Metabolomic and BH3 profiling of esophageal cancers: novel assessment methods for precision therapy.

BACKGROUND: Esophageal cancers accounted for nearly 16,000 deaths in 2016. The number of patients with esophageal cancers increases every year. Neoadjuvant chemoradiotherapy (nCRT) prior to esophagectomy is a standard treatment for esophageal cancers. The patients who have no residual tumor (pathological complete response (pCR)) at surgery are the most likely to experience long term survival. Accurately determining which patients will have a pCR will improve prognostic information for patients and families, confirm lack of response to nCRT, or avoid surgery if no residual tumor is present. Imaging, endoscopy, and liquid biomarkers have all failed to detect pCR without performing an esophagectomy. METHODS: In this study, we are enrolling patients with esophageal adenocarcinoma and squamous cell carcinoma. Patients will undergo standard evaluation including CT scans, laboratory tests, endoscopy with biopsies, and evaluation by a thoracic surgeon. Tissue biopsy is required for enrollment that will be sent for BH3 profiling and metabolomics. Patients will be treated with standard nCRT followed by surgery. Patients with metastatic disease are not eligible. Surgery at the National Cancer Institute will be minimally-invasive robotic surgery. Patients will remain on study indefinitely with regular clinic visits and imaging tests. DISCUSSION: The mitochondria are critically involved in the intrinsic pathway apoptosis. Bcl-2 homology domain 3 (BH3) profiling is a technique to measure a cell's susceptibility to apoptosis. BH3 profiling measures the relative interactions of proteins that induce or block apoptosis. The collective balance of these proteins determines whether a cell is near the threshold to undergo apoptosis. If the cell is near this threshold, then the tumor may be more likely to die when treated with nCRT. The mitochondria secrete metabolites that may be detectable as biomarkers. Metabolomics is a global assessment of all metabolite changes that has been performed for detection, monitoring, prognosis, and treatment response in cancers. Stratification of patients based on whether pCR occurs or not may elucidate metabolomic signatures that may be associated with response. We are asking whether BH3 profiling or a metabolomic signature will correlate with tumor death after nCRT for esophageal cancer. TRIAL REGISTRATION: NCT03223662 ; Clinicaltrials.gov. July 21, 2017.

Randomized phase II trial of a first-in-human cancer cell lysate vaccine in patients with thoracic malignancies.

BACKGROUND: Although most malignancies express cancer-testis antigens (CTA), immune responses to these proteins are limited in thoracic oncology patients. This trial was undertaken to examine if a cancer cell lysate vaccine could induce immunity to CTA, and to ascertain if metronomic cyclophosphamide and celecoxib enhances vaccine-induced immune responses. METHODS: Eleven patients with primary thoracic malignancies and 10 patients with extrathoracic neoplasms metastatic to the chest rendered NED by conventional therapies were randomized to receive H1299 lung cancer cell lysates (10 mg protein/vaccine) with Iscomatrix™ adjuvant via deep intradermal injection q 4 weeks ×6 with or without daily oral metronomic cyclophosphamide/celecoxib. The primary endpoint was serologic response to purified CTA assessed 1 month after the 6th vaccination. Secondary endpoints included assessment of the effects of cyclophosphamide and celecoxib on frequency and magnitude of vaccine-induced immune responses to CTA. Exploratory endpoints included evaluation of the effects of the vaccine regimens on peripheral immune subsets. Standard of care imaging studies were obtained at baseline and 1 month after the 3rd and 6th vaccinations. RESULTS: All patients exhibited local and systemic inflammatory responses lasting 72-96 hours following vaccinations. There were no dose limiting treatment related toxicities. Fourteen patients (67%) completed all six vaccinations. Eight of 14 patients (57%) exhibited serologic responses to NY-ESO-1. One patient developed antibodies to GAGE7; several patients exhibited reactivity to XAGE and MAGE-C2. Vaccine therapy decreased the percent of Tregs (P=0.0068), PD-1 expression on Tregs (P=0.0027), PD-L1 expression on CD14+ monocytes (P=0.0089), PD-L1 expression on classical monocytes (P=0.016), and PD-L1 expression on intermediate monocytes (P=0.0031). Cyclophosphamide/celecoxib did not appear to increase immune responses or enhance vaccine-induced alterations in peripheral immune subsets. CONCLUSIONS: H1299 lysate vaccines with Iscomatrix™ induce immune responses to CTA and modulate peripheral immune subsets in a manner that may enhance antitumor immunity in patients with thoracic malignancies.

Clinical and molecular responses in lung cancer patients receiving Romidepsin.

