Search

Purpose: We previously demonstrated an association between decreased SMAD4 expression and cetuximab resistance in head and neck squamous cell carcinoma (HNSCC). The purpose of this study was to further elucidate the clinical relevance of SMAD4 loss in HNSCC.Experimental Design: SMAD4 expression was assessed by IHC in 130 newly diagnosed and 43 patients with recurrent HNSCC. Correlative statistical analysis with clinicopathologic data was also performed. OncoFinder, a bioinformatics tool, was used to analyze molecular signaling in TCGA tumors with low or high SMAD4 mRNA levels. The role of SMAD4 was investigated by shRNA knockdown and gene reconstitution of HPV-negative HNSCC cell lines in vitro and in vivo.Results: Our analysis revealed that SMAD4 loss was associated with an aggressive, HPV-negative, cetuximab-resistant phenotype. We found a signature of prosurvival and antiapoptotic pathways that were commonly dysregulated in SMAD4-low cases derived from TCGA-HNSCC dataset and an independent oral cavity squamous cell carcinoma (OSCC) cohort obtained from GEO. We show that SMAD4 depletion in an HNSCC cell line induces cetuximab resistance and results in worse survival in an orthotopic mouse model in vivo. We implicate JNK and MAPK activation as mediators of cetuximab resistance and provide the foundation for the concomitant EGFR and JNK/MAPK inhibition as a potential strategy for overcoming cetuximab resistance in HNSCCs with SMAD4 loss.Conclusions: Our study demonstrates that loss of SMAD4 expression is a signature characterizing the cetuximab-resistant phenotype and suggests that SMAD4 expression may be a determinant of sensitivity/resistance to EGFR/MAPK or EGFR/JNK inhibition in HPV-negative HNSCC tumors. Clin Cancer Res; 23(17); 5162–75. ©2017 AACR.

Supplementary Figure from Phase II Multi-institutional Clinical Trial Result of Concurrent Cetuximab and Nivolumab in Recurrent and/or Metastatic Head and Neck Squamous Cell Carcinoma

Supplementary Figure 1 from Gene Expression Differences Associated with Human Papillomavirus Status in Head and Neck Squamous Cell Carcinoma

Purpose: Activating PIK3CA genomic alterations are frequent in head and neck squamous cell carcinoma (HNSCC), and there is an association between phosphoinositide 3-kinase (PI3K) signaling and radioresistance. Hence, we investigated the therapeutic efficacy of inhibiting PI3K with GDC-0032, a PI3K inhibitor with potent activity against p110α, in combination with radiation in HNSCC.Experimental Design: The efficacy of GDC-0032 was assessed in vitro in 26 HNSCC cell lines with crystal violet proliferation assays, and changes in PI3K signaling were measured by Western blot analysis. Cytotoxicity and radiosensitization were assessed with Annexin V staining via flow cytometry and clonogenic survival assays, respectively. DNA damage repair was assessed with immunofluorescence for γH2AX foci, and cell cycle analysis was performed with flow cytometry. In vivo efficacy of GDC-0032 and radiation was assessed in xenografts implanted into nude mice.Results: GDC-0032 inhibited potently PI3K signaling and displayed greater antiproliferative activity in HNSCC cell lines with PIK3CA mutations or amplification, whereas cell lines with PTEN alterations were relatively resistant to its effects. Pretreatment with GDC-0032 radiosensitized PIK3CA-mutant HNSCC cells, enhanced radiation-induced apoptosis, impaired DNA damage repair, and prolonged G2–M arrest following irradiation. Furthermore, combined GDC-0032 and radiation was more effective than either treatment alone in vivo in subcutaneous xenograft models.Conclusions: GDC-0032 has increased potency in HNSCC cell lines harboring PIK3CA-activating aberrations. Further, combined GDC-0032 and radiotherapy was more efficacious than either treatment alone in PIK3CA-altered HNSCC in vitro and in vivo. This strategy warrants further clinical investigation. Clin Cancer Res; 22(8); 2009–19. ©2015 AACR.

Human papillomavirus (HPV) is associated with a subset of head and neck squamous cell carcinoma (HNSCC). Between 15% and 35% of HNSCCs harbor HPV DNA. Demographic and exposure differences between HPV-positive (HPV+) and negative (HPV−) HNSCCs suggest that HPV+ tumors may constitute a subclass with different biology, whereas clinical differences have also been observed. Gene expression profiles of HPV+ and HPV− tumors were compared with further exploration of the biological effect of HPV in HNSCC. Thirty-six HNSCC tumors were analyzed using Affymetrix Human 133U Plus 2.0 GeneChip and for HPV by PCR and real-time PCR. Eight of 36 (22%) tumors were positive for HPV subtype 16. Statistical analysis using Significance Analysis of Microarrays based on HPV status as a supervising variable resulted in a list of 91 genes that were differentially expressed with statistical significance. Results for a subset of these genes were verified by real-time PCR. Genes highly expressed in HPV+ samples included cell cycle regulators (p16INK4A, p18, and CDC7) and transcription factors (TAF7L, RFC4, RPA2, and TFDP2). The microarray data were also investigated by mapping genes by chromosomal location (DIGMAP). A large number of genes on chromosome 3q24-qter had high levels of expression in HPV+ tumors. Further investigation of differentially expressed genes may reveal the unique pathways in HPV+ tumors that may explain the different natural history and biological properties of these tumors. These properties may be exploited as a target of novel therapeutic agents in HNSCC treatment.

