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Head and neck squamous cell carcinoma (HNSCC) has one of the most hypoxic and immunosuppressive tumor microenvironments (TME) among solid tumors. However, there is no proven therapeutic strategy to remodel the TME to be less hypoxic and proinflammatory. In this study, we classified tumors according to a Hypoxia-Immune signature, characterized the immune cells in each subgroup, and analyzed the signaling pathways to identify a potential therapeutic target that can remodel the TME. We confirmed that hypoxic tumors had significantly higher numbers of immunosuppressive cells, as evidenced by a lower ratio of CD8+ T cells to FOXP3+ regulatory T cells, compared with nonhypoxic tumors. Patients with hypoxic tumors had worse outcomes after treatment with pembrolizumab or nivolumab, anti-programmed cell death-1 inhibitors. Our expression analysis also indicated that hypoxic tumors predominantly increased the expression of the EGFR and TGFβ pathway genes. Cetuximab, an anti-EGFR inhibitor, decreased the expression of hypoxia signature genes, suggesting that it may alleviate the effects of hypoxia and remodel the TME to become more proinflammatory. Our study provides a rationale for treatment strategies combining EGFR-targeted agents and immunotherapy in the management of hypoxic HNSCC. Significance: While the hypoxic and immunosuppressive TME of HNSCC has been well described, comprehensive evaluation of the immune cell components and signaling pathways contributing to immunotherapy resistance has been poorly characterized. We further identified additional molecular determinants and potential therapeutic targets of the hypoxic TME to fully leverage currently available targeted therapies that can be administered with immunotherapy.

TP53 mutation is the most frequent genetic event in head and neck squamous cell carcinoma (HNSCC), found in more than 80% of patients with human papillomavirus–negative disease. As mutations in the TP53 gene are associated with worse outcomes in HNSCC, novel therapeutic approaches are needed for patients with TP53-mutated tumors. The National Cancer Institute sponsored a Clinical Trials Planning Meeting to address the issues of identifying and developing clinical trials for patients with TP53 mutations. Subcommittees, or breakout groups, were tasked with developing clinical studies in both the locally advanced and recurrent and/or metastatic (R/M) disease settings as well as considering signal-seeking trial designs. A fourth breakout group was focused on identifying and standardizing biomarker integration into trial design; this information was provided to the other breakout groups prior to the meeting to aid in study development. A total of 4 concepts were prioritized to move forward for further development and implementation. This article summarizes the proceedings of the Clinical Trials Planning Meeting with the goal of developing clinical trials for patients with TP53-mutant HNSCC that can be conducted within the National Clinical Trials Network.

