John I. Song

STAT signaling in head and neck cancer

The upper aerodigestive tract is predisposed to the formation of multiple primary tumors due to field cancerization. TGF-α/EGFR autocrine signaling appears to play an important role in squamous cell carcinoma of the head and neck (SCCHN) and upregulation of TGF-α and EGFR is an early event in SCCHN carcinogenesis. STAT proteins, including Stat3, are activated by TGF-α and EGFR and strategies that downmodulate TGF-α or EGFR inhibit SCCHN cell proliferation and abrogate Stat3 activation. Targeting Stat3 leads to SCCHN growth inhibition, increases apoptosis and a downmodulation of Bcl-xL expression in head and neck tumors. These studies support the role of Stat3 as an oncogene, which is activated early in SCCHN carcinogenesis, and efforts to understand EGFR-mediated Stat3 signaling could facilitate novel strategies that will interfere with this growth promoting pathway.

Postoperative medical complications: Not microsurgical complications: Negatively influence the morbidity, mortality, and true costs after microsurgical reconstruction for head and neck cancer

Background: Immediate reconstruction of composite head and neck defects using free tissue transfer is an accepted treatment standard. There remains, however, ongoing debate on whether the costs associated with this reconstructive approach merit its selection, especially considering poor patient prognoses and the high cost of care. Methods: A retrospective review of the last 100 consecutive patients undergoing microsurgical reconstruction for head and neck cancer by the two senior surgeons was performed to determine whether microsurgical complications or postoperative medical complications had the more profound influence on morbidity and mortality outcomes and the true costs of these reconstructions. Results: Two patients required re-exploration of the microsurgical anastomoses, for a re-exploration rate of 2 percent, and one flap failed, for a flap success rate of 99 percent. The major surgical complication rate requiring a second operative procedure was 6 percent. Sixteen percent had minor surgical complications related to the donor site. Major medical complications, defined as a significant risk to the patient’s life, occurred in 5 percent of the patients, but there was a 37 percent incidence of “minor” medical complications primarily caused by pulmonary problems and alcohol withdrawal. Postsurgical complications almost doubled the average hospital stay from 13.5 days for those patients without complications to 24 days for patients with complications. Thirty-six percent of the true cost of microsurgical reconstruction of head and neck cancer was due to the intensive care unit and hospital room costs, and 24 percent was due to operating room costs. Postsurgical complications resulted in a 70.7 percent increase in true costs, reflecting a prolonged stay in the intensive care unit and not an increase in operating room costs or regular hospital room costs. Conclusion: Postoperative medical complications in these elderly, debilitated patients related to pulmonary problems and alcohol withdrawal were statistically far more important in negatively affecting the outcomes and true costs of microsurgical reconstruction.

A patient tumor transplant model of squamous cell cancer identifies PI3K inhibitors as candidate therapeutics in defined molecular bins

Targeted therapy development in head and neck squamous cell carcinoma (HNSCC) is challenging given the rarity of activating mutations. Additionally, HNSCC incidence is increasing related to human papillomavirus (HPV). We sought to develop an in vivo model derived from patients reflecting the evolving HNSCC epidemiologic landscape, and use it to identify new therapies. Primary and relapsed tumors from HNSCC patients, both HPV+ and HPV−, were implanted on mice, giving rise to 25 strains. Resulting xenografts were characterized by detecting key mutations, measuring protein expression by IHC and gene expression/pathway analysis by mRNA‐sequencing. Drug efficacy studies were run with representative xenografts using the approved drug cetuximab as well as the new PI3K inhibitor PX‐866. Tumors maintained their original morphology, genetic profiles and drug susceptibilities through serial passaging. The genetic makeup of these tumors was consistent with known frequencies of TP53, PI3KCA, NOTCH1 and NOTCH2 mutations. Because the EGFR inhibitor cetuximab is a standard HNSCC therapy, we tested its efficacy and observed a wide spectrum of efficacy. Cetuximab‐resistant strains had higher PI3K/Akt pathway gene expression and protein activation than cetuximab‐sensitive strains. The PI3K inhibitor PX‐866 had anti‐tumor efficacy in HNSCC models with PIK3CA alterations. Finally, PI3K inhibition was effective in two cases with NOTCH1 inactivating mutations. In summary, we have developed an HNSCC model covering its clinical spectrum whose major genetic alterations and susceptibility to anticancer agents represent contemporary HNSCC. This model enables to prospectively test therapeutic‐oriented hypotheses leading to personalized medicine.