PURPOSE: Our preclinical experiments indicated that Romidepsin (Depsipeptide FK228; DP) mediates growth arrest and apoptosis in cultured lung cancer cells. A phase II trial was done to examine clinical and molecular responses mediated by this histone deacetylase inhibitor in lung cancer patients. EXPERIMENTAL DESIGN: Nineteen patients with neoplasms refractory to standard therapy received 4-h DP infusions (17.8 mg/m(2)) on days 1 and 7 of a 21-day cycle. Each full course of therapy consisted of two identical 21-day cycles. Plasma DP levels were evaluated by liquid chromatography-mass spectrometry techniques. A variety of molecular end points were assessed in tumor biopsies via immunohistochemistry techniques. Long oligo arrays were used to examine gene expression profiles in laser-captured tumor cells before and after DP exposure, relative to lung cancer cells and adjacent normal bronchial epithelia from patients undergoing pulmonary resections. RESULTS: Nineteen patients were evaluable for toxicity assessment; 18 were evaluable for treatment response. Myelosuppression was dose limiting in one individual. No significant cardiac toxicities were observed. Maximum steady-state plasma DP concentrations ranged from 384 to 1,114 ng/mL. No objective responses were observed. Transient stabilization of disease was noted in nine patients. DP enhanced acetylation of histone H4, increased p21 expression in lung cancer cells, and seemed to shift global gene expression profiles in these cells toward those detected in normal bronchial epithelia. CONCLUSION: Although exhibiting minimal clinical efficacy at this dose and schedule, DP mediates biological effects that may warrant further evaluation of this histone deacetylase inhibitor in combination with novel-targeted agents in lung cancer patients.

Phase I study of decitabine-mediated gene expression in patients with cancers involving the lungs, esophagus, or pleura.

PURPOSE: The DNA methylation paradox, manifested as derepression of cancer-testis antigens, and silencing of tumor suppressors during malignant transformation, provides the rationale for the utilization of chromatin remodeling agents for cancer therapy. A phase I trial was done to examine pharmacokinetics, toxicities, and gene expression mediated by 5-aza-2'-deoxycytidine (DAC) in patients with thoracic malignancies. EXPERIMENTAL DESIGN: Thirty-five patients with cancers refractory to standard therapy received continuous 72-hour DAC infusions using a phase I dose-escalation schema. Each full course of therapy consisted of two identical 35-day cycles. Plasma DAC levels were evaluated by liquid chromatography-mass spectrometry techniques. Quantitative reverse transcription-PCR, methylation-specific PCR, and immunohistochemical techniques were used to evaluate NY-ESO-1, MAGE-3, and p16 expression in tumor biopsies. Long oligonucleotide arrays were used to evaluate gene expression profiles in laser-captured tumor cells before and after DAC exposure. RESULTS: Thirty-five patients were evaluable for toxicities; 25 were evaluable for treatment response. Myelosuppression constituted dose-limiting toxicity. The maximum tolerated dose of DAC was 60 to 75 mg/m(2) depending on the number of prior cytotoxic chemotherapy regimens. No objective responses were observed. Plasma DAC concentrations approximated thresholds for gene induction in cultured cancer cells. Target gene induction was observed in 36% of patients. Posttreatment antibodies to NY-ESO-1 were detected in three patients exhibiting NY-ESO-1 induction in their tumor tissues. Complex, heterogeneous gene expression profiles were observed in pretreatment and posttreatment tissues. CONCLUSION: Prolonged DAC infusions can modulate gene expression in primary thoracic malignancies. These findings support further evaluation of DNA-demethylating agents alone or in combination with other regimens targeting induced gene products for the treatment of these neoplasms.

Comparative evaluation of super high-resolution CT scan and virtual bronchoscopy for the detection of tracheobronchial malignancies.

OBJECTIVES: Novel imaging modalities are currently available for the noninvasive evaluation of the tracheobronchial tree. This study was undertaken to compare the diagnostic potentials of conventional CT scanning, super high-resolution CT (SHR-CT) scanning, and virtual bronchoscopy (VB) directly with fiberoptic bronchoscopy (FB) for the detection of tracheobronchial neoplasms. DESIGN: Prospective observer study, in which 44 consecutive patients with thoracic malignancies were evaluated using several diagnostic imaging modalities. Images of the thorax were interpreted by individuals blind to the results of FB for the detection of endoluminal, obstructive, or mucosal lesions. MEASUREMENTS AND RESULTS: Image acquisition and simulation of the tracheobronchial anatomy were created successfully in all patients. Thirty-two patients who underwent both SHR-CT scanning and VB had correlative FBs within 1 month. In all nine patients who had a normal anatomy, SHR-CT scanning and VB accurately correlated with the FB findings. However, CT scanning demonstrated two false-positive obstructive lesions in one patient. Twenty-three patients had a total of 35 abnormal FB findings. The sensitivities of SHR-CT scanning and VB for the detection of endoluminal, obstructive, and mucosal lesions were 90%, 100%, and 16%, respectively. The overall sensitivities and specificities of SHR-CT scanning and VB were 83% and 100%, respectively. In contrast, CT scanning had sensitivities of 50%, 72%, and 0% for the detection of endoluminal, obstructive, and mucosal lesions with an overall sensitivity and specificity of 59%, and 85%, respectively. There was no case in which conventional CT scanning was better at detecting lesions than either SHR-CT scanning or VB. CONCLUSIONS: SHR-CT scanning and VB are accurate, noninvasive methods for identifying obstructions and endoluminal lesions within the respiratory tract. Thus, these novel imaging techniques are valuable as complementary modalities to FB, providing information that is useful for the detection and management of airway malignancies.

