzer). The stimulation of TLR3 by its synthetic agonist Poly(A:U) resulted in a faster growth of HNC cells under low foetal calf serum conditions. Targeted analysis of glucose metabolism pathways demonstrated a tendency towards a shift from tricarboxylic acid cycle (Krebs cycle) to glycolysis and anabolic reactions in cells treated with Poly(A:U). Glycolysis assays confirmed that TLR3 stimulation enhanced the capacity of malignant cells to switch from oxidative phosphorylation to extra-mitochondrial glycolysis. We found evidence that HIF-1α is involved in this process: addition of the TLR3 agonist resulted in a higher cell concentration of the HIF-1α protein, even in normoxia, whereas knocking-down TLR3 resulted in a lower concentration, even in hypoxia. Finally, we assessed TLR3 expression by immunohistochemistry in a series of 7 HNSCC specimens and found that TLR3 was detected at higher levels in tumors displaying a hypoxic staining pattern. Overall, our results demonstrate that TLR3 stimulation induces the Warburg effect in HNC cells in vitro, and suggest that TLR3 may play a role in tumor adaptation to hypoxia.

Abstract 2988: Integrated functional RNAi screening and structural genomics identify cell cycle and kinetochore components co-modulating NF-κB activity as potential therapeutic targets in head and neck squamous cell carcinoma

Abstract Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, with over 600,000 new cases diagnosed each year, and a five year survival of ∼50%. Recent findings in The Cancer Genome Atlas (TCGA) analysis of 279 HNSCC patient samples revealed that components of the NF-κB/REL signaling pathways are aberrantly expressed in about ∼70% of cases, and implicated in expression of pro-proliferative, inflammatory, angiogenic, and therapeutic resistance genes. Alterations are prevalent in this pathway across patient samples; however, the detailed mechanisms that increase NF-κB signaling in HNSCC are not well understood. Recently, we established an NF-κB β-lactamase reporter UM-SCC-1 cell line to identify key regulators of this NF-κB oncogenic signaling. We performed genome-wide RNAi screening using the NF-κB reporter line, and upon validation, most structural components of the TNF receptor complex and downstream NF-κB pathway genes, such as IKKs, were scored highly in the screening. We integrated the functional genomics data with structural mutation and expression data from TCGA to prioritize genes that are frequently deregulated in HNSCC specimens. WNT9/FZD6, NOTCH2, and TGFβR2 and several receptors were identified to cross-activate NF-κB signaling. In addition, we observed novel genes that co-regulate NF-κB activity, which are involved in the cell cycle, such as CDC2, CDC7, WEE1 and CALM2, as well as components of the kinetochore such as NDC80, PLK1, TTK, and AURKA. Among these targets WEE1 kinase is currently under investigation as a therapeutic target in HNSCC using the WEE1 inhibitor AZD1775. To further investigate potential cross-talk between WEE1 cell cycle regulation and NF-κB activation, we have shown that treatment of HNSCC cells with AZD1775 decreases NF-κB transcriptional activation which corresponds to decrease in TNF-inducible phosphorylation of RELA. These changes also correlate with alteration in cell cycle regulation detected by flow cytometry. Mechanistic studies are currently underway to understand how WEE1 signaling connects to NF-κB activation, and what role inhibition of NF-κB signaling plays in AZD1775 activity as a sensitizer to chemo-or radiation therapy for HNSCC. Supported by NIDCD intramural projects ZIA-DC-000073,74 Citation Format: Anthony D. Saleh, Sophie Carlson, Shaleeka Cornelius, Hui Cheng, Scott Martin, Pinar Ormanoglu. Integrated functional RNAi screening and structural genomics identify cell cycle and kinetochore components co-modulating NF-κB activity as potential therapeutic targets in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2988.