Supplementary Figure Legends

Representative SMAD4 IHC, FaDu negative control, SCC25/SCC61 positive control.

Matrigel colony formation assay of FaDu-mock and FaDu-SMAD4 cells treated with PBS or Cetuximab in combination with JNKi (SP600125) or MEKi (U0126) at indicated concentrations for 7 days. (*p

Purpose:Patients with head and neck squamous cell carcinoma (HNSCC) who actively smoke during treatment have worse survival compared with never-smokers and former-smokers. We hypothesize the poor prognosis in tobacco smokers with HNSCC is, at least in part, due to ongoing suppression of immune response. We characterized the tumor immune microenvironment (TIM) of HNSCC in a retrospective cohort of 177 current, former, and never smokers.Experimental Design:Tumor specimens were subjected to analysis of CD3, CD8, FOXP3, PD-1, PD-L1, and pancytokeratin by multiplex immunofluorescence, whole-exome sequencing, and RNA sequencing. Immune markers were measured in tumor core, tumor margin, and stroma.Results:Our data indicate that current smokers have significantly lower numbers of CD8+ cytotoxic T cells and PD-L1+ cells in the TIM compared with never- and former-smokers. While tumor mutation burden and mutant allele tumor heterogeneity score do not associate with smoking status, gene-set enrichment analyses reveal significant suppression of IFNα and IFNγ response pathways in current smokers. Gene expression of canonical IFN response chemokines, CXCL9, CXCL10, and CXCL11, are lower in current smokers than in former smokers, suggesting a mechanism for the decreased immune cell migration to tumor sites.Conclusions:These results suggest active tobacco use in HNSCC has an immunosuppressive effect through inhibition of tumor infiltration of cytotoxic T cells, likely as a result of suppression of IFN response pathways. Our study highlights the importance of understanding the interaction between smoking and TIM in light of emerging immune modulators for cancer management.

Supplementary Table from Phase II Multi-institutional Clinical Trial Result of Concurrent Cetuximab and Nivolumab in Recurrent and/or Metastatic Head and Neck Squamous Cell Carcinoma

Background: We hypothesized that a serum proteomic profile predictive of survival benefit in non–small cell lung cancer patients treated with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI) reflects tumor EGFR dependency regardless of site of origin or class of therapeutic agent.Methods: Pretreatment serum or plasma from 230 patients treated with cetuximab, EGFR-TKIs, or chemotherapy for recurrent/metastatic head and neck squamous cell carcinoma (HNSCC) or colorectal cancer (CRC) were analyzed by mass spectrometry. Each sample was classified into “good” or “poor” groups using VeriStrat, and survival analyses of each cohort were done based on this classification. For the CRC cohort, this classification was correlated with the tumor EGFR ligand levels and KRAS mutation status.Results: In the EGFR inhibitor–treated cohorts, the classification predicted survival (HNSCC: gefitinib, P = 0.007 and erlotinib/bevacizumab, P = 0.02; CRC: cetuximab, P = 0.0065) whereas the chemotherapy cohort showed no survival difference. For CRC patients, tumor EGFR ligand RNA levels were significantly associated with the proteomic classification, and combined KRAS and proteomic classification provided improved survival classification.Conclusions: Serum proteomic profiling can detect clinically significant tumor dependence on the EGFR pathway in non–small cell lung cancer, HNSCC, and CRC patients treated with either EGFR-TKIs or cetuximab. This classification is correlated with tumor EGFR ligand levels and provides a clinically practical way to identify patients with diverse cancer types most likely to benefit from EGFR inhibitors. Prospective studies are necessary to confirm these findings. Cancer Epidemiol Biomarkers Prev; 19(2); 358–65

(A) Western blot confirming the knockdown of SMAD4 levels by stable expression of short-hairpin RNA in SCC25 cell line. (SC: scrambled control shRNA, SMAD4KD: shRNA against SMAD4). (B) Matrigel colony formation assay was performed for SCC25 cell line (controls and SMAD4 knock-downs) treated with PBS, 100nM or 1000nM cetuximab. (C) Western blot showing MAPK and JNK activity in SMAD4-depleted or control SCC25 cells.

CCR Translation for This Article from Phase II Efficacy and Pharmacogenomic Study of Selumetinib (AZD6244; ARRY-142886) in Iodine-131 Refractory Papillary Thyroid Carcinoma with or without Follicular Elements

Western blot analysis of SCC61L-SC and SMAD4KD cells for phospho-SMAD2 and total SMAD2/3 expression levels. Actin was used as a loading control.

