Roy H. Decker

Ultrasensitive Measurement of Hotspot Mutations in Tumor DNA in Blood Using Error-Suppressed Multiplexed Deep Sequencing

Detection of cell-free tumor DNA in the blood has offered promise as a cancer biomarker, but practical clinical implementations have been impeded by the lack of a sensitive and accurate method for quantitation that is also simple, inexpensive, and readily scalable. Here we present an approach that uses next-generation sequencing to quantify the small fraction of DNA molecules that contain tumor-specific mutations within a background of normal DNA in plasma. Using layers of sequence redundancy designed to distinguish true mutations from sequencer misreads and PCR misincorporations, we achieved a detection sensitivity of approximately 1 variant in 5,000 molecules. In addition, the attachment of modular barcode tags to the DNA fragments to be sequenced facilitated the simultaneous analysis of more than 100 patient samples. As proof-of-principle, we showed the successful use of this method to follow treatment-associated changes in circulating tumor DNA levels in patients with non-small cell lung cancer. Our findings suggest that the deep sequencing approach described here may be applied to the development of a practical diagnostic test that measures tumor-derived DNA levels in blood.

Stereotactic Body Radiotherapy for Central Lung Tumors

Introduction: Patients with centrally located lung tumors have been reported to have a higher risk of toxicity when treated with stereotactic body radiotherapy (SBRT) compared with patients with peripheral tumors. The optimal SBRT fractionation schedule for treatment of central tumors is unknown. The primary purpose of this study was to assess toxicity in patients with central lesions treated with SBRT at our institution, the majority of whom were treated with four fractions. Methods: Forty-seven patients with 51 central lesions, either primary lung cancer or lung metastases, were treated with SBRT at the Department of Therapeutic Radiology, Yale University School of Medicine/Yale Cancer Center from 2007 to 2011. The patients were treated with three to five fractions with the majority of patients receiving 50 Gy in four fractions of 12.5 Gy. Forty of the lesions were located within 2 cm of the proximal tracheobronchial tree whereas 11 were located within 2 cm of other mediastinal structures. Toxicity data were collected and analyzed according to pretreatment and tumor characteristics and dosimetric parameters. Lobar control data were compiled. Results: With a median follow-up of 11.3 months (range, 4.8–40.8), four patients experienced grade 3 dyspnea and one patient developed hemoptysis that contributed to respiratory failure and subsequent death. Grade 2 toxicity included fatigue (n = 3), dyspnea (n = 3), chest-wall pain (n = 1), and cough (n = 1). Patients with grade 3+ toxicity had larger maximum tumor diameters compared with those patients without grade 3+ toxicity (median diameter 4.3 cm versus 2.9 cm, p = 0.02). There were no detectable significant differences between the two groups with respect to baseline pulmonary function tests, distance to tracheobronchial tree, maximum point dose to the tracheobronchial tree, maximum dose to 5 cc of the tracheobronchial tree, mean lung dose, and volume of lung receiving 5 Gy, 10 Gy, and 20 Gy. There were two patients who experienced local recurrences. The median biological equivalent dose (linear quadratic formula, &agr;/&bgr; = 10) for patients with local recurrence was 76 Gy compared with 112.5 Gy for patients without local recurrence (2-tailed t test, p = 0.04). The 2-year actuarial lobar local control for the entire cohort was 94%. The 2-year lobar local-control rate for patients receiving a biological equivalent dose of 100 Gy or more was 100% and for those receiving less than 100 Gy was 80% (log rank, p = 0.02). Conclusion: SBRT for central lung tumors seems to be safe, although treatment of larger tumors does carry an increased risk of high-grade toxicity. Efforts to decrease the toxicity risk by decreasing the biologically equivalent dose resulted in increased local failure.