Abstract 1724: Integrated functional RNAi screening and structural genomics identify inverse co-modulators of TP53 family and NF-κB transitional activation as potential therapeutic targets in head and neck squamous cell carcinoma

Abstract Head and neck squamous cell carcinoma (HNSCC) is the 6th most common cancer worldwide with a 50-60% mortality rate. Deregulation of p53 family members in HNSCC occurs in over 90% of cases, preventing transcription of growth arrest and apoptosis genes. Conversely, members of the NF-κB/REL family are aberrantly activated in about ∼70% of cases, and drive expression of pro-proliferation, inflammation, angiogenesis, and therapeutic resistance genes. The function of different TP53 and NF-κB family members are inversely modulated within two major subsets of HNSCC, suggesting that common molecules and pathways coordinate this modulation. To test this hypothesis, we have developed HNSCC stable cell lines that report transcriptional activation of TP53 or NF-κB individually through a β-lactamase reporter. Whole genome siRNA screening in our NF-κB reporter cell line has been performed. Screening was performed by 56-hour knockdown with siRNAs, followed by stimulation with TNF-α. The results have revealed known and novel targets that maintain oncogenic NF-κB signaling in HNSCC. Validating the assay, most structural components of the TNF receptor complex and downstream NF-κB pathway genes, such as IKKs, scored highly in the screen. We integrated this functional genomics data with structural mutation and expression data from TCGA to prioritize genes that are frequently deregulated in HNSCC specimens from patients. Several co-receptors that may support NF-κB signaling were identified such as WNT9/FZD6, NOTCH2, and TGFβR2. Our primary novel finding was the identification of genes that regulate mitosis, such as CDC2, CDC7, CHEK1, WEE1 and CALM2. Several components of the kinetochore were also identified including NDC80, PLK1,TTK and AURKA. To validate our screening data, individual assays were performed with two siRNAs for a panel of 27 genes from the pathways mentioned above. &amp;gt;95% of siRNAs tested validated our screening data. Genes scoring well in our analysis were also assayed in our p53 reporter cell line using cisplatin and TNF-α as stimuli. Genes that inversely activate p53 were identified. To further investigate the relationship between kinetochore function and NF-κB activation, we treated cells with various G2/M inhibitors such as docetaxel and showed decreased NF-κB signaling. Future experiments are underway to determine the signaling mechanism that connects the kinetochore to NF-κB regulators. Experiments are also ongoing to determine if aberrant activation of NF-κB can serve as a biomarker for sensitivity to cell cycle inhibitors. Citation Format: Anthony Saleh, Shaleeka Cornelius, Scott Martin, Pinar Ormanoglu, Hui Cheng, Rita Das, Xinping Yang, Zhong Chen, Carter Van Waes. Integrated functional RNAi screening and structural genomics identify inverse co-modulators of TP53 family and NF-κB transitional activation as potential therapeutic targets in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1724. doi:10.1158/1538-7445.AM2015-1724

Abstract 4211: The proteomic characterization of HNSCC patient-derived xenografts

Abstract Head and neck squamous cell carcinoma (HNSCC) is the fifth most common cancer in the US and a leading cancer worldwide. There has been little change in the prognosis for these patients despite extensive research and FDA approval of the targeted therapy cetuximab. Cetuximab has been effective in immortalized HNSCC cell lines but demonstrates only a modest benefit for a subset of patients. This gap between pre-clinical discovery and testing and final clinical efficacy can be narrowed improving the translational relevance of preclinical model systems. Patient derived xenografts (PDXs) have emerged as a promising approach in several cancers. We sought to define and compare the protein expression patterns between primary HNSCC and PDXs derived from 13 patient tumors and test predictive biomarkers to cetuximab therapy. We compared 13 PDXs propagated at the University of Pittsburgh and analyzed for the expression levels of 190 proteins by reverse-phase protein array (RPPA) to 212 HNSCC specimens and 4566 cancer specimens from TCGA also analyzed by RPPA. We found that 12/13 PDXs were more like HNSCC than any of 14 other cancer types analyzed. Unsupervised clustering of the primary HNSCC and the PDXs demonstrated that PDXs clustered together, indicating some differences in protein expression between HNSCC and PDXs. Further analysis demonstrated that a subset of the proteins were different between the mouse derived PDXs and the primary HNSCC while a second subset were similar between mouse derived PDXs and primary HNSCC. Proteins that were different between these two groups may be poor candidates for translational models, but proteins that were similar may prove useful. We tested the different responses to cetuximab using the PDXs. We found that a PDX with high EGFR/pEGFR expression was more sensitive to cetuximab treatment than a PDX with low EGFR/pEGFR expression. These results suggest that improved model systems used for translational may predict clinical responses. RPPA measurement of protein expression in PDXs and primary cancers demonstrates that PDXs do represent HNSCC overall. Comparison of the PDXs specifically with primary HNSCC reveals that PDXs are not an appropriate model system for all targeted therapies/studies but provide an effective model for many studies of interest in the field. Citation Format: Hua Li, Sarah E. Wheeler, Yongseok Park, Zhenlin Ju, Ann Marie Egloff, Michele Fichera, Sufi Thomas, Gordon B. Mills, Jennifer R. Grandis. The proteomic characterization of HNSCC patient-derived xenografts. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4211. doi:10.1158/1538-7445.AM2015-4211