Supplementary Figure S1. GDC-0032 downregulated PI3K signaling in a dose dependent fashion. Supplementary Figure S2. BYL719 enhances radiation induced cell death in PIK3CA activated cell lines. Supplementary Figure S3. A66 enhances radiation induced cell death in PIK3CA activated cell lines Supplementary Figure S4. PI3K pathway inhibition enhances cell death in HSC-2 (PIK3CA mutated) following irradiation. Cells were treated with the given drugs for 24 hours, irradiated with 4 Gy, then assessed for Annexin V staining 72 hours after irradiation. Supplementary Figure S5. GDC-0032 radiosensitizes LB-771 (PIK3CA amplified) cells in vitro as assessed via the clonogenic survival assay. Supplementary Figure S6. GDC-0032 enhances DNA damage and apoptosis following irradiation. Supplementary Figure S7. GDC-0032 does not significantly alter the early G2/M checkpoint. Supplementary Figure S8. ATR inhibition partially blocks the early G2/M checkpoint. Supplementary Figure S9. Wee1 inhibition partially blocks the early G2/M checkpoint. Supplementary Figure S10. Wee1 inhibition doesn't reverse the enhanced G2/M checkpoint induced by GDC-0032 following irradiation. 3 Supplementary Figure S11. GDC-0032 and radiation is superior to either treatment alone in an HPV(+) oropharyngeal squamous cell carcinoma patient derived xenograft.

Purpose: A multicenter, open-label, phase II trial was conducted to evaluate the efficacy, safety, and tolerability of selumetinib in iodine-refractory papillary thyroid cancer (IRPTC).Experimental Design: Patients with advanced IRPTC with or without follicular elements and documented disease progression within the preceding 12 months were eligible to receive selumetinib at a dose of 100 mg twice daily. The primary endpoint was objective response rate using Response Evaluation Criteria in Solid Tumors. Secondary endpoints were safety, overall survival, and progression-free survival (PFS). Tumor genotype including mutations in BRAF, NRAS, and HRAS was assessed.Results: Best responses in 32 evaluable patients out of 39 enrolled were 1 partial response (3%), 21 stable disease (54%), and 11 progressive disease (28%). Disease stability maintenance occurred for 16 weeks in 49%, 24 weeks in 36%. Median PFS was 32 weeks. BRAF V600E mutants (12 of 26 evaluated, 46%) had a longer median PFS compared with patients with BRAF wild-type (WT) tumors (33 versus 11 weeks, respectively, HR = 0.6, not significant, P = 0.3). The most common adverse events and grades 3 to 4 toxicities included rash, fatigue, diarrhea, and peripheral edema. Two pulmonary deaths occurred in the study and were judged unlikely to be related to the study drug.Conclusions: Selumetinib was well tolerated but the study was negative with regard to the primary outcome. Secondary analyses suggest that future studies of selumetinib and other mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK; MEK) inhibitors in IRPTC should consider BRAF V600E mutation status in the trial design based on differential trends in outcome. Clin Cancer Res; 18(7); 2056–65. ©2012 AACR.

Supplemental Table 1. Summary of patient characteristics with newly diagnosed HNSCC. Supplemental Table 2. Summary of patient characteristics with recurrent and/or metastatic HNSCC. Supplemental Table 3. Injection of FaDu-SMAD4 cells results in lower take rate in mice compared to the control cell line and shows no signs of metastatic lesion.

PDF file - 116K

NOTCH1 mutations have been reported to occur in 10% to 15% of head and neck squamous cell carcinomas (HNSCC). To determine the significance of these mutations, we embarked upon a comprehensive study of NOTCH signaling in a cohort of 44 HNSCC tumors and 25 normal mucosal samples through a set of expression, copy number, methylation, and mutation analyses. Copy number increases were identified in NOTCH pathway genes, including the NOTCH ligand JAG1. Gene set analysis defined a differential expression of the NOTCH signaling pathway in HNSCC relative to normal tissues. Analysis of individual pathway-related genes revealed overexpression of ligands JAG1 and JAG2 and receptor NOTCH3. In 32% of the HNSCC examined, activation of the downstream NOTCH effectors HES1/HEY1 was documented. Notably, exomic sequencing identified 5 novel inactivating NOTCH1 mutations in 4 of the 37 tumors analyzed, with none of these tumors exhibiting HES1/HEY1 overexpression. Our results revealed a bimodal pattern of NOTCH pathway alterations in HNSCC, with a smaller subset exhibiting inactivating NOTCH1 receptor mutations but a larger subset exhibiting other NOTCH1 pathway alterations, including increases in expression or gene copy number of the receptor or ligands as well as downstream pathway activation. Our results imply that therapies that target the NOTCH pathway may be more widely suitable for HNSCC treatment than appreciated currently. Cancer Res; 74(4); 1091–104. ©2013 AACR.