Merkel cell carcinoma is among the most aggressive and lethal of primary skin cancers, with a high rate of distant metastasis. Anti-programmed death receptor 1 (anti-PD-1) and programmed death ligand 1 (PD-L1) monotherapy is currently standard of care for unresectable, recurrent, or metastatic Merkel cell carcinoma. We assessed treatment with combined nivolumab plus ipilimumab, with or without stereotactic body radiotherapy (SBRT) in patients with advanced Merkel cell carcinoma as a first-line therapy or following previous treatment with anti-PD-1 and PD-L1 monotherapy.In this randomised, open label, phase 2 trial, we randomly assigned adults from two cancer sites in the USA (one in Florida and one in Ohio) to group A (combined nivolumab and ipilimumab) or group B (combined nivolumab and ipilimumab plus SBRT) in a 1:1 ratio. Eligible patients were aged at least 18 years with histologically proven advanced stage (unresectable, recurrent, or stage IV) Merkel cell carcinoma, a minimum of two tumour lesions measureable by CT, MRI or clinical exam, and tumour tissue available for exploratory biomarker analysis. Patients were stratified by previous immune-checkpoint inhibitor (ICI) status to receive nivolumab 240 mg intravenously every 2 weeks plus ipilimumab 1 mg/kg intravenously every 6 weeks (group A) or the same schedule of combined nivolumab and ipilimumab with the addition of SBRT to at least one tumour site (24 Gy in three fractions at week 2; group B). Patients had to have at least two measurable sites of disease so one non-irradiated site could be followed for response. The primary endpoint was objective response rate (ORR) in all randomly assigned patients who received at least one dose of combined nivolumab and ipilimumab. ORR was defined as the proportion of patients with a complete response or partial response per immune-related Response Evaluation Criteria in Solid Tumours. Response was assessed every 12 weeks. Safety was assessed in all patients. This trial is registered with ClinicalTrials.gov, NCT03071406.50 patients (25 in both group A and group B) were enrolled between March 14, 2017, and Dec 21, 2021, including 24 ICI-naive patients (13 [52%] of 25 group A patients and 11 [44%] of 25 group B patients]) and 26 patients with previous ICI (12 [48%] of 25 group A patients and 14 [56%] of 25 group B patients]). One patient in group B did not receive SBRT due to concerns about excess toxicity. Median follow-up was 14·6 months (IQR 9·1-26·5). Two patients in group B were excluded from the analysis of the primary endpoint because the target lesions were irradiated and so the patients were deemed non-evaluable. Of the ICI-naive patients, 22 (100%) of 22 (95% CI 82-100) had an objective response, including nine (41% [95% CI 21-63]) with complete response. Of the patients who had previously had ICI exposure, eight (31%) of 26 patients (95% CI 15-52) had an objective response and four (15% [5-36]) had a complete response. No significant differences in ORR were observed between groups A (18 [72%] of 25 patients) and B (12 [52%] of 23 patients; p=0·26). Grade 3 or 4 treatment-related adverse events were observed in 10 (40%) of 25 patients in group A and 8 (32%) of 25 patients in group B.First-line combined nivolumab and ipilimumab in patients with advanced Merkel cell carcinoma showed a high ORR with durable responses and an expected safety profile. Combined nivolumab and ipilimumab also showed clinical benefit in patients with previous anti-PD-1 and PD-L1 treatment. Addition of SBRT did not improve efficacy of combined nivolumab and ipilimumab. The combination of nivolumab and ipilimumab represents a new first-line and salvage therapeutic option for advanced Merkel cell carcinoma.Bristol Myers Squibb Rare Population Malignancy Program.

GSEA of hallmark pathways in TCGA and Moffitt cohorts. TCGA (A–C) and Moffitt head and neck squamous cell carcinoma (D–F) cohorts. The gene expressions were compared for pathways enriched in: (A, D) Immune versus Hypoxia. Red: enriched in Immune, Blue: enriched in Hypoxia. B and E, Immune versus Mixture. Red: enriched in Immune, Blue: enriched in Mixture. C and F, Hypoxia versus Mixture. Red: enriched in Hypoxia, Blue: enriched in Mixture. Indicated are the top 10 most differentially expressed pathways in Cancer Hallmarks gene sets. NES: Normalized enrichment score.

Supplementary Table S1. TCGA Cohort

Patient characteristics in Moffitt cohort

(A, B) Representative mIHC images showing staining of nuclei (blue), tumor cell (orange - PCK) and selected immune checkpoint markers in each molecular subgroup. (A) Images show four lymphoid markers - CD3+ T-cells (Green), CD8+ cytotoxic T-cells (Yellow), CD69+ activated T-cells (Red) and CD103+ resident memory T-cells (Cyan). (B) Images show three myeloid markers – HLA-DR+ APCs (Green), MHCII+ APCs (Cyan) and CD163+ M2 macrophages (Yellow). (C) Linear model showing correlation between PD-L1 CPS and Immune, Mixture and Hypoxia subgroups. (D-E) Boxplots showing cell counts distribution of different immune markers among the primary (n = 63) and recurrent (n = 20) tumors in TMA mIHC cohort. Students t-tests were used to test for significant differences between the groups.

Hypoxia subgroup has immunosuppressive TME. Boxplots showing cell counts distribution of different immune markers CAIX+ cells representing hypoxia marker (A), CD3+ T cells (B), CD8+ cytotoxic T cells (C), PCK+ tumor cells expressing PD-L1 (D), HLA-DR+ APCs (E), and CD8+ T cells/FOXP3+ Tregs (F), among the three subgroups in the TMA mIHC cohort (n = 90). Statistical analysis was performed by ANOVA test and followed by Tukey post hoc tests. G, Representative mIHC images showing that the average numbers of immune cells expressing CD3+ T cells (green color), CD8+ cytotoxic T cells (yellow color), CD69+ activated T cells (red color), CD103+ resident memory T cells (cyan color) within 20 μm of a PCK+ cell (orange color) was statistically significantly lowest in the Hypoxia subgroup compared with the Mixture and Immune subgroups. Nuclei are shown in blue. Individual TMA core was considered as a region of interest (ROI). Bottom panel represents the zoom-in distance proximity and spatial distribution between a tumor cell and an immune cell within the TME.

