Penny Fang

Combining Radiation Therapy with Immune Checkpoint Blockade for Central Nervous System Malignancies

Malignancies of the central nervous system (CNS), particularly glioblastoma and brain metastases from a variety of disease sites, are difficult to treat despite advances in multimodality approaches consisting of surgery, chemotherapy, and radiation therapy (RT). Recent successes of immunotherapeutic strategies including immune checkpoint blockade (ICB) via anti-PD-1 and anti-CTLA-4 antibodies against aggressive cancers, such as melanoma, non-small cell lung cancer, and renal cell carcinoma, have presented an exciting opportunity to translate these strategies for CNS malignancies. Moreover, via both localized cytotoxicity and systemic proinflammatory effects, the role of RT in enhancing antitumor immune response and, therefore, promoting tumor control is being re-examined, with several preclinical and clinical studies demonstrating potential synergistic effect of RT with ICB in the treatment of primary and metastatic CNS tumors. In this review, we highlight the preclinical evidence supporting the immunomodulatory effect of RT and discuss the rationales for its combination with ICB to promote antitumor immune response. We then outline the current clinical experience of combining RT with ICB in the treatment of multiple primary and metastatic brain tumors. Finally, we review advances in characterizing and modifying tumor radioimmunotherapy responses using biomarkers and microRNA (miRNA) that may potentially be used to guide clinical decision-making in the near future.

Stereotactic radiosurgery of early melanoma brain metastases after initiation of anti-CTLA-4 treatment is associated with improved intracranial control

BACKGROUND Numerous studies suggest that radiation can boost antitumor immune response by stimulating release of tumor-specific antigens. However, the optimal timing between radiotherapy and immune checkpoint blockade to achieve potentially synergistic benefits is unclear. MATERIAL AND METHODS Multi-institutional retrospective analysis was conducted of ninety-nine metastatic melanoma patients from 2007 to 2014 treated with ipilimumab who later received stereotactic radiosurgery (SRS) for new brain metastases that developed after starting immunotherapy. All patients had complete blood count acquired before SRS. Primary outcomes were intracranial disease control and overall survival (OS). RESULTS The median follow-up time was 15.5months. In the MD Anderson cohort, patients who received SRS after 5.5months (n=20) of their last dose of ipilimumab had significantly worse intracranial control than patients who received SRS within 5.5months (n=51) (median 3.63 vs. 8.09months; hazard ratio [HR] 2.07, 95% confidence interval [CI] 1.03-4.16, p=0.041). OS was not different between the two arms. The improvement in intracranial control was confirmed in an independent validation cohort of 28 patients treated at Yale-New Haven Hospital. Circulating absolute lymphocyte count before SRS predicted for treatment response as those with baseline counts >1000/µL had reduced risk of intracranial recurrence compared with those with ≤1000/µL (HR 0.46, 95% CI 0.0.23-0.94, p=0.03). CONCLUSIONS In this multi-institutional study, patients who received SRS for new brain metastases within 5.5months after ipilimumab therapy had better intracranial disease control than those who received SRS later. Moreover, higher circulating lymphocyte count was associated with improved intracranial disease control.

Spatial interaction of tumor cells and regulatory T cells correlates with survival in non-small cell lung cancer

OBJECTIVES To determine the prognostic significance of spatial proximity of lung cancer cells and specific immune cells in the tumor microenvironment. MATERIALS AND METHODS We probed formalin-fixed, paraffin-embedded (FFPE) tissue microarrays using a novel tyramide signal amplification multiplexing technique labelling CD8, CD4, Foxp3, and CD68+ cells. Each multiplex stained immunohistochemistry slide was digitally processed by Vectra INFORMS software, and an X- and Y-coordinate assigned to each labeled cell type. The abundance and spatial location of each cell type and their proximity to one another was analyzed using a novel application of the G-cross spatial distance distribution method which computes the probability of finding at least one immune cell of any given type within a rμm radius of a tumor cell. Cox proportional hazards multiple regression was used for multivariate analysis of the influence of proximity of lymphocyte types. RESULTS Pathologic tumor specimens from 120 NSCLC patients with pathologic tumor stage I-III disease were analyzed. On univariate analysis, age (P = .0007) and number of positive nodes (P = .0014) were associated with overall survival. Greater area under the curve (AUC) of the G-cross function for tumor cell-Treg interactions was significantly associated with worse survival adjusting for age and number of positive nodes (HR 1.52 (1.11-2.07), P = .009). Greater G-cross AUC for T-reg-CD8 was significantly associated with better survival adjusting for age and number of positive lymph nodes (HR 0.96 (0.92-0.99), P = .042). CONCLUSION Increased infiltration of regulatory T cells into core tumor regions is an independent predictor of worse overall survival in NSCLC. However, increased infiltration of CD8+ cytotoxic T cells among regulatory T cells seems to mitigate this effect and was significantly associated with better survival. Validation of the G-cross method of measuring spatial proximity between tumor and immune cell types and exploration of its use as a prognostic factor in lung cancer treatment is warranted.