Supplementary Figure 1 from A Feed-Forward Loop Involving Protein Kinase Cα and MicroRNAs Regulates Tumor Cell Cycle

Supplementary Table 2 from Gene Expression Profiles Identify Epithelial-to-Mesenchymal Transition and Activation of Nuclear Factor-κB Signaling as Characteristics of a High-risk Head and Neck Squamous Cell Carcinoma

Supplementary Figure 3 from A Feed-Forward Loop Involving Protein Kinase Cα and MicroRNAs Regulates Tumor Cell Cycle

PDF file - 82K, Growth effects of NOTCH1 siRNA inhibition in HNSCC cells with wildtype NOTCH1 status. NOTCH1 was downregulated in UPCI-SCC090 (090, top, NOTCH1 wt) and SCC61 (bottom, NOTCH1 wt) by siRNA as described in methods. The effect of NOTCH1 downregulation on cell proliferation for these two cell lines is shown in the middle. The effect of siNOTCH1 on NOTCH1 expression (left) or expression of its targets (HES1 and HEY1, right) is also shown. The mRNA expression levels were calculated relative to GAPDH and were normalized to 1 in the control experiments with scrambled siRNA sequences. Experiments were performed in pentaplicates, mean plus-minus SEM were shown. *, p-value < 0.05.

Supplementary Figure 2 from A Feed-Forward Loop Involving Protein Kinase Cα and MicroRNAs Regulates Tumor Cell Cycle

PDF file - 73K, Significant overexpression of JAG1 (A) and PSEN1 (B) in the tumors with increased copy number gains in comparison to that in the normal tissues in the cohort of 44 HNSCC tumors and 25 normal mucosa. N, normal mucosa; T, HNSCC tumors; -, without JAG1(A) or PSEN1 (B) copy number gain; +, with JAG1 (A) or PSEN1 (B) copy number gain. Boxes represent the interquartile range (25th-75th percentile) and horizontal lines inside the boxes indicate median. Whiskers indicate the minimum and maximum values. P value was calculated by using t test.

Supplementary Table 1 from Gene Expression Profiles Identify Epithelial-to-Mesenchymal Transition and Activation of Nuclear Factor-κB Signaling as Characteristics of a High-risk Head and Neck Squamous Cell Carcinoma

PDF file - 169K, NOTCH pathway activation in HNSCC. A, Immunohistochemical analysis of NOTCH1, HES1 and HEY1 protein expression in HNSCC. Tissue microarray arrays with quadruplicates of samples from each of 56 HNSCC tumor and 11 non-tumor patients were stained with anti-NOTCH1, anti-HES1 or anti-HEY1 antibody, as described in methods. Top, the heat-map of immunohistochemical analysis for tissue microarray slides. Rows, genes; column, each individual sample. The average staining for four tissues was categorized to Strong (brown), Moderate (grey) or Weak/Negative (blue) staining. Samples are ranked by the pathology ID numbers in tumor (left) and normal (right) groups. Bottom: quantification of immunohistochemical data. The number and percentage of tumor (Tu) and normal (No) patient in strong, moderate and weak/negative category was quantified. P-values were calculated by comparing numeric scores in tumor vs normal sample by independent t test. The samples 57964, 58602, 22146, 5931, 26514 represent tumor samples X6, X7, X11, X28 and X34 from the original discovery cohort of 44 HNSCC tumors. B, Representative immunohistochemical results. The representative slides of positive or moderate staining are shown for tumors and compared to normal tissue. Bar, 100 um equivalent. Tumor (T) and normal (N) tissues on each sample are labeled. ID 22146 represented X11 samples, which were used both in array and in immunohistochemical analysis, where protein and RNA expression strongly correlate.

PDF file - 111K

PDF file - 54K, Effect of gamma-secretase inhibitor on the NOTCH1 pathway genes and cell growth in HNSCC cells with NOTCH1 wildtype status. UPCI-SCC090 cells were growing on the media supplemented with 0.2 uM gamma-secretase inhibitor XXI (GSI-XXI) for up to 72 hours. The effect of GSI-XXI on the NOTCH1 targets HES1 and HEY1 is shown on the left for UPSI-SCC090 cell line.

Gene expression signatures generated from DNA microarray analyses have shown promise as predictive biomarkers of clinical outcome. In this study, we determined a high-risk signature for disease recurrence using formalin-fixed head and neck squamous cell carcinoma (HNSCC) tumors and compared the results with an independent data set obtained from fresh frozen tumors. We also showed that genes involved in epithelial-to-mesenchymal transition (EMT) and nuclear factor-κB (NF-κB) signaling deregulation are the most prominent molecular characteristics of the high-risk tumors. Gene expression was determined in 40 samples, including 34 formalin-fixed tissues and 6 matched frozen tissues, from 29 HNSCC patients. A 75-gene list predictive of disease recurrence was determined by training on the formalin-fixed tumor data set and tested on data from the independent frozen tumor set from 60 HNSCC patients. The difference in recurrence-free survival (RFS) between the high-risk versus low-risk groups in the training and test sets was statistically significant (P = 0.002 and 0.03, respectively, log-rank test). In addition, the gene expression data was interrogated using Gene Set Enrichment Analysis to determine biological significance. The most significant sets of genes enriched in the high-risk tumors were genes involving EMT, NF-κB activation, and cell adhesion. In conclusion, global gene expression analysis is feasible using formalin-fixed tissue. The 75-gene list can be used as a prognostic biomarker of recurrence, and our data suggest that the molecular determinants of EMT and NF-κB activation can be targeted as the novel therapy in the identified high-risk patients. (Cancer Res 2006; 66(16): 8210-8)

