Matthew E. Witek

A Validated Quantitative Assay to Detect Occult Micrometastases by Reverse Transcriptase-Polymerase Chain Reaction of Guanylyl Cyclase C in Patients with Colorectal Cancer

Purpose: Guanylyl cyclase C (GCC), a receptor for bacterial diarrheagenic enterotoxins, may be a prognostic and predictive marker to detect occult micrometastases in patients undergoing staging for colorectal cancer. However, quantification of GCC expression in tissues by the quantitative reverse transcription-PCR (qRT-PCR) has not undergone analytic and clinicopathologic validation. Experimental Design: A technique to quantify GCC mRNA in tissues employing RT-PCR was developed and validated employing external calibration standards of RNA complementary to GCC. Results: GCC qRT-PCR exhibited reaction efficiencies >92%, coefficients of variations <5%, linearity >6 orders of magnitude, and a limit of quantification of >25 copies of GCC cRNA. This assay confirmed that GCC mRNA was overexpressed by colorectal tumors from 41 patients, which correlated with increased GCC protein quantified by immunohistochemistry. Analyses obtained with 164 lymph nodes from patients free of cancer and 15 nodes harboring metastases established a threshold for metastatic disease of ∼200 GCC mRNA copies/μg total RNA, with a sensitivity of 93% and specificity of 97%. GCC mRNA above that threshold was detected in 76 of 367 (∼21%) nodes free of disease by histopathology from 6 of 23 (26%) patients, suggesting the presence of occult micrometastases. Conclusions: Quantifying GCC mRNA in tissues by RT-PCR employing external calibration standards is analytically robust and reproducible, with high clinicopathologic sensitivity and specificity. This validated assay is being applied to ∼10,000 lymph nodes in a prospective trial to define the sensitivity of GCC qRT-PCR for staging patients with colorectal cancer.

The Putative Tumor Suppressor Cdx2 Is Overexpressed by Human Colorectal Adenocarcinomas

Purpose: The current paradigm suggests that the homeodomain transcription factor Cdx2, which directs the development and maintenance of the intestinal epithelium, is a tumor suppressor in the colon and rectum. Although a cardinal property of tumor suppressors is their inactivation during carcinogenesis, the expression of Cdx2 in colorectal tumors has not been compared with that in normal mucosa. Here, Cdx2 expression and function was quantified in tumors and matched normal mucosa from patients with colorectal cancer. Experimental Design: Cdx2 expression was quantified by reverse transcription-PCR, immunoblot analysis, and immunohistochemistry. Transcriptional activity was explored by quantifying expression of an endogenous downstream target of Cdx2, guanylyl cyclase C (GCC), in tissues by quantitative reverse transcription-PCR and expression of exogenous Cdx2-specific luciferase promoter constructs in epithelial cells isolated from tumors and normal mucosa. Results: Most (>80%) colorectal tumors overexpressed Cdx2 mRNA and protein compared with normal mucosa, with median fold increases of 3.6 and 1.4, respectively (P < 0.002). Concomitantly, immunohistochemistry revealed elevated levels of Cdx2 in nuclei of tumor cells compared with normal epithelial cells. Further, tumors exhibited increased expression of GCC compared with normal mucosa. Moreover, cells isolated from tumors overexpressed a Cdx2-specific luciferase promoter construct compared with normal mucosal cells. Conclusion: These observations show, for the first time, the structural and functional overexpression of Cdx2 by human colorectal tumors compared with matched normal mucosa. They suggest that loss of Cdx2 expression or transcriptional activity is an infrequent event during tumorigenesis, which does not contribute to molecular mechanisms underlying initiation and progression of most colorectal tumors.

NCCN Guidelines Insights: Head and Neck Cancers, Version 2.2017

The NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines) for Head and Neck Cancers provide treatment recommendations for cancers of the lip, oral cavity, pharynx, larynx, ethmoid and maxillary sinuses, and salivary glands. Recommendations are also provided for occult primary of the head and neck (H&N), and separate algorithms have been developed by the panel for very advanced H&N cancers. These NCCN Guidelines Insights summarize the panels discussion and most recent recommendations regarding the increase in human papillomavirus-associated oropharyngeal cancer and the availability of immunotherapy agents for treatment of patients with recurrent or metastatic H&N cancer.