Severe lymphopenia during neoadjuvant chemoradiation for esophageal cancer: A propensity matched analysis of the relative risk of proton versus photon-based radiation therapy

BACKGROUND AND PURPOSE Circulating lymphocytes are exquisitely sensitive to radiation exposure, even to low scattered doses which can vary drastically between radiation modalities. We compared the relative risk of radiation-induced lymphopenia between intensity modulated radiation therapy (IMRT) or proton beam therapy (PBT) in esophageal cancer (EC) patients undergoing neoadjuvant chemoradiation therapy (nCRT). MATERIAL AND METHODS EC patients treated with IMRT and PBT were propensity matched based on key clinical variables. Treatment-associated lymphopenia was graded using CTCAE v.4.0. Using matched cohorts, univariate and multivariable multiple logistic regression was used to identify factors associated with increased risk of grade 4 lymphopenia as well as characterize their relative contributions. RESULTS Among the 480 patients treated with nCRT, 136 IMRT patients were propensity score matched with 136 PBT patients. In the matched groups, a greater proportion of the IMRT patients (55/136, 40.4%) developed grade 4 lymphopenia during nCRT compared with the PBT patients (24/136, 17.6%, P < 0.0001). On multivariable analysis, PBT was significantly associated with a reduction in grade 4 lymphopenia risk (odds ratio, 0.29; 95% confidence interval, 0.16-0.52; P < 0.0001). CONCLUSION PBT is associated with significant risk reduction in grade 4 lymphopenia during nCRT in esophageal cancer.

Prediction and diagnosis of interval metastasis after neoadjuvant chemoradiotherapy for oesophageal cancer using 18F-FDG PET/CT

ObjectiveDuring neoadjuvant chemoradiotherapy for oesophageal cancer, or in the interval prior to surgery, some patients develop systemic metastasis. This study aimed to evaluate the diagnostic performance of 18F-FDG PET/CT for the detection of interval metastasis and to identify predictors of interval metastases in a large cohort of oesophageal cancer patients.MethodsIn total, 783 consecutive patients with potentially resectable oesophageal cancer who underwent chemoradiotherapy and pre- and post-treatment 18F-FDG PET/CT between 2006 and 2015 were analyzed from a prospectively maintained database. Diagnostic accuracy measures were calculated on a per-patient basis using histological verification or clinical follow-up as a reference standard. Multivariable logistic regression analysis was performed to determine pre-treatment predictors of interval metastasis. A prediction score was developed to predict the probability of interval metastasis.ResultsOf 783 patients that underwent 18F-FDG PET/CT restaging, 65 (8.3%) were found to have interval metastasis and 44 (5.6%) were deemed to have false positive lesions. The resulting sensitivity and specificity was 74.7% (95% CI: 64.3–83.4%) and 93.7% (95% CI: 91.6–95.4%), respectively. Multivariable analysis revealed that tumor length, cN status, squamous cell tumor histology, and baseline SUVmax were associated with interval metastasis. Based on these criteria, a prediction score was developed with an optimism adjusted C-index of 0.67 that demonstrated accurate calibration.Conclusions18F-FDG PET/CT restaging detects distant interval metastases in 8.3% of patients after chemoradiotherapy for oesophageal cancer. The provided prediction score may stratify risk of developing interval metastasis, and could be used to prioritize additional restaging modalities for patients most likely to benefit.