Phase I Trial of Gefitinib in Combination With Radiation or Chemoradiation for Patients With Locally Advanced Squamous Cell Head and Neck Cancer

PURPOSE To establish the safety and toxicity profile of daily gefitinib with radiation alone or with concurrent chemoradiotherapy in previously untreated patients with locally advanced squamous cell head and neck cancer (LAHNC). PATIENTS AND METHODS Patients with intermediate-stage LAHNC were treated with concomitant boost radiation (RT) alone with escalating doses of daily gefitinib (250 or 500 mg; cohort I). Once a safety profile was determined with RT alone, patients with high-risk disease were then treated with daily gefitinib (250 or 500 mg), weekly cisplatin (CDDP; 30 mg/m2), and once-daily RT (cohort II). Patients also received post-RT gefitinib at 250 mg daily for a period of up to 2 years. RESULTS Twenty-three patients were enrolled and assessable for toxicity. No dose-limiting toxicities (DLTs) were observed in patients treated in cohort I at either 250 or 500 mg of gefitinib daily with concomitant boost RT to 72 Gy. In patients receiving chemoradiotherapy and gefitinib (cohort II), DLTs included one grade 4 diarrhea and one grade 4 neutropenic fever. Fifteen patients started maintenance gefitinib, and eight (53%) experienced grade 1 to 2 acne-like skin rash and diarrhea, but no grade 3 or 4 toxicity occurred. CONCLUSION Gefitinib (250 or 500 mg daily) was well tolerated with concomitant boost RT or concurrent chemoradiotherapy with weekly CDDP. Protracted administration of gefitinib for up to 2 years at 250 mg daily was also tolerated well.

Methylation of microRNA-9 is a specific and sensitive biomarker for oral and oropharyngeal squamous cell carcinomas

Detection of DNA methylation has produced promising results as biomarkers for head and neck squamous cell carcinoma (HNSCC). However, current panels are limited by an insufficient number of sensitive and specific tumor markers. MicroRNAs (miR) play an important role in tumorigenesis, and may represent a novel panel of molecules for the development of cancer biomarkers. We investigated methylation of three miRNA promoter sites of miR-9 (miR-9-1, miR-9-2, miR-9-3) in 107 human head and neck tissue samples and controls. We found methylations of miR-9-1 and miR-9-3 were higher in oral and oropharyngeal carcinomas than that in laryngeal carcinoma, achieving a combined sensitivity of 63% and 56%, respectively, for these two tumor types, compared to 21% for the laryngeal carcinoma. Quantitative PCR of miR-9 showed reduced expression associated with methylation of miR-9 in tumor tissues. To investigate the functional consequences of miR-9 methylation, we found that miR-9 methylation is correlated with miR-9 expression level in human HNSCC cell lines. Demethylation treatment using 5-aza-deoxycytidine restored its expression in a miR-9 methylated human HNSCC cell line UM-SCC22A. Furthermore, cell proliferation and viability was significantly inhibited, while PTEN expression was elevated after transfection of miR-9 into the UM-SCC22A cell line. In summary, our results suggest that methylations of miR-9-1 and miR-9-3 are sensitive and specific biomarkers for HNSCC, particularly for oral and oropharyngeal squamous cell carcinomas. In addition, miR-9 may function as a tumor suppressor in HNSCC through inhibition of cell proliferation and elevation of tumor suppressor PTEN.

Effect of Radiation Techniques in Treatment of Oropharynx Cancer

Objectives: To compare the toxicity and outcomes of three radiotherapy techniques—three‐dimensional conformal (3D‐RT), accelerated fractionation with concomitant boost (AFxCB), and intensity modulated radiotherapy (IMRT)—in the combined modality treatment of stage III–IV squamous cell carcinoma (SCC) of the oropharynx.

Epithelial stem cell mutations that promote squamous cell carcinoma metastasis.

Squamous cell carcinomas (SCCs) originate in stratified epithelia, with a small subset becoming metastatic. Epithelial stem cells are targets for driver mutations that give rise to SCCs, but it is unknown whether they contribute to oncogenic multipotency and metastasis. We developed a mouse model of SCC by targeting two frequent genetic mutations in human SCCs, oncogene Kras(G12D) activation and Smad4 deletion, to mouse keratin 15-expressing (K15+) stem cells. We show that transgenic mice developed multilineage tumors, including metastatic SCCs. Among cancer stem cell-enriched (CSC-enriched) populations, those with increased side population (SP) cells correlated with epithelial-mesenchymal transition (EMT) and lung metastasis. We show that microRNA-9 (miR-9) contributed to SP expansion and metastasis, and miR-9 inhibition reduced the number of SP cells and metastasis. Increased miR-9 was detected in metastatic human primary SCCs and SCC metastases, and miR-9-transduced human SCC cells exhibited increased invasion. We identified α-catenin as a predominant miR-9 target. Increased miR-9 in human SCC metastases correlated with α-catenin loss but not E-cadherin loss. Our results demonstrate that stem cells with Kras(G12D) activation and Smad4 depletion can produce tumors that are multipotent and susceptible to EMT and metastasis. Additionally, tumor initiation and metastatic properties of CSCs can be uncoupled, with miR-9 regulating the expansion of metastatic CSCs.