Hypoxia subgroup has an enrichment of EGFR pathway. Pearson correlation coefficient for the TIMEx scores for the EGFR pathway and the Hypoxia gene signature among the TCGA (A), CPTAC (B), and Moffitt (C) HNSCC datasets. Expression distribution of EGFR RNA (D), protein (E), and phosphoprotein (F) levels among the three subgroups in the CPTAC HNSCC cohort.

Cetuximab treatment benefits Hypoxia subgroup. A, Heatmap of gene expression data from pre- and post-cetuximab (CTX) treated patient samples. RNA expression data (GSE109756) was z-normalized, each row represents a single gene in the hypoxia-immune signature gene list, each column represents a patient sample. Hierarchical clustering was performed as detailed in Materials and Methods. Samples were reordered according to its molecular subgroup: Immune (blue), mixture (black), and Hypoxia (red) with pre-cetuximab treated samples labeled BC and post-cetuximab labeled OC. TIMEx scores for the Hypoxia gene signature among the 15 patients classified into the three subgroups (B) and GSEA comparing gene expression changes in the pre- and post-cetuximab data using CIBERSORT gene sets: higher in post-cetuximab (red) and higher in pre-ceuximab (blue) (C).

Disease control given pembrolizumab or nivolumab monotherapy in Moffitt retrospective study cohort

Molecular classification and validation of hypoxia-immune signature in HNSCC. A, Heatmap of molecular subgroups in the Moffitt (n = 228) HNSCC cohort. RNA expression data were z-normalized, each row represents a single gene in the hypoxia-immune signature gene list, each column represents a patient sample. Samples were reordered according to its molecular subgroup: Immune (blue), Mixture (black), and Hypoxia (red). B, Kaplan–Meier survival plots for overall survival stratified according to the Hypoxia-Immune signature for the Moffitt (n = 184) HNSCC patient cohort.

Head and neck squamous cell carcinoma (HNSCC) has one of the most hypoxic and immunosuppressive tumor microenvironments (TME) among solid tumors. However, there is no proven therapeutic strategy to remodel the TME to be less hypoxic and proinflammatory. In this study, we classified tumors according to a Hypoxia-Immune signature, characterized the immune cells in each subgroup, and analyzed the signaling pathways to identify a potential therapeutic target that can remodel the TME. We confirmed that hypoxic tumors had significantly higher numbers of immunosuppressive cells, as evidenced by a lower ratio of CD8+ T cells to FOXP3+ regulatory T cells, compared with nonhypoxic tumors. Patients with hypoxic tumors had worse outcomes after treatment with pembrolizumab or nivolumab, anti-programmed cell death-1 inhibitors. Our expression analysis also indicated that hypoxic tumors predominantly increased the expression of the EGFR and TGFβ pathway genes. Cetuximab, an anti-EGFR inhibitor, decreased the expression of hypoxia signature genes, suggesting that it may alleviate the effects of hypoxia and remodel the TME to become more proinflammatory. Our study provides a rationale for treatment strategies combining EGFR-targeted agents and immunotherapy in the management of hypoxic HNSCC.Significance:While the hypoxic and immunosuppressive TME of HNSCC has been well described, comprehensive evaluation of the immune cell components and signaling pathways contributing to immunotherapy resistance has been poorly characterized. We further identified additional molecular determinants and potential therapeutic targets of the hypoxic TME to fully leverage currently available targeted therapies that can be administered with immunotherapy.