Surveillance Epidemiology and End Results Evaluation of the Role of Surgery for Stage I Small Cell Lung Cancer

Introduction: This study was performed to evaluate the clinical outcomes of surgery for stage I small cell lung cancer (SCLC). Methods: The National Cancer Institute Surveillance Epidemiology and End Results (SEER) database was analyzed to evaluate outcomes for patients with SCLC treated from 1988 to 2004. Patients with stage I disease were selected. Kaplan-Meier survival curves were constructed for overall survival (OS) and cause-specific survival for patient strata based on type of surgery and radiation use or nonuse. Although SEER does not provide chemotherapy details, it is assumed that most, if not all, of these patients received systemic therapy. Results: A total of 1560 patients were identified as having stage I SCLC. Median age was 70 years (range 27–94 years). Two hundred forty-seven patients underwent lobectomy, 121 had local tumor excision/ablation, 10 had a pneumonectomy, and surgery was unknown in 21. One thousand one hundred sixty-one did not have any cancer-directed surgery. Of those who had lobectomy, 205 (83%) did not receive radiation therapy (RT), 38 (15%) did receive RT, and use of RT was unknown in 4 (2%). For those who had lobectomy without RT (n = 205), 3- and 5-year OS was 58.1% (95% confidence interval [CI] 51.1–64.5%) and 50.3% (95% CI 43.1–57.1%), respectively. For those patients who had a lobectomy with RT (n = 38), 3- and 5-year OS was 64.9% (95% CI 45.5–78.9%) and 57.1% (95% CI 37.4–72.7%), respectively. Conclusions: Surgery without RT seems to offer reasonable OS outcomes in a cohort of stage I patients who undergo lobectomy. These results should be considered with the understanding that systemic therapy information and margin status are not available from the SEER database.

Determinants and patterns of survival in adenoid cystic carcinoma of the head and neck, including an analysis of adjuvant radiation therapy.

Objectives: The effect of adjuvant radiation therapy (RT) on survival in patients with adenoid cystic carcinoma (ACC) is much debated. Studies with large numbers of patients and long follow-up are lacking in the literature. Methods: We identified 2286 single-primary ACCs of the head and neck treated with surgery in the National Cancer Institutes surveillance, epidemiology, and end results database. The determinants of survival, including the effect of adjuvant RT, were analyzed with a Cox Proportional Hazards Model. Results: The overall 5-, 10-, 15-, 20-, and 25-year survival rates for patients receiving surgical resection were 77.3%, 59.6%, 44.9%, 35.0%, 25.5%, respectively. In the interval between 10 and 30 years after diagnosis, 111 patients died of ACC, and 137 died of all competing causes combined. Increasing stage caused a decrease in survival that was proportional over time. On multivariable analysis, distant metastasis, lymph node involvement, higher T classification, increasing age, and submandibular gland, or sinus/nasal cavity subsites were each independently associated with decreased overall and cause-specific survival. Adjuvant RT failed to improve overall or cause-specific survival. Conclusions: Cause-specific survival continues to decline up to 30 years after diagnosis in ACC of the head and neck. In the interval between 10 and 30 years after diagnosis, patients are nearly as likely to die of ACC as from all competing causes combined. Certain clinicopathological factors are associated with decreased survival. There is no evidence of increased survival in patients receiving adjuvant RT.

Whole Pelvic Radiotherapy Versus Prostate Only Radiotherapy in the Management of Locally Advanced or Aggressive Prostate Adenocarcinoma

PURPOSE To determine whether whole pelvic radiotherapy (WPRT) or prostate-only radiotherapy (PORT) yields improved biochemical disease-free survival (BDFS) in patients with advanced or aggressive prostate adenocarcinoma. METHODS AND MATERIALS Between 2000 and 2007, a consecutive sample of 277 patients with prostate adenocarcinoma and at least a 15% likelihood of lymph node involvement who had undergone WPRT (n = 68) or PORT (n = 209) at two referral centers was analyzed. The median radiation dose in both arms was 75.6 Gy. The outcome measure was BDFS, as determined using the prostate-specific antigen nadir + 2 ng/mL definition of failure. BDFS was calculated using the Kaplan-Meier method and compared with the log-rank test. A multivariate analysis was performed to assess for confounding. Treatment-related toxicity was assessed using the National Cancer Institutes Common Terminology Criteria for Adverse Events guidelines. The median follow-up was 30 months. RESULTS WPRT patients had more advanced and aggressive disease at baseline (p < .001). The 4-year BDFS rate was 69.4% in the PORT cohort and 86.3% in the WPRT cohort (p = .02). Within the entire cohort, after adjustment for confounding variables, the pretreatment prostate-specific antigen (p < .001), Gleason score (p < .001), use of hormonal therapy (p = .002), and use of WPRT (vs. PORT, p = .006) predicted for BDFS. Patients undergoing WPRT had increased acute gastrointestinal toxicity (p = .048), but no significant difference in acute genitourinary toxicity was seen (p = .09). No difference in late toxicity was found. CONCLUSION WPRT may yield improved BDFS in patients with advanced or aggressive prostate adenocarcinoma, but results in a greater incidence of acute toxicity.