Tumor radiation therapy creates therapeutic vaccine responses to the colorectal cancer antigen GUCY2C.

PURPOSE Radiation therapy (RT) is thought to produce clinical responses in cancer patients, not only through direct toxicity to cancer cells and supporting tumor stroma cells, but also through activation of immunologic effectors. More recently, RT has potentiated the local and systemic effects of cancer immunotherapy (IT). However, combination regimens that maximize immunologic and clinical efficacy remain undefined. METHODS AND MATERIALS We evaluated the impact of local RT on adenoviral-mediated vaccination against the colorectal cancer antigen GUCY2C (Ad5-GUCY2C) in a murine subcutaneous tumor model using mouse CT26 colon cancer cells (CT26-GUCY2C). Immune responses were assessed by ELISpot, and clinical responses were assessed by tumor size and incidence. RESULTS The specific sequence of tumor-directed RT preceding Ad5-GUCY2C IT transformed inactive therapeutic Ad5-GUCY2C vaccination into a curative vaccine. GUCY2C-specific T cell responses were amplified (P<.05), tumor eradication was maximized (P<.01), and tumor volumes were minimized (P<.001) in mice whose tumors were irradiated before, compared with after, Ad5-GUCY2C vaccination. The immunologic and antitumor efficacy of Ad5-GUCY2C was amplified comparably by unfractionated (8 Gy × 1), or biologically equivalent doses of fractionated (3.5 Gy × 3), RT. The antitumor effects of sequential RT and IT (RT-IT) depended on expression of GUCY2C by tumor cells and the adenoviral vaccine vector, and tumor volumes were inversely related to the magnitude of GUCY2C-specific T cell responses. Moreover, mice cured of CT26-GUCY2C tumors by RT-IT showed long-lasting antigen-dependent protection, resisting tumors formed by GUCY2C-expressing 4T1 breast cancer cells inoculated 50 days after CT26 cells. CONCLUSIONS Optimal sequencing of RT and IT amplifies antigen-specific local and systemic immune responses, revealing novel acute and long-term therapeutic antitumor protection. These observations underscore the importance of modality sequence optimization before the initiation of clinical trials of RT and IT to maximize immune and antitumor responses.

Molecular markers to predict clinical outcome and radiation induced toxicity in lung cancer

The elucidation of driver mutations involved in the molecular pathogenesis of cancer has led to a surge in the application of novel targeted therapeutics in lung cancer. Novel oncologic research continues to lead investigators towards targeting personalized tumor characteristics rather than applying targeted therapy to broad patient populations. Several driver genes, in particular epidermal growth factor receptor (EGFR) and ALK fusions, are the earliest to have made their way into clinical trials. The avant-garde role of genomic profiling has led to important clinical challenges when adapting current standard treatments to personalized oncologic care. This new frontier of medicine requires newer biomarkers for toxicity that will identify patients at risk, as well as, new molecular markers to predict and assess clinical outcomes. Thus far, several signature genes have been developed to predict outcome as well as genetic factors related to inflammation to predict toxicity.

Treatment-related acute esophagitis for patients with locoregionally advanced non-small cell lung cancer treated with involved-field radiotherapy and concurrent chemotherapy.