(A, B) Representative mIHC images showing staining of nuclei (blue), tumor cell (orange - PCK) and selected immune checkpoint markers in each molecular subgroup. (A) Images show four lymphoid markers - CD3+ T-cells (Green), CD8+ cytotoxic T-cells (Yellow), CD69+ activated T-cells (Red) and CD103+ resident memory T-cells (Cyan). (B) Images show three myeloid markers – HLA-DR+ APCs (Green), MHCII+ APCs (Cyan) and CD163+ M2 macrophages (Yellow). (C) Linear model showing correlation between PD-L1 CPS and Immune, Mixture and Hypoxia subgroups. (D-E) Boxplots showing cell counts distribution of different immune markers among the primary (n = 63) and recurrent (n = 20) tumors in TMA mIHC cohort. Students t-tests were used to test for significant differences between the groups.

Supplementary Table S2. CPTAC Cohort

Thyroid cancer is the most common endocrine malignancy with almost one million people living with thyroid cancer in the United States. Although early-stage well-differentiated thyroid cancers account for the majority of thyroid cancers on diagnosis and have excellent survival rates, the incidence of advanced-stage disease has increased over the past few years and confers poorer prognosis. Until recently, patients with advanced thyroid cancer had limited therapeutic options. However, the landscape of thyroid cancer treatment has dramatically changed in the past decade with the current availability of several novel effective therapeutic options, leading to significant advances and improved patient outcomes in the management of advanced disease. In this review, we summarize the current status of advanced thyroid cancer treatment options and discuss recent advances made in targeted therapies that have proven promising to clinically benefit patients with advanced thyroid cancer.

PDF file - 1004K, S1. The Twist box domain is required for full Twist1 transcriptional activity. S2. Quantification of immunofluorescence from isogenic prostate cancer cell lines stably expressing Twist1 and Twist1-F191G. S3. The Twist box domain is required for full Twist1-induced EMT marker phenotypes of prostate cancer cells. S4. Twist1 overexpression induces temporal changes in the material properties of prostate cancer cells during their migration in a wound healing assay. S5. The overexpression of Twist1 or Twist1-F191G does not increase cellular proliferation of prostate cancer cells in vitro. S6. The Twist box domain is required for full Twist1-induced cellular migration in PC3 cells. S7. Twist1 overexpression increases cell traction forces of individual androgenindependent PC3 prostate cancer cells. S8. Twist1 overexpression confers radioresistance to prostate cancer cells which is attenuated by mutation of the Twist box domain. S9. The Twist box domain is required for Twist1-induced soft agar anchorageindependent growth of 22Rv1 prostate cancer cells. S10. Twist1 overexpression does not confer prostate cancer cells increased primary tumorigenicity and slows primary tumor cell growth in vivo. S11. The Twist box domain is required for full Twist1-induced expression of Hoxa9/HOXA9.

EGF receptor (EGFR)–targeted monoclonal antibodies (mAb), such as cetuximab, execute their antitumor effect in vivo via blockade of receptor–ligand interactions and engagement of Fcγ receptors on immune effector cells that trigger antibody-dependent cell-mediated cytotoxicity (ADCC). We show that tumors counteract the in vivo antitumor activity of anti-EGFR mAbs by increasing tumor cell-autonomous expression of TGF-β. We show that TGF-β suppresses the expression of key molecular effectors of immune cell–mediated cytotoxicity, including Apo2L/TRAIL, CD95L/FasL, granzyme B, and IFN-γ. In addition to exerting an extrinsic inhibition of the cytotoxic function of immune effectors, TGF-β–mediated activation of AKT provides an intrinsic EGFR-independent survival signal that protects tumor cells from immune cell–mediated apoptosis. Treatment of mice-bearing xenografts of human head and neck squamous cell carcinoma with cetuximab resulted in emergence of resistant tumor cells that expressed relatively higher levels of TGF-β compared with untreated tumor-bearing mice. Although treatment with cetuximab alone forced the natural selection of TGF-β–overexpressing tumor cells in nonregressing tumors, combinatorial treatment with cetuximab and a TGF-β–blocking antibody prevented the emergence of such resistant tumor cells and induced complete tumor regression. Therefore, elevated levels of TGF-β in the tumor microenvironment enable tumor cells to evade ADCC and resist the antitumor activity of cetuximab in vivo. Our results show that TGF-β is a key molecular determinant of the de novo and acquired resistance of cancers to EGFR-targeted mAbs, and provide a rationale for combinatorial targeting of TGF-β to improve anti-EGFR–specific antibody therapy of EGFR-expressing cancers. Mol Cancer Ther; 11(11); 2429–39. ©2012 AACR.