Aurora-A signaling is activated in advanced stage of squamous cell carcinoma of head and neck cancer and requires osteopontin to stimulate invasive behavior.

The clinical significances, cellular effects, and molecular mechanisms by which Aurora-A mediate its invasive effects in HNSCC are still unclear. Here, we found that Aurora-A expression is significantly higher in tumor tissues on 14-microarray of HNSCC in Oncomine-databases. The activity of Aurora-A was not only found in HNSCC specimens, but also significantly correlated with advanced-T-classification, positive-N-classification, TNM-stage and the poor 5-year survival rate. HNSCC-microarray profile showed that osteopontin and Aurora-A exhibited positive correlation. Stimulation of HNC cells with osteopontin results in an increase in Aurora-A expression where localized at the centrosome. Functionally, Aurora-A had the abilities to stimulate cell motility in HNC cells through increase ERK1/2 activity under osteopontin stimulation. Conversely, depletion of Aurora-A expression by siRNAs suppressed ERK1/2 activity as well as inhibition of cell invasiveness. Treatment with anti-CD44 antibodies in HNC cells not only caused a decrease of mRNA/protein of Aurora-A and ERK1/2 activity upon osteopontin stimulation, but also affected the abilities of Aurora-A-elicited cell motility. Finally, immunohistochemical/Western-blotting analysis of human aggressive HNSCC specimens showed a significant positively correlation between osteopontin-Aurora-A and ERK1/2. These findings suggest that Aurora-A is not only an important prognostic factor but also a new therapeutic target in the osteopontin/CD44/ERK pathway for HNSCC treatment.

Genetic alterations in HRAS gene in relation to outcome and response to cetuximab in head and neck squamous cell carcinoma.

5574 Background: Aberrant signaling through RAS/MAPK pathway is implicated in resistance to EGFR-targeted agents in cancer. Genetic alterations in Hras gene such as mutations and specific polymorphisms are associated with aggressive phenotype in several smoking-related malignancies. We sought to determine the impact of Hras genetic alterations on response to cetuximab and prognosis in HNSCC. Methods: Clinical outcome according to Hras status was investigated in a retrospective cohort of 140 HNSCC specimens. Primary endpoints were overall survival (OS) and disease-free survival (DFS) and secondary endpoint was treatment response. For statistical analysis, T-test was used for continuous data and x2 –test for categorical data. Cetuximab-resistant cell lines harboring mutant Hras (BB49, T24) were infected with lentivirus expressing shRNA targeting the Hras or a scrambled- shRNA. MTT assay was used to determine the effect of cetuximab on growth of lentivirus infected cells. Biochemical analysis involved immunoblotting for pERK1/2. Results: Mutationanalysis of tumor samples showed that 5.7% participants harbored Hras mutations and 16.42% harbored Hras polymorphisms (rs12628, rs41258054) that are associated with tumorigenesis. Patients bearing tumors with mutated Hras had inferior mean OS ( 22.13vs 35.20, p=0.02) and a non-significant trend for inferior mean DFS. Patients with tumors containing Hras genetic alterations (mutation or polymorphism) had significantly inferior mean OS (p=0.02) compared to those harboring wt Hras and trended towards inferior DFS (p=0.07). Patients had received various treatments such as surgery plus/minus RT and various chemotherapy regimens. A subgroup analysis of 38 patients treated with cetuximab-based regimens showed that wt Hras was associated with higher likelihood of attaining CR or PR to treatment of borderline significance (p=0.06) due to small sample size. Silencing of Hras in Hras-mutant cell lines restored sensitivity to cetuximab and caused a direct downregulation of pERK1/2 levels. Conclusions: Hras genetic alterations are associated with aggressive clinical course and may affect response to cetuximab in HNSCC.