Purpose:To explore the incidence and risk factors for treatment-related acute esophagitis associated with involved-field radiation therapy (RT) delivered concurrently with chemotherapy for patients with locoregionally advanced non–small cell lung cancer. Materials and Methods:Forty-nine consecutive patients diagnosed with locoregionally advanced non–small cell lung cancer were treated using involved-field RT. Radiotherapy target volumes included the primary lung tumor and involved mediastinal lymphadenopathy as defined on imaging studies including computed tomography of the chest and [18F] fluorodeoxyglucose-positron emission tomography/computed tomography. The patients were treated to a median total dose of 63 Gy (range, 55.8 to 74 Gy) using daily fractions of 1.8 or 2.0 Gy. No elective radiotherapy of mediastinal lymph nodes was used. Concurrent platinum-based chemotherapy was delivered to all patients. Treatment-related toxicity was evaluated during the course of RT and subsequent follow-up visits. Results:Thirty-one (63%) patients were female and 18 (37%) were male. Median age at the time of diagnosis was 68 years (range, 36 to 83 y). Thirty-one patients (63%) developed treatment-related acute esophagitis: 24 patients (49%) grade 2 and 7 (14%) patients grade 3 esophagitis, with the peak occurring during the seventh week of radiotherapy. No grade ≥4 esophagitis was seen in this cohort. Eighteen patients (37%) did not develop radiation-induced esophagitis associated with their course of chemoradiotherapy. In the univariate analysis, age at the time of diagnosis, radiation dose per fraction, and total volume of the esophagus were significantly associated with the risk of acute esophagitis. Increasing age reduced the risk of acute esophagitis (odds ratio [OR] for 10-y increase=0.40) as did increasing total esophagus volume (OR for 10-U increase=0.27). Dose per fraction of 1.8 Gy was associated with lower risk of acute esophagitis when compared with dose per fraction of 2 Gy (OR=0.19). Marginal associations were observed for all of the volume variables. Higher volume variable values had a nonsignificant association with an increase in risk of acute esophagitis. However, only the total volume of the esophagus (P=0.0032) and larger dose per fraction (2 vs. 1.8 Gy) (P=0.011) remained significantly associated with higher risk of developing grade ≥2 acute esophagitis in the multivariate analysis. Conclusions:Higher risk of grade ≥2 treatment-related esophagitis was associated with lower total esophageal volume and higher radiotherapy dose per fraction and should be taken into consideration during patient treatment planning. Inclusion of total esophageal volume and dose per fraction into future clinical protocols may further help our understanding of treatment-related esophagitis and enable the development of novel preventative approaches.

Optimizing patient positioning for intensity modulated radiation therapy in hippocampal-sparing whole brain radiation therapy

PURPOSE Sparing the hippocampus during whole brain radiation therapy (WBRT) offers potential neurocognitive benefits. However, previously reported intensity modulated radiation therapy (IMRT) plans use multiple noncoplanar beams for treatment delivery. An optimized coplanar IMRT template for hippocampal-sparing WBRT would assist in clinical workflow and minimize resource utilization. In this study, we sought to determine the optimal patient position to facilitate coplanar treatment planning and delivery of hippocampal-sparing WBRT using IMRT. METHODS AND MATERIALS A variable angle, inclined board was utilized for patient positioning. An anthropomorphic phantom underwent computed tomography simulation at various head angles. The IMRT goals were designed to achieve target coverage of the brain while maintaining hippocampal dose-volume constraints designed to conform to the Radiation Therapy Oncology Group 0933 protocol. Optimal head angle was then verified using data from 8 patients comparing coplanar and noncoplanar WBRT IMRT plans. RESULTS Hippocampal, hippocampal avoidance region, and whole brain mean volumes were 1.1 cm(3), 12.5 cm(3), and 1185.1 cm(3), respectively. The hippocampal avoidance region occupied 1.1% of the whole brain planning volume. For the 30-degree head angle, a 7-field coplanar IMRT plan was generated, sparing the hippocampus to a maximum dose of 14.7 Gy; D100% of the hippocampus was 7.4 Gy and mean hippocampal dose was 9.3 Gy. In comparison, for flat head positioning the hippocampal Dmax was 22.9 Gy with a D100% of 9.2 Gy and mean dose of 11.7 Gy. Target coverage and dose homogeneity was comparable with previously published noncoplanar IMRT plans. CONCLUSIONS Compared with conventional supine positioning, an inclined head board at 30 degrees optimizes coplanar whole brain IMRT treatment planning. Clinically acceptable hippocampal-sparing WBRT dosimetry can be obtained using a simplified coplanar plan at a 30-degree head angle, thus obviating the need for complex and time consuming noncoplanar IMRT plans.