PDF file - 256K, Clinical characteristics of patients with HNSCC

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Trop2, an oncogenic cell surface protein under investigation as a therapeutic target, is commonly overexpressed in several epithelial tumor types yet its function in tumor biology remains relatively unexplored. To investigate the role of Trop2 in epithelial carcinogenesis, we generated Trop2−/− mice, which are viable and possess a normal lifespan. Contrary to expectations, Trop2 loss fails to suppress keratinocyte transformation. Instead, ras-transformed Trop2−/− keratinocytes preferentially pass through an epithelial to mesenchymal transition (EMT) and form tumors with spindle cell histology. Furthermore, Trop2 loss renders Arf-null mice susceptible to the formation of biphasic sarcomatoid carcinomas containing both squamous and spindle cell components upon carcinogen exposure in an otherwise skin cancer–resistant strain (C57BL/6). Immortalized keratinocytes derived from Trop2−/−Arf−/− mice exhibit enhanced proliferative and migratory capacity as well as increased activation of mitogen-activated protein kinase and Src prior to transformation. The clinical relevance of these findings was supported by studying the molecular epidemiology of Trop2 in primary head and neck squamous cell carcinomas. This analysis revealed that Trop2 mRNA levels are decreased in a subset of tumors with features of EMT, and total loss of Trop2 protein expression is observed in the spindle cell component of sarcomatoid carcinomas. Therefore, while previous studies have emphasized the potential importance of Trop2 gain of function, these results uncover a role for Trop2 loss in tumorigenesis and the mesenchymal transdifferentiation observed in a subset of squamous cell carcinomas. Mol Cancer Res; 9(12); 1686–95. ©2011 AACR.

The Notch pathway is frequently altered in head and neck squamous cell carcinomas (HNSCC); however, the clinical significance of NOTCH1 dysregulation is poorly understood. This study was designed to characterize expression of the transcriptionally active NOTCH1 intracellular domain (NICD1) in HNSCCs and evaluate its association with NOTCH1 mutation status and clinical parameters. IHC for NICD1 was performed on 79 previously sequenced archival HNSCCs with known NOTCH1 mutation status. Three distinct immunohistochemical staining patterns were identified: positive/peripheral (47%), positive/nonperipheral (34%), and negative (19%). NICD1 expression was associated with NOTCH1 mutation status (P < 0.001). Most NOTCH1–wild-type tumors were peripheral (55%), whereas mutated NOTCH1 tumors were most commonly negative (47%). Nonperipheral tumors were more likely than peripheral tumors to have extracapsular spread [adjusted odds ratio (aOR), 16.01; 95% confidence interval (CI), 1.92–133.46; P = 0.010] and poor differentiation (aOR, 5.27; 95% CI, 0.90–30.86; P = 0.066). Negative staining tumors tended to be poorly differentiated (aOR, 24.71; 95% CI, 1.53–399.33; P = 0.024) and were less likely to be human papillomavirus (HPV) positive (aOR, 0.043; 95% CI, 0.001–1.59; P = 0.087). NOTCH1 mutagenesis was significantly associated with HPV status, with NOTCH1–wild-type tumors more likely to be HPV positive than NOTCH1-mutated tumors (aOR, 19.06; 95% CI, 1.31–276.15; P = 0.031). TP53 disruptive mutations were not associated with NICD1 expression or NOTCH1 mutation. In conclusion, NICD1 is expressed in three distinct patterns in HNSCC that are significantly associated with high-risk features. These findings further support a dual role for NOTCH1 as both tumor suppressor and oncogene in HNSCC. Further research is necessary to clarify the role of NOTCH1 in HNSCC and understand the clinical and therapeutic implications therein. Cancer Prev Res; 8(4); 287–95. ©2015 AACR.

PDF file - 116K, Supplemental Table S2 - Myc-CaP + Twist1-F191G versus Myc-CaP + Vector Differentially.

PDF file - 384K, Supplemental Table S1 - Myc-CaP + Twist1 versus Myc-CaP + Vector Differentially.