Telomerase variant A279T induces telomere dysfunction and inhibits non-canonical telomerase activity in esophageal carcinomas.

BACKGROUND: Although implicated in the pathogenesis of several chronic inflammatory disorders and hematologic malignancies, telomerase mutations have not been thoroughly characterized in human cancers. The present study was performed to examine the frequency and potential clinical relevance of telomerase mutations in esophageal carcinomas. METHODS: Sequencing techniques were used to evaluate mutational status of telomerase reverse transcriptase (TERT) and telomerase RNA component (TERC) in neoplastic and adjacent normal mucosa from 143 esophageal cancer (EsC) patients. MTS, flow cytometry, time lapse microscopy, and murine xenograft techniques were used to assess proliferation, apoptosis, chemotaxis, and tumorigenicity of EsC cells expressing either wtTERT or TERT variants. Immunoprecipitation, immunoblot, immunofluorescence, promoter-reporter and qRT-PCR techniques were used to evaluate interactions of TERT and several TERT variants with BRG-1 and ß-catenin, and to assess expression of cytoskeletal proteins, and cell signaling. Fluorescence in-situ hybridization and spectral karyotyping techniques were used to examine telomere length and chromosomal stability. RESULTS: Sequencing analysis revealed one deletion involving TERC (TERC del 341-360), and two non-synonymous TERT variants [A279T (2 homozygous, 9 heterozygous); A1062T (4 heterozygous)]. The minor allele frequency of the A279T variant was five-fold higher in EsC patients compared to healthy blood donors (p<0.01). Relative to wtTERT, A279T decreased telomere length, destabilized TERT-BRG-1-ß-catenin complex, markedly depleted ß-catenin, and down-regulated canonical Wnt signaling in cancer cells; these phenomena coincided with decreased proliferation, depletion of additional cytoskeletal proteins, impaired chemotaxis, increased chemosensitivity, and significantly decreased tumorigenicity of EsC cells. A279T expression significantly increased chromosomal aberrations in mouse embryonic fibroblasts (MEFs) following Zeocin™ exposure, as well as Li Fraumeni fibroblasts in the absence of pharmacologically-induced DNA damage. CONCLUSIONS: A279T induces telomere dysfunction and inhibits non-canonical telomerase activity in esophageal cancer cells. These findings warrant further analysis of A279T expression in esophageal cancers and premalignant esophageal lesions.

Cigarette smoke mediates epigenetic repression of miR-487b during pulmonary carcinogenesis.

MicroRNAs are critical mediators of stem cell pluripotency, differentiation, and malignancy. Limited information exists regarding microRNA alterations that facilitate initiation and progression of human lung cancers. In this study, array techniques were used to evaluate microRNA expression in normal human respiratory epithelia and lung cancer cells cultured in the presence or absence of cigarette smoke condensate (CSC). Under relevant exposure conditions, CSC significantly repressed miR-487b. Subsequent experiments demonstrated that miR-487b directly targeted SUZ12, BMI1, WNT5A, MYC, and KRAS. Repression of miR-487b correlated with overexpression of these targets in primary lung cancers and coincided with DNA methylation, de novo nucleosome occupancy, and decreased H2AZ and TCF1 levels within the miR-487b genomic locus. Deoxy-azacytidine derepressed miR-487b and attenuated CSC-mediated silencing of miR-487b. Constitutive expression of miR-487b abrogated Wnt signaling, inhibited in vitro proliferation and invasion of lung cancer cells mediated by CSC or overexpression of miR-487b targets, and decreased growth and metastatic potential of lung cancer cells in vivo. Collectively, these findings indicate that miR-487b is a tumor suppressor microRNA silenced by epigenetic mechanisms during tobacco-induced pulmonary carcinogenesis and suggest that DNA demethylating agents may be useful for activating miR-487b for lung cancer therapy.