Incorporating Bevacizumab and Erlotinib in the Combined-Modality Treatment of Stage III Non–Small-Cell Lung Cancer: Results of a Phase I/II Trial

PURPOSE Bevacizumab and erlotinib have been shown to improve survival in stage IV non-small-cell lung cancer (NSCLC). This phase I/II trial was designed to incorporate these agents with induction and concurrent chemoradiotherapy in stage III NSCLC. PATIENTS AND METHODS Patients received induction chemotherapy (carboplatin area under the curve [AUC] 6, paclitaxel 225 mg/m(2), and bevacizumab 15 mg/kg on days 1 and 22) followed by concurrent chemotherapy (carboplatin AUC 2 and paclitaxel 45 mg/m(2) weekly with bevacizumab 10 mg/kg every other week for four doses) and thoracic conformal radiation therapy (TCRT) to 74 Gy. In the phase I portion, cohort 1 received no erlotinib, whereas cohorts 2 and 3 received erlotinib at 100 and 150 mg, respectively, Tuesday through Friday, during TCRT. Consolidation therapy with erlotinib (150 mg daily) and bevacizumab (15 mg/kg every 3 weeks) was planned 3 to 6 weeks later for six cycles. RESULTS Forty-five eligible patients were enrolled. The objective response rates to induction and overall treatment were 39% (95% CI, 24% to 55%) and 60% (95% CI, 44% to 75%), respectively. The median progression-free and overall survival times were 10.2 months (95% CI, 8.4 to 18.3 months) and 18.4 months (95% CI, 13.4 to 31.7 months), respectively. The principal toxicity was esophagitis (29% grade 3 or 4 esophagitis, with one patient with grade 3 tracheoesophageal fistula), which was often prolonged. Consolidation therapy with bevacizumab and erlotinib was not feasible. CONCLUSION The use of bevacizumab and erlotinib as administered in this trial is not recommended given the lack of an efficacy signal and the substantial risk of esophageal toxicity.

Radical prostatectomy vs. intensity-modulated radiation therapy in the management of localized prostate adenocarcinoma

BACKGROUND AND PURPOSE To determine whether radical prostatectomy (RP) or intensity-modulated radiation therapy (IMRT) to > or =72 Gy, plus hormonal therapy if indicated, results in improved biochemical disease-free survival (BDFS) in localized prostate adenocarcinoma. MATERIALS AND METHODS Between 1997 and 2005, a consecutive sample of 556 patients who underwent RP (n=204) or IMRT (n=352) at two referral centers was analyzed. The patients were stratified into prognostic groups based on clinical stage, Gleason score, and pretreatment prostate-specific antigen (PSA). The outcome measure was BDFS. RESULTS IMRT patients had more advanced disease at baseline (p<.001). There was no difference in five-year BDFS rates between RP and IMRT in the favorable (92.8% vs. 85.3%, p=.20) or intermediate prognosis (86.7% vs. 82.2%, p=.46) subsets. A difference favoring IMRT plus hormonal therapy was seen in the poor prognosis (38.4% vs. 62.2%, p<.001) subset. Within the entire cohort, after adjustment for confounding variables, Gleason score (p<.001) and clinical stage (p<.001) predicted BDFS, but treatment modality (p=.06) did not. Within the poor prognosis subset, treatment modality (p=.006) predicted BDFS. CONCLUSIONS BDFS is similar between RP and IMRT for patients with a favorable or intermediate prognosis. Patients with a poor prognosis display higher BDFS when treated with IMRT to > or =72 Gy plus hormonal therapy.

Surveillance, Epidemiology, and End Results (SEER) database analysis of microcystic adnexal carcinoma (sclerosing sweat duct carcinoma) of the skin.