Supplementary Figure 4 from A Feed-Forward Loop Involving Protein Kinase Cα and MicroRNAs Regulates Tumor Cell Cycle

PDF file - 68K, Growth effects of HEY1 siRNA inhibition in HNSCC cells with wildtype NOTCH1 status. HEY1 was downregulated in UPCI-SCC090 and SCC61 by siRNA as described in methods. The effect of siHEY1 on the expression of HEY1 is shown on the left. The effect of HEY1 downregulation on cell proliferation for these two cell lines is shown on the right. Note, there is over 90% transfection efficiency of HEY1 downregulation for each cell line, however the strong growth inhibition effect was shown only for NOTCH1 wildtype 090 cell line.

PDF file - 108K, Validation of JAG1, JAG2, NOTCH1 and PSEN1 overexpression in TCGA HNSCC cohort. A, Overexpression of JAG1, JAG2 and NOTCH1 in tumors in comparison to that in normal tissues in the TCGA HNSCC cohort. B, Overexpression of JAG1 and PSEN1 in the tumors with increased copy number gains in comparison to that in the normal tissues as well as to that in the tumors without copy number gains in the TCGA HNSCC cohort. N, normal mucosa; T, HNSCC tumors; -, without JAG1 or PSEN1 copy number gain; +, with JAG1 or PSEN1 copy number gain. Dots, relative expression levels in different tissue samples. Boxes represent the interquartile range (25th-75th percentile) and horizontal lines inside the boxes indicate median. Whiskers indicate the minimum and maximum values. P value was calculated by using t test.

PDF file - 88K, Supplementary Table I. Baseline characteristics of the HNSCC patients for microarray analysis. Supplementary Table II. Short Tandem Repeat Profile of the 2 HNSCC Cell Lines. Supplementary Table III. NOTCH Signaling Pathway Genes assigned in KEGG pathway database and shown on expression microarrays. Supplementary Table IV. Summary of the mRNA levels of NOTCH signaling pathway genes in the cohort of 44 HNSCC tumors and 25 normal mucosas measured by Affymetrix expression array.

Supplementary Figure Legends 1-4 from A Feed-Forward Loop Involving Protein Kinase Cα and MicroRNAs Regulates Tumor Cell Cycle

PURPOSE Primary or acquired resistance to cetuximab, an antiepidermal growth factor receptor monoclonal antibody (mAb), minimizes its utility in recurrent/metastatic head and neck squamous cell carcinoma (HNSCC). Aberrant hepatocyte growth factor/cMet pathway activation is an established resistance mechanism. Dual pathway targeting may overcome resistance. PATIENTS AND METHODS This multicenter, randomized, noncomparative phase II study evaluated ficlatuzumab, an antihepatocyte growth factor mAb, with or without cetuximab in recurrent/metastatic HNSCC. The primary end point was median progression-free survival (PFS); an arm met significance criteria if the lower bound of the 90% CI excluded the historical control of 2 months. Key eligibility criteria were HNSCC with known human papillomavirus (HPV) status, cetuximab resistance (progression within 6 months of exposure in the definitive or recurrent/metastatic setting), and resistance to platinum and anti–PD-1 mAb. Secondary end points included objective response rate (ORR), toxicity, and the association of HPV status and cMet overexpression with efficacy. Continuous Bayesian futility monitoring was used. RESULTS From 2018 to 2020, 60 patients were randomly assigned and 58 were treated. Twenty-seven versus 33 patients were allocated to monotherapy versus combination. Arms were balanced for major prognostic factors. The monotherapy arm closed early for futility. The combination arm met prespecified significance criteria with a median PFS of 3.7 months (lower bound 90% CI, 2.3 months; P = .04); the ORR was 6 of 32 (19%), including two complete and four partial responses. Exploratory analyses were limited to the combination arm: the median PFS was 2.3 versus 4.1 months ( P = .03) and the ORR was 0 of 16 (0%) versus 6 of 16 (38%; P = .02) in the HPV-positive versus HPV-negative subgroups, respectively. cMet overexpression was associated with reduced hazard of progression in HPV-negative but not HPV-positive disease ( P interaction = .02). CONCLUSION The ficlatuzumab-cetuximab arm met significance criteria for PFS and warrants phase III development. HPV-negative HNSCC merits consideration as a selection criterion.