PDF file - 70K, A, Serum levels of the indicated cytokines in patients with HNSCC and pleomorphic adenoma (non-cancer control). B, Comparison of serum TGF-beta1 levels in non-cancer controls and patients with HNSCC at the time of new diagnosis and at the time of recurrence. C, Comparison of serum levels of TGF-beta1 in patients with HPV-negative HNSCC at the time of diagnosis, and at two time points after the completion of treatment

Supplementary Table 1. Clinicopathological characteristics of tumors with NICD1 IHC available compared with unavailable.

Anti-programmed cell death protein 1 (PD-1) therapy is a standard of care in recurrent metastatic head and neck squamous cell carcinoma (RMHNSCC). Vascular endothelial growth factor inhibitors, including tyrosine kinase inhibitors, have immunomodulatory properties and have offered promising results when combined with anti-PD-1 agents. We conducted a phase 2, multicenter, single-arm trial of pembrolizumab and cabozantinib in patients with RMHNSCC who had Response Evaluation Criteria in Solid Tumors v.1.1 measurable disease and no contraindications to either agent. We assessed the primary end points of tolerability and overall response rate to the combination with secondary end points of progression-free survival and overall survival and performed correlative studies with PDL-1 and combined positive score, CD8(+) T cell infiltration and tumor mutational burden. A total of 50 patients were screened and 36 were enrolled with 33 evaluable for response. The primary end point was met, with 17 out of 33 patients having a partial response (52%) and 13 (39%) stable disease with an overall clinical benefit rate of 91%. Median and 1-year overall survival were 22.3 months (95% confidence interval (CI) = 11.7–32.9) and 68.4% (95 % CI = 45.1%–83.5%), respectively. Median and 1-year progression-free survival were 14.6 months (95% CI = 8.2–19.6) and 54% (95% CI = 31.5%–72%), respectively. Grade 3 or higher treatment-related adverse events included increased aspartate aminotransferase (n = 2, 5.6%). In 16 patients (44.4%), the dose of cabozantinib was reduced to 20 mg daily. The overall response rate correlated positively with baseline CD8(+) T cell infiltration. There was no observed correlation between tumor mutational burden and clinical outcome. Pembrolizumab and cabozantinib were well tolerated and showed promising clinical activity in patients with RMHNSCC. Further investigation of similar combinations are needed in RMHNSCC. The trial is registered at ClinicalTrials.gov under registration no. NCT03468218.

Demographic and expression data

Purpose:A phase II multi-institutional clinical trial was conducted to determine overall survival (OS) in patients with recurrent and/or metastatic (R/M) head and neck squamous cell carcinoma (HNSCC) treated with a combination of cetuximab and nivolumab.Patients and Methods:Patients with R/M HNSCC were treated with cetuximab 500 mg/m2 i.v. on day 14 as a lead-in followed by cetuximab 500 mg/m2 i.v. and nivolumab 240 mg i.v. on day 1 and day 15 of each 28-day cycle. Expression of p16 and programmed cell death-ligand 1 (PD-L1) in archived tumors were determined. Tumor-tissue–modified human papillomavirus (TTMV) DNA was quantified in plasma.Results:Ninety-five patients were enrolled, and 88 patients were evaluable for OS with a median follow-up of 15.9 months. Median OS in the 45 patients who had prior therapy for R/M HNSCC (cohort A) was 11.4 months, with a 1 year OS 50% [90% confidence interval (CI), 0.43–0.57]. Median OS in the 43 patients who had no prior therapy (cohort B) was 20.2 months, with a 1-year OS 66% (90% CI, 0.59–0.71). In the combined cohorts, the p16-negative immunostaining was associated with higher response rate (RR; P = 0.02) but did not impact survival while higher PD-L1 combined positive score was associated with higher RR (P = 0.03) and longer OS (log-rank P = 0.04). In the p16-positive patients, lower median (1,230 copies/mL) TTMV DNA counts were associated with higher RR (P = 0.01) and longer OS compared with higher median (log-rank P = 0.05).Conclusions:The combination of cetuximab and nivolumab is effective in patients with both previously treated and untreated R/M HNSCC and warrants further evaluation.

Supplemental figure and table legends