Abstract 786: Alterations of TGFβ/Smad signaling in human head and neck squamous cell carcinomas

Abstract Head-and-neck squamous cell carcinoma (HNSCC) ranks as the sixth most frequent cancer worldwide. Based on earlier in vitro studies of HNSCC cell lines and genetic analysis of human HNSCC specimens, we postulated that disruptions of Transforming Growth Factor- β (TGF-β) signaling contribute to the development and progression of HNSCC. Smad proteins are the intracellular components of the TGF-β signaling pathway. In a previous immunohistochemical study of the expression and activation of Smads in 170 HNSCC specimens assembled in a tissue microarray, phospho-Smad2 (pSmad2), an active form of Smad2 and Smad4 were absent in 14% and 22% of cases respectively. Moreover, there was a trend towards improved overall survival (OS) among patients with pSmad2-negative tumors. In the present study, we analyzed the pattern of expression of (p)Smad2, (p)Smad3 and Smad4 in a much larger 4-fold redundant tissue array of 798 HNSCC tumor samples obtained from 346 patients. In addition, we compared a new automated scoring method to standard visual scoring of immunostained slides. Each tissue core was visually scored as positive or negative, and the four scores averaged to derive a visual score (VS). Automated scores (AS) were obtained by scanning all slides using a Zeiss MedMicro whole slide scanner at 40×equivalent resolution. Images of individual cores were extracted from the scanned archive and staining maps of chromogen (brown) were isolated from the counter stain using our previously reported color decomposition algorithm. AS was computed based on the staining characteristic of each core and a final score for each specimen was obtained by averaging the scores of four matched cores. Receiver operating characteristic (ROC) curves demonstrated that the AS scoring method performed extremely well compared the VS “gold standard”, with area under the curve (AUC) values of &amp;gt;80% for all 5 Smad immunostains. We found that 19% (95% CI: 16-21%), 40% (95% CI: 36-43%) and 12% (95% CI: 10-15%) of HNSCC specimens failed to express pSmad2, pSmad3 or Smad4, respectively. Loss of TGF-β receptor signaling, as indicated by both pSmad2 and −3 negativity, was observed in 11% (95% CI: 9-13%) of specimens. In addition, 10% (95% CI: 8-12%) of specimens failed to express Smad4. These results are consistent with our previous findings in a smaller sample set. Moreover, among 198 patients with survival information, patients with pSmad2/3 negative tumors had a significantly higher OS when compared with patients with pSmad2/3 positive tumors (p=0.0090). In contrast, loss of Smad4 expression was not associated with poor survival. These results indicate that inactivation of TGF-β receptor signaling or of Smad4 in HNSCC have different clinical implications and may reflect underlying differences in biological significance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 786.

Squamous Carcinoma Cells Influence Monocyte Phenotype and Suppress Lipopolysaccharide-Induced TNF-alpha in Monocytes

Bacteria and chronic inflammation are present in squamous cell carcinoma of the head and neck (HNSCC), but their roles in the pathogenesis of HNSCC are unclear. Our studies described here revealed that human monocytes co-cultured short term with HNSCC cells were more likely to express CD16, and CD16(+) small mononuclear cells were common in HNSCC specimens. In addition, we identified monocytes as the primary source of LPS-induced IL-6 and TNF-alpha in the monocyte-HNSCC co-cultures. Remarkably, relative to LPS-stimulated monocytes cultured alone, HNSCC cells profoundly suppressed LPS-induced TNF-alpha in monocytes, without compromising IL-6 production. High levels of cytoprotective factors like IL-6 and low levels of TNF-alpha are important for the tumor microenvironment that enables tumor cell survival, affects monocyte differentiation and may contribute to tumor colonization by bacteria. This study provides novel observations that HNSCC cells affect monocyte phenotype and function, which are relevant to the regulation of the HNSCC microenvironment.

Overexpression and nuclear translocation of hypoxia-inducible factor prolyl hydroxylase PHD2 in head and neck squamous cell carcinoma is associated with tumor aggressiveness.