As oropharyngeal cancer (OPC) associated with human papillomavirus (HPV) increases in men, the need for a screening test to diagnose OPC early is crucial. While HPV-associated OPC has a favorable prognosis, recurrence is likely, and metastatic OPC is often incurable regardless of HPV status. Our previous study of pretreatment, male OPC cases (n = 101) and age- and smoking-matched controls (n = 101) found methylation of the host EPB41L3 tumor suppressor gene and HPV16 in the oral gargle was correlated with these biomarkers in the tumor. Methylation of these genes in the oral gargle was significantly (p 0.0001) higher among cases compared to controls. To further study the utility of HPV16/EPB41L3 methylation, we expanded the sample size and specifically increased the number of early OPC cases (T1-T2, N0-N1; small tumors with a single ipsilateral node3 cm) to evaluate these biomarkers in early and late OPC. This study included 228 OPC cases, 92 of which were early cases and frequency matched to 142 healthy controls. In logistic regression, the AUC for HPV16/EPB41L3 methylation for all OPC cases was 0.82. Among early and late OPC cases, the AUC was 0.78 and 0.85, respectively. For early cases, 76% sensitivity was achieved, replicating results from our prior study, with a specificity of 65%, indicating room for improvement. The ability of HPV16/EPB41L3 methylation to distinguish OPC from healthy controls highlights its utility as a potential biomarker for OPC. However, the inability to predict early OPC better than late stage OPC indicates the need for additional biomarkers to improve screening performance.

Background Cigarette smoking is related to greater cancer incidence, worse cancer-related clinical outcomes, and worse patient quality of life. Few studies have evaluated the role of smoking in patients’ experiences of cancer-related symptom burden. This study examined relationships between smoking and total symptom burden as well as the incidence of severe symptoms among adult cancer patients. Patients and Methods Patients at Moffitt Cancer Center completed self-report surveys as part of routine cancer care. Symptom burden was evaluated as the sum of individual symptom ratings (total symptom burden) and the number of symptoms rated severe (incidence of severe symptoms). Zero-inflated negative binomial modeling was used to evaluate the relationships between smoking status (ever vs never smoker) and symptom burden outcomes controlling for relevant sociodemographic and clinical covariates and accounting for the proportion of participants reporting no symptom burden. Results This study included 12 571 cancer patients. More than half reported a history of cigarette smoking (n = 6771, 55%). Relative to never smokers, participants with a smoking history had 15% worse expected total symptom burden (ratio = 1.15, 95% confidence interval [CI] 1.11-1.20, P < .001) and 13% more expected severe symptoms (ratio = 1.13, 95% CI 1.05-1.21, P = .001) above and beyond the effects of relevant sociodemographic and clinical characteristics. Conclusion Results provide support that smoking is associated with worse cancer symptom burden. More research is needed to evaluate how smoking history (ie, current vs former smoker) and smoking cessation influence cancer symptom burden.

Nucleus segmentation represents the initial step for histopathological image analysis pipelines, and it remains a challenge in many quantitative analysis methods in terms of accuracy and speed. Recently, deep learning nucleus segmentation methods have demonstrated to outperform previous intensity- or pattern-based methods. However, the heavy computation of deep learning provides impression of lagging response in real time and hampered the adoptability of these models in routine research. We developed and implemented NuKit a deep learning platform, which accelerates nucleus segmentation and provides prompt results to the users. NuKit platform consists of two deep learning models coupled with an interactive graphical user interface (GUI) to provide fast and automatic nucleus segmentation “on the fly”. Both deep learning models provide complementary tasks in nucleus segmentation. The whole image segmentation model performs whole image nucleus whereas the click segmentation model supplements the nucleus segmentation with user-driven input to edits the segmented nuclei. We trained the NuKit whole image segmentation model on a large public training data set and tested its performance in seven independent public image data sets. The whole image segmentation model achieves average [Formula: see text] and [Formula: see text]. The outputs could be exported into different file formats, as well as provides seamless integration with other image analysis tools such as QuPath. NuKit can be executed on Windows, Mac, and Linux using personal computers.

The current paradigm of clinical trials treating patients until disease progression using maximum tolerated dose does not account for the dynamic tumor-host-drug interactions that result in acquired resistance. Here, we present the concept of an Evolutionary Tumor Board (ETB) and report interim results from a prospective, non-interventional pilot study in which novel therapeutic strategies based on evolutionary principles were developed under the ETB framework. The ETB approach relies on an interdisciplinary team that integrates clinical, preclinical, and theoretical knowledge and the application of mathematical modeling to predict patient responses to different therapies, including novel approaches derived from eco-evolutionary first principles. We have previously proposed several evolutionary therapies that aim to enhance the efficacy of an overall treatment regimen, using existing agents for a given disease. Key among these evolutionary therapies is the idea of “first-strike second-strike”, where different agents are administered in sequence, and new strikes are applied as soon as the efficacy of the previous strike is nearing a minimum, as opposed to waiting until progression is identified on periodic imaging. This approach requires careful analysis of longitudinal patient data coupled with predictive dynamics generated by mathematical models. Here we describe the ETB process and the interim results from 15 patients enrolled in the feasibility trial. In addition, we describe the challenges faced as well as the solutions that can be implemented via improved modeling approaches, better patient data collection, and a reassessment of how we understand tumor dynamics in the light of evolutionary principles.