Bile acids initiate lineage-addicted gastroesophageal tumorigenesis by suppressing the EGF receptor-AKT axis.

While bile acids are a risk factor for tumorigenesis induced by reflux disease, the mechanisms by which they contribute to neoplasia remain undefined. Here, we reveal that in gastroesophageal junction (GEJ) cells bile acids activate a tissue‐specific developmental program defining the intestinal epithelial cell phenotype characterizing GEJ metaplasia. Deoxycholic acid (DCA) inhibited phosphorylation of EGF receptors (EGFRs) suppressing the proto‐oncogene AKT. Suppression of EGFRs and AKT by DCA actuated an intestine‐specific cascade in which NF‐κB transactivated the tissue‐specifi c transcription factor CDX2. In turn, CDX2 orchestrated a lineage‐specific differentiation program encompassing genes characterizing intestinal epithelial cells. Conversely, progression from metaplasia to invasive carcinoma in patients, universally associated with autonomous activation of EGFRs and/or AKT, was coupled with loss of this intestinal program. Thus, bile acids induce intestinal metaplasia at the GEJ by activating the lineage‐specifi c differentiation program involving suppression of EGFR and AKT, activating the NF‐κB‐CDX2 axis. Induction of this axis provides the context for lineage‐addicted tumorigenesis, in which autonomous activation of AKT corrupts adaptive intestinal NF‐κB signaling, amplifying tumorigenic programs.

Small cell carcinoma of the head and neck: An analysis of the National Cancer Database

PURPOSE/OBJECTIVE(S) To evaluate treatment trends and overall survival of patients with small cell carcinoma of the head and neck region. MATERIALS/METHODS Patients from 2004 to 2012 were identified from the National Cancer Database. Patient demographics and overall survival were analyzed. Multivariable analysis was used to identify predictors of survival. RESULTS Among 347,252 head and neck patients a total of 1042 (0.3%) patients with small cell carcinoma were identified. 17% of patients were diagnosed as stage I/II, 61% as stage III/IVA/IVB and 22% as stage IVC disease. The distribution by anatomic site was 9% oral cavity, 12% oropharynx, 35% larynx, 4% hypopharynx, 10% nasopharynx and 30% nasal cavity and paranasal sinuses. The median overall survival by anatomical site was 20.8months for oral cavity, 23.7months for oropharynx, 17.9months for larynx/hypopharynx, 15.1months for nasopharynx and 36.4months for nasal cavity primary tumors. On multivariable analysis across stage, patients with nasal cavity and paranasal sinuses tumors had the best survival and patients with nasopharynx primaries had the worst survival. In stage I/II patients, type of treatment delivered resulted in no overall survival difference (p=0.78). In patients with locally advanced disease, there was no difference in survival between those treated with combined surgery, radiotherapy and chemotherapy compared to those treated only with radiotherapy and chemotherapy (p=0.46). The addition of radiotherapy to chemotherapy in the metastatic setting did not result in improved survival (p=0.14). CONCLUSIONS Small cell carcinoma of the head and neck is a rare malignancy with a poor prognosis. The addition of surgery to radiotherapy and chemotherapy did not improve survival in patients with locally advanced disease.

NCCN guidelines® insights: Head and neck cancers, version 1.2018 featured updates to the NCCN guidelines

The NCCN Guidelines for Head and Neck (H&N) Cancers provide treatment recommendations for cancers of the lip, oral cavity, pharynx, larynx, ethmoid and maxillary sinuses, and salivary glands. Recommendations are also provided for occult primary of the H&N, and separate algorithms have been developed by the panel for very advanced H&N cancers. These NCCN Guidelines Insights summarize the panels discussion and most recent recommendations regarding evaluation and treatment of nasopharyngeal carcinoma.