Hypoxia in tumors is associated with poor prognosis and resistance to treatment. The outcome of hypoxia is largely regulated by the hypoxia-inducible factors (HIF-1alpha and HIF-2alpha). HIFs in turn are negatively regulated by a family of prolyl hydroxylases (PHD1-3). The PHD2 isoform is the main down-regulator of HIFs in normoxia and mild hypoxia. This study was designed to analyze the correlation of the expression and subcellular localization of PHD2 with the pathologic features of human carcinomas and HIF-1alpha expression. The expression of PHD2 was studied from paraffin-embedded normal tissue (n = 21) and head and neck squamous cell carcinoma (HNSCC; n = 44) by immunohistochemistry. Further studies included PHD2 mRNA detection and HIF-1alpha immunohistochemistry from HNSCC specimens as well as PHD2 immunocytochemistry from HNSCC-derived cell lines. In noncancerous tissue, PHD2 is robustly expressed by endothelial cells. In epithelium, the basal proliferating layer also shows strong expression, whereas the more differentiated epithelium shows little or no PHD2 expression. In HNSCC, PHD2 shows strongly elevated expression both at the mRNA and protein level. Moreover, PHD2 expression increases in less differentiated phenotypes and partially relocalizes from the cytoplasm into the nucleus. Endogenously high nuclear PHD2 is seen in a subset of HNSCC-derived cell lines. Finally, although most of the tumor regions with high PHD2 expression show down-regulated HIF-1alpha, regions with simultaneous HIF-1alpha and PHD2 expression could be detected. Our results show that increased levels and nuclear translocation of the cellular oxygen sensor, PHD2, are associated with less differentiated and strongly proliferating tumors. Furthermore, they imply that even the elevated PHD2 levels are not sufficient to down-regulate HIF-1alpha in some tumors.

Motility-related protein-1/CD9 expression in head and neck squamous cell carcinoma.

Motility-related protein (MRP)-1/CD9 is implicated in cell adhesion and motility and was shown to be clearly involved in tumor prognosis and angiogenesis. Elevated MRP-1/CD9 expression on tumor cells has been linked to a favorable prognosis in breast cancer, colon cancer, lung cancer, and HNSCC. Because MRP-1/CD9 is associated with angiogenesis, it might play a role in tumor angiogenesis as well. We analyzed MRP-1/CD9 expression in HNSCC specimens and cell lines by real-time RT-PCR and in HNSCC biopsy specimens and stromal vessels by immunohistochemistry. Kruskal Wallis and Chi2 test, univariate and multivariate Cox regression, and Kaplan-Meier methods were used for statistical analysis. Real-time and PCR RT showed elevated expression of MRP-1/CD9 in one (SCC25) of four HNSCC cell lines and two of six HNSCC patients, whereas two cell lines (SCC9 and JPPA) and one HNSCC patient had lower MRP-1/CD9 levels compared with other specimens. Immunohistochemistry demonstrated strong MRP-1/CD9 IR expression on tumor cells in 13 patients (39%), whereas 21 patients (61%) had less to medium MRP-1/CD9 IR expression. Increased MRP-1/CD9 expression on tumor cells was correlated with prolonged patient survival (p =.02) and a longer disease-free interval (p =.004), a diminished recurrence rate (p =.02), and lower stages of neck lymph nodes (p =.04). MRP-1/CD9 IR was also found in a subpopulation of vessels that seem to be less in tumor specimens than in normal mucosa (p <.0001). MRP-1/CD9+ vessels are podoplanin+ and are therefore regarded as lymphatic vessels. Our results revealed that elevated MRP-1/CD9 expression on HNSCC is linked to a favorable clinical outcome and confirmed reports of MRP-1/CD9 expression in other carcinomas. MRP-1/CD9+ vessels were found to be lymphatic in nature. The number and staining intensity of these vessels is decreased in tumor tissue, which suggests a stabilizing role for this protein in lymphangiogenesis.

Vascular endothelial growth factor C and vascular endothelial growth factor receptor 3 expression in squamous cell carcinomas of the head and neck.