Subglottic squamous cell carcinoma (SCC) represents less than 5% of all laryngeal cancers. Our objective was to better characterize survival using the National Cancer Database (NCDB) registry from 2004 to 2015. 403 patients met inclusion criteria. 63.8% presented with advanced-stage disease. Treatment regimens were as follows: 15.9% underwent surgery alone, 16.9% underwent surgery followed by adjuvant therapy, and 67.2% underwent primary chemo/radiation (C/RT). Five-year overall survival (OS) was 58.6% for Stage I and II patients, 49.1% for Stage III, and 36.3% for stage IV. Adjusted OS for all-stage patients was worse with C/RT compared to upfront surgery (40.6% vs. 58.4%; HR 1.83 [95%CI 1.29–2.61] p

Hematoxylin and eosin (H&E) staining is the most common type of histopathological images used for quantitative data analysis. For histopathological image analysis, nucleus segmentation represents one of the initial steps in quantitative data analysis pipelines. Recently, deep learning methods for nucleus segmentation on histopathological images become the mainstream in digital pathology. However, adopting these pre-trained models in digital pathology or clinical research remains limited. Two main factors limit the usage of deep learning methods in routine research: 1) many models required some technical background to execute the programs, and 2) the speed of returning the results to the users. To overcome these limitations, we have developed and implemented NuKit, a deep learning platform which accelerates nucleus segmentation and provides prompt results to the users. NuKit platform consists of two deep learning models coupled with an interactive graphical user interface (GUI) to provide fast and automatic nucleus segmentation “on the fly”. The two deep learning models are: 1) the whole image segmentation model and 2) the click segmentation model. Both deep learning models provide complementary tasks in nucleus segmentation in the NuKit platform. The whole image segmentation model performs whole image nucleus whereas the click segmentation model supplements the nucleus segmentation with user-driven input to edits the segmented nuclei. We used PanNuke as the training data set which contains 160,368 nuclei extracted from 7,901 image tiles. The sources of this data set were curated from The Cancer Genome Atlas (TCGA) and included a very few in-house data. For test sets, we used several popular data sets such as TCGA (not used in the training set). We used DICE coefficient as the metric for comparisons of NuKit with other deep learning methods. For the results, we observed that NuKit achieved comparable results with the state-of-the-art deep learning methods and outperforms those non-deep learning methods. For TNBC and CoNSeP data sets, NuKit achieved DICE 0.793 and 0.854 comparing to HoVer-Net 0.749 and 0.664. For CoNSeP test data set, NuKit achieved DICE 0.860 comparing to Cell Profiler 0.434 and QuPath 0.588. In addition, both pre-trained models were embedded in the NuKit GUI, which provides interactive and prompt response to users in analyzing their histopathological images. The outputs of NuKit are interoperable with other quantitative image analysis tools to facilitate the ecosystem of computational pathology to many formats to combine with other software to construct pipelines. In summary, we have introduced NuKit, an innovation platform which combines two deep learning models coupled with interactive GUI to accelerate nucleus segmentation task in histopathological images. We believe that NuKit provides a new platform to bridge the pre-trained deep learning models into digital pathology and clinical usage. Citation Format: Ching-Nung Lin, Christine H. Chung, Aik Choon Tan. NuKit: a deep learning platform for histopathological Images. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5409.

Prognosis for patients with recurrent and/or metastatic head and neck squamous cell carcinoma (HNSCC) remains poor. Development of more effective and less toxic targeted therapies is necessary for HNSCC patients. Checkpoint kinase 1 (CHK1) plays a vital role in cell cycle regulation and is a promising therapeutic target in HNSCC. Prexasertib, a CHK1 inhibitor, induces DNA damage and cell death, however, its effect on the tumor immune microenvironment (TIME) is largely unknown. Therefore, we evaluated a short-term and long-term effects of prexasertib in HNSCC and its TIME. Prexasertib caused increased DNA damage and cell death in vitro and significant tumor regression and improved survival in vivo. The gene expression and multiplex immunohistochemistry (mIHC) analyses of the in vivo tumors demonstrated increased expression of genes that are related to T-cell activation and increased immune cell trafficking, and decreased expression of genes that related to immunosuppression. However, increased expression of genes related to immunosuppression emerged over time suggesting evasion of immune surveillances. These findings in gene expression analyses were confirmed using mIHC which showed differential modulation of TIME in the tumor margins and as well as cores over time. These results suggest that evasion of immune surveillance, at least in part, may contribute to the acquired resistance to prexasertib in HNSCC.