Hedgehog Signaling Alters Reliance on EGF Receptor Signaling and Mediates Anti-EGFR Therapeutic Resistance in Head and Neck Cancer

The EGF receptor (EGFR)-directed monoclonal antibody cetuximab is the only targeted therapy approved for the treatment of squamous cell carcinoma of the head and neck (HNSCC) but is only effective in a minority of patients. Epithelial-to-mesenchymal transition (EMT) has been implicated as a drug resistance mechanism in multiple cancers, and the EGFR and Hedgehog pathways (HhP) are relevant to this process, but the interplay between the two pathways has not been defined in HNSCC. Here, we show that HNSCC cells that were naturally sensitive to EGFR inhibition over time developed increased expression of the HhP transcription factor GLI1 as they became resistant after long-term EGFR inhibitor exposure. This robustly correlated with an increase in vimentin expression. Conversely, the HhP negatively regulated an EGFR-dependent, EMT-like state in HNSCC cells, and pharmacologic or genetic inhibition of HhP signaling pushed cells further into an EGFR-dependent phenotype, increasing expression of ZEB1 and VIM. In vivo treatment with cetuximab resulted in tumor shrinkage in four of six HNSCC patient-derived xenografts; however, they eventually regrew. Cetuximab in combination with the HhP inhibitor IPI-926 eliminated tumors in two cases and significantly delayed regrowth in the other two cases. Expression of EMT genes TWIST and ZEB2 was increased in sensitive xenografts, suggesting a possible resistant mesenchymal population. In summary, we report that EGFR-dependent HNSCC cells can undergo both EGFR-dependent and -independent EMT and HhP signaling is a regulator in both processes. Cetuximab plus IPI-926 forces tumor cells into an EGFR-dependent state, delaying or completely blocking tumor recurrence.

Survival and Patterns of Relapse in Patients With Oral Tongue Cancer

PURPOSE To evaluate the survival and patterns of relapse for patients with squamous cell carcinoma (SCC) of the oral tongue. PATIENTS AND METHODS Between 1999 and 2007, 50 patients with SCC of the oral tongue were treated at the University of Colorado Denver. Of the 50 patients, 38 had newly diagnosed SCC of the oral tongue (13 with stage I-II and 25 with stage III-IV disease), and 12 presented with locally recurrent SCC. Of the 50 patients, 49 were treated with initial surgery and 1 with definitive chemoradiotherapy. Adjuvant radiotherapy or chemoradiotherapy was administered to 42 patients after surgery. Of the 13 patients with newly diagnosed stage I-II disease, 7 did not receive adjuvant therapy. The actuarial locoregional control, freedom from distant relapse, and survival were determined using the Kaplan-Meier method, and comparisons were made using the log-rank test. RESULTS The median follow-up was 29 months (range 4 to 95) for living patients. The 2-year locoregional control and freedom from distant relapse rate was 58% and 83%, respectively. Locoregional control was particularly low among patients with stage I-II disease, for whom the 2-year locoregional control rate was only 35%. The median survival time and 2-year survival rate for all patients was 42 months and 65%, respectively. The 2-year survival rate for patients with stage I-II oral tongue cancer was 77% compared with 52% for patients with stage III-IV disease (P = .04). CONCLUSIONS Despite aggressive therapy, patients with SCC of the oral tongue have a low rate of local tumor control and survival, particularly among those with stage I-II disease. These patients should be considered for inclusion in clinical trials evaluating novel postoperative therapies.

XactMice: humanizing mouse bone marrow enables microenvironment reconstitution in a patient-derived xenograft model of head and neck cancer

The limitations of cancer cell lines have led to the development of direct patient-derived xenograft models. However, the interplay between the implanted human cancer cells and recruited mouse stromal and immune cells alters the tumor microenvironment and limits the value of these models. To overcome these constraints, we have developed a technique to expand human hematopoietic stem and progenitor cells (HSPCs) and use them to reconstitute the radiation-depleted bone marrow of a NOD/SCID/IL2rg−/− (NSG) mouse on which a patient’s tumor is then transplanted (XactMice). The human HSPCs produce immune cells that home into the tumor and help replicate its natural microenvironment. Despite previous passage on nude mice, the expression of epithelial, stromal and immune genes in XactMice tumors aligns more closely to that of the patient tumor than to those grown in non-humanized mice—an effect partially facilitated by human cytokines expressed by both the HSPC progeny and the tumor cells. The human immune and stromal cells produced in the XactMice can help recapitulate the microenvironment of an implanted xenograft, reverse the initial genetic drift seen after passage on non-humanized mice and provide a more accurate tumor model to guide patient treatment.