VEGF proteins and their receptors are involved in tumor vessel neoformation. The third VEGF receptor, VEGFR3 (flt-4) is important during both blood vessel development and lymphatic vessel formation. Because HNSCC preferentially metastasizes to regional lymph nodes, we investigated the expression of VEGFR3 and its ligand VEGF-C in head and neck squamous cell carcinomas by semiquantitative RT-PCR (4 HNSCC cells lines and 6 HNSCC specimens) and by immunohistochemistry (18 HNSCC specimens). VEGFR3 protein expression was confirmed by Western blotting in four HNSCC cell lines and six HNSCC specimens. Semiquantitative mRNA analysis showed VEGF-C mRNA expression in three (SCC9, SCC25, LFFR) of four HNSCC cell lines and all six HNSCC specimens. VEGFR3 mRNA was found in two HNSCC cell lines (JPPA and SCC25) and only weakly detected in the other two HNSCC cell lines (SCC9 and LFFR). High amounts of VEGFR3 mRNA were shown in all six patients' tumor specimens. VEGFR3 Western blot analysis yielded a distinct band at the predicted size of 210 kD in JPPA and SCC9 and hardly detectable bands in SCC25 and LFFR cell lines. All six HNSCC specimens displayed strong VEGFR3 protein bands. Immunohistochemistry in 18 HNSCC specimens assigned strong to mediate VEGF-C IR and minor VEGFR3 IR to tumor cells and strong VEGF-C and VEGFR3 IR to tumor surrounding vessels. In addition, intense VEGF-C immunostaining was observed on perivascular and mononuclear cells in the tumor surrounding stroma. Subtyping of VEGFR3+ microvascular tumor vessels revealed partially double immunolabeling with CD34 and flk-1, indicating a common origin of blood and lymphatic vessels. The expression of VEGF-C on tumor cells could be correlated with recurrences, and larger primary tumors had more VEGF-C-positive vessels. The broad expression of VEGF C and VEGFR3 in HNSCC suggests involvement in tumor lymph angiogenesis and vascular angiogenesis, promoting tumor growth and propagation of cancer cells. This implies that inhibitors of lymph angiogenesis could become effective therapeutic options similar to classical angiogenesis inhibitors.

Comparison of retroviral p15E-related factors and interferon alpha in head and neck cancer.

Head and neck squamous cell carcinomas (HNscc) produce low-molecular-mass factors (low-M(r) factors, M(r) < or = 25,000), which are antigenically related to the immunosuppressive retroviral transmembrane envelope protein p15E. These P15E-related tumour factors are thought to be responsible for some immunological impairments found in these patients (particularly the defective monocyte chemotaxis). A sequential and functional homology has been reported to exist between a bioactive fragment of interferon alpha (IFN alpha) and the putative immunosuppressive region of retroviral p15E (CKS-17). In this study we investigated (a) a possible functional and structural relationship between p15E and IFN alpha, and (b) the presence of and the relationship between p15E-related low-M(r) factors and IFN alpha in HNscc patients. We report the following results. (a) Recombinant human (rhu) IFN alpha was able to inhibit monocyte chemotaxis. (b) The anti-p15E antibodies crossreacted with rhuIFN alpha in a dot-blot technique; however, the anti-IFN alpha antibodies did not crossreact with disrupted murine leukaemia virus (p15E source). (c) Low-M(r) factors (n = 8-11) prepared from the sera of HNscc patients, which inhibit the monocyte chemotactic responsiveness, could be adsorbed by the anti-p15E antibodies as well as by the anti-IFN alpha antibodies. However, the abilities of the factors to adsorb to the two categories of antibodies (namely, anti-p15E and anti-IFN alpha) did not correlate. (d) Immunohistochemically we found IFN alpha-related epitopes, in almost all HNscc specimens studied (17/18), in locations distinctive from those of p15E-related factors. The anti-IFN alpha antibodies used in this study mainly reacted with basal epithelial cells close to the basal membrane, the prickle and granular cells of the squamous cell carcinomas. The anti-p15E antibodies mainly reacted with corneal layers, the granular and prickle cells, and did not react with basal epithelial cells. Our findings suggest that the immunosuppressive factors produced by HNscc cells are heterogeneous and p15E- and/or IFN alpha-related.