Background:Microcystic adnexal carcinoma (MAC) is a very rare cancer of the skin. It has only been described previously in case reports and small retrospective series. Objective:To analyze and summarize data from the National Cancer Institute, Surveillance, Epidemiology, and End Results (SEER) database regarding MAC. Methods:The SEER 1973 to 2004 database was investigated, and patients with MAC were identified. A statistical analysis was performed. Results:Two hundred twenty-three patients were identified. Predominant site of disease was the head and neck skin (74%). There was only 1 case of recorded metastatic disease. Lymph nodes were pathologically involved in 1%. The 10-year overall survival was 86.4% (Standard Error [SE]: 3.3%). US census population-matched relative survival was 97.7% at 10 years (SE: 5.2%). Limitations:This study is limited by the retrospective nature of the SEER database. Conclusions:MAC is locally invasive, and rarely metastasizes to lymph nodes. Overall and population-matched relative survival is excellent.

Inhibitors of MEK1/2 Interact with UCN-01 to Induce Apoptosis and Reduce Colony Formation in Mammary and Prostate Carcinoma Cells

Recent studies have suggested that inhibition of the mitogen activated protein kinase (MAPK) pathway as well as abrogation of cell cycle check-point control can potentiate the lethal actions of chemotherapeutic drugs and radiation. We therefore investigated the impact of combined exposure to the check-point abrogator (UCN-01) in conjunction with MEK1/2 inhibitors upon survival of breast and prostate carcinoma cells. Treatment of cells with UCN-01 alone resulted in prolonged activation of the MAPK pathway. Inhibition of MEK1/2 caused modest reductions in basal MAPK activity and transiently suppressed UCN-01-stimulated MAPK activity below that of MEK1/2 inhibitor alone. Significantly, combined, but not individual, exposure of cells to UCN-01 and MEK1/2 inhibitors enhanced BAX association with mitochondria and triggered release of cytochrome c into the cytosol, accompanied by activation of effector pro-caspases, resulting in a greater than additive potentiation of apoptosis within 18-24h. Radiation exposure of drug treated cells did not further enhance apoptosis. Treatment of cells with both caspase 9 and caspase 8 inhibitors was required to completely inhibit apoptosis in carcinoma cells. Over- expression of Bcl-xL blocked cytochrome c release and cell killing induced by the drug combination. Colony forming assays demonstrated that cells exposed to both agents exhibited a substantial reduction in clonogenic survival compared to either drug alone; moreover, radiation further reduced clonogenic survival despite failing to promote additional apoptosis. Collectively, these data demonstrate that combined exposure of carcinoma cells to UCN-01 and MEK1/2 inhibitors induces apoptosis and interacts with radiation to further reduce clonogenic survival.

A Clinical Model for Identifying Radiosensitive Tumor Genotypes in Non-Small Cell Lung Cancer

Purpose: Non–small cell lung cancer (NSCLC) includes a spectrum of radiosensitive and radioresistant tumors. However, little is known about the molecular determinants of cellular radiation responses. We examined clinical outcomes after gamma knife radiotherapy for NSCLC intracranial metastases to evaluate the use of this model for determining radiosensitive tumor genotypes. Experimental Design: Between 2005 and 2012, 239 patients with NSCLC were enrolled in a prospective gamma knife data repository. Molecular pathology regarding EGF receptor (EGFR), ALK, and KRAS mutation status was available for 81 patients. Local and distant brain control was determined for 79 patients with 469 brain metastases. Modified Cox proportional hazards models were established to evaluate local control for treated lesions after serial gamma knife treatments. Results: In total, 11% of patients developed in-field recurrence. No patients with metastases from tumors with EGFR mutations (0/164 lesions) or EML4-ALK translocations (0/61 lesions) recurred in-field. In contrast, 19% of patients without these mutations and 18% of patients with KRAS mutations recurred in-field (10/139 and 3/105 lesions, respectively). Rates of distant brain recurrence did not significantly differ across tumor genotypes. The predicted median in-field local control was significantly longer for EGFR-mutant and ALK-translocated tumors compared with other patients with NSCLC (P < 0.001), whereas distant brain recurrence time was equivalent (P = 0.97). On multivariate analysis, EGFR mutation, ALK translocation, and metastasis size were independent predictors for superior local control after gamma knife treatment. Conclusions: This study suggests that EGFR kinase domain mutations and EML4-ALK translocations are radiosensitive NSCLC genotypes, and proposes a novel model to identify radiosensitive subtypes of NSCLC. Clin Cancer Res; 19(19); 5523–32. ©2013 AACR.