This study aimed to describe considerations for developing supportive care interventions targeted to head and neck cancer (HNC) survivors.One-time semi-structured interviews (N = 33) were conducted with HNC survivors who had recently finished treatment (n = 20) and HNC providers (e.g., physicians, nurses; n = 13). Interviews were transcribed verbatim and coded using inductive applied thematic analysis techniques to identify themes.HNC survivors (75% male; M = 61 years old) and providers (54% physicians; 62% female) were unanimously supportive of developing HNC-specific supportive care interventions. Participants described potential benefits of offering interventions at various points throughout the HNC treatment and survivorship trajectory rather than at a single critical time. Many participants preferred group-based interventions because of the high value of peer-support. Others described how group interventions may not be appropriate for all HNC survivors due to risks for negative social comparisons and exacerbated anxiety. Participants suggested topics that should be addressed in HNC-specific interventions including education about acute and long-term side effects, symptom management, nutritional support, relationship/social role changes, grief/loss, and fear of recurrence.HNC-specific supportive care interventions are critically needed, as survivors experience persistent symptoms and distinct psychosocial concerns that impact quality of life. Findings from this study can inform the development of supportive care interventions targeted to the unique psychosocial concerns of HNC survivors.

Background Early-phase clinical trials are critical to the advancement of cancer care, especially in patients with pancreatic ductal adenocarcinoma, given its aggressive nature and limited available therapeutic options. Methods A retrospective chart review of all patients with refractory or metastatic pancreatic ductal adenocarcinoma, referred to the Sarah Cannon Research Institute at HealthONE between 2014 and 2019, were reviewed. Patients who completed genomic profiling and qualified for a phase 1 trial (primarily 1a but some 1b) were identified to assess barriers to trial enrollment. Results Of 74 identified patients, 54 patients (73%) qualified for at least 1 clinical trial based on eligibility criteria and alterations detected via molecular profiling. Up to 40 industry-sponsored clinical trials were available during this time for consideration. Of the 54, 28 patients (52%) enrolled in a clinical trial, while 26 (48%) did not enroll. The most frequently cited barriers to enrollment were concerns regarding time commitment (12%), prolonged wait time for enrollment (12%), and fear of adverse events (8%). Seven of the 26 patients (27%) were lost to follow-up or had no stated reason for declining enrollment; others did not go on trial due to death/transition to hospice (n=5; 19%) or progression of disease/declining functional status (n=4; 15%). There were few statistically significant differences between patients who chose to go on trial and those who declined. Conclusions An understanding of why eligible patients elect not to participate in early-phase clinical trials provides insight into the patient experience and can help identify misperceptions and areas for improvement in education and the clinical trial enrollment process.

AMP-activated protein kinase (AMPK in human and AAK in C. elegans) is a master regulator of metabolism. It has many isotypes, but its isotype-dependent functions are largely unknown. By developing ...

Purpose: Patients with head and neck squamous cell carcinoma (HNSCC) who actively smoke during treatment have worse survival compared with never-smokers and former-smokers. We hypothesize the poor prognosis in tobacco smokers with HNSCC is, at least in part, due to ongoing suppression of immune response. We characterized the tumor immune microenvironment (TIM) of HNSCC in a retrospective cohort of 177 current, former, and never smokers. Experimental Design: Tumor specimens were subjected to analysis of CD3, CD8, FOXP3, PD-1, PD-L1, and pancytokeratin by multiplex immunofluorescence, whole-exome sequencing, and RNA sequencing. Immune markers were measured in tumor core, tumor margin, and stroma. Results: Our data indicate that current smokers have significantly lower numbers of CD8+ cytotoxic T cells and PD-L1+ cells in the TIM compared with never- and former-smokers. While tumor mutation burden and mutant allele tumor heterogeneity score do not associate with smoking status, gene-set enrichment analyses reveal significant suppression of IFNα and IFNγ response pathways in current smokers. Gene expression of canonical IFN response chemokines, CXCL9, CXCL10, and CXCL11, are lower in current smokers than in former smokers, suggesting a mechanism for the decreased immune cell migration to tumor sites. Conclusions: These results suggest active tobacco use in HNSCC has an immunosuppressive effect through inhibition of tumor infiltration of cytotoxic T cells, likely as a result of suppression of IFN response pathways. Our study highlights the importance of understanding the interaction between smoking and TIM in light of emerging immune modulators for cancer management.

The promise of precision medicine has been limited by the pervasive therapeutic resistance to many targeted therapies for cancer. Inferring the timing (i.e., pre-existing or acquired) and mechanism (i.e., drug-induced) of such resistance is crucial for designing effective new therapeutics. This paper studies the mechanism and timing of cetuximab resistance in head and neck squamous cell carcinoma (HNSCC) using tumor volume data obtained from patient-derived tumor xenografts. We propose a family of mathematical models, with each member of the family assuming a different timing and mechanism of resistance. We present a method for fitting these models to individual volumetric data, and utilize model selection and parameter sensitivity analyses to ask: which member of the family of models best describes HNSCC response to cetuximab, and what does that tell us about the timing and mechanisms driving resistance? We find that along with time-course volumetric data to a single dose of cetuximab, the initial resistance fraction and, in some instances, dose escalation volumetric data are required to distinguish among the family of models and thereby infer the mechanisms of resistance. These findings can inform future experimental design so that we can best leverage the synergy of wet laboratory experimentation and mathematical modeling in the study of novel targeted cancer therapeutics.