Outcomes and toxicity following high-dose radiation therapy in 15 fractions for non-small cell lung cancer

PURPOSE Accelerated hypofractionated radiation therapy (AHRT) is increasingly used for select lung cancer patients. We evaluated clinical outcomes and predictors of pulmonary/esophageal toxicity in patients treated with ≥52.5 Gy in 15 fractions. METHODS AND MATERIALS We evaluated 229 patients treated with radiation therapy doses ≥52.5 Gy in 15 fractions for non-small cell lung cancer from January 2009 through January 2016. Toxicity was scored using Common Terminology Criteria for Adverse Events, v4.0. Univariate and multivariate logistic regression was used to identify predictors of toxicity. Overall survival, progression-free survival, and local control were estimated using the Kaplan-Meier method. Predictors of clinical outcome were modeled using Cox proportional hazards regression. RESULTS Median follow-up was 7 months. Forty-two patients (19%) developed grade ≥2 pneumonitis, and 9 (4%) developed grade ≥3 esophagitis. In multivariate analysis, age >75 years (odds ratio [OR], 2.56; 95% confidence interval [CI], 1.24-5.25; P = .01) and percentage of lung volume receiving doses of >10 Gy higher than 32% were associated with grade ≥2 pneumonitis (OR, 2.79; 95% CI, 1.39-5.79; P = .005). On univariate analysis, esophagus mean dose ≥17 Gy (OR, 10.14; 95% CI, 1.82-189.8; P = .006), gross tumor volume size ≥71 cm3 (P = .002), and planning target volume size ≥409 cm3 (P = .02) were associated with development of grade ≥3 esophagitis. In patients with stage II/III disease (n = 73), median local control was not reached, median overall survival was 14 months, and median progression-free survival was 6 months. CONCLUSIONS AHRT in 15 fractions can be safe and effective. Consideration for using AHRT with immunotherapy and sequential chemotherapy for improved out-of-radiation field and distant control is warranted.

Abstract 4990: Sequential tracking of PD-L1 expression and RAD50 induction in CTCs and circulating stromal cells of lung cancer patients during treatment with radiotherapy

Background: PD-L1 expression can be induced by radiotherapy (RT) and may be an immune escape mechanism after RT, and has the potential to predict for responsiveness to immune checkpoint blockade immunotherapy. However, repetitive biopsies for monitoring PD-L1 expression in tumor and/or stroma are not feasible. Sequential assessment of PD-L1 expression on cancer associated circulating cells during treatment may be a way to measure the efficacy of combining RT and immunotherapy. RAD50 is a DNA repair gene that can be used in tracking tumor cell response to radiation. We therefore evaluated PD-L1 expression and RAD50 induction in CTCs and Cancer Associated Macrophage-Like Cells (CAMLs) in lung cancer patients (pts) before and during RT to track expression changes of these markers. Methods: Twenty-nine pts with stage I-IV lung cancer were included in this prospective pilot study. Four pts received radiation therapy for stage I disease and 25 other pts received chemoradiation for stage II-IV disease. A baseline blood sample (7.5 ml) was drawn prior to the start of RT (T0), and a second blood sample was drawn at a treatment visit during RT (T1) for a total of 58 samples. Blood was processed using CellSieve™ microfiltration (Creatv MicroTech), stained for cytokeratin 8, 18 & 19 and CD45, and imaged. Using the QUASR (Quench, Underivatize, Amine-Strip and Restain) technique to remove fluoresce signal, all cells were restained for RAD50-AlexaFluor550 and PD-L1-AlexaFluor 488, along with DAPI nuclear stain. The RAD50 foci were quantified within nuclear regions and PD-L1 pixel intensity measured and grouped into 4 IHC groups: 0-negative (pixel average 0-215), 1-low (pixel average 216-300), 2-medium (pixel average 301-750), and 3-high (pixel average 751+). Results: There was at least one cytokeratin positive cell (i.e. CTC or CAMLs) found in all but 2 samples (97%). CTCs were found in 69% of T0 and 72% of T1 samples, and CAMLs in 79% of T0 and 97% of T1 samples. PD-L1 expression ranged from 34-2552 pixel intensity, with an average of 285 at T0 and 525 at T1 (p = 0.07). Interestingly, 9 pts had no PD-L1 expression at T0 but an increase to 2 to 3+ at T1, 12 pts with low/no PD-L1 expression remained low at T1, and 8 pts had high PD-L1 expression that remained high or decreased at T1. RAD50 foci ranged from 0-16 per cell, with an average of 0.56 at T0 that increased to 4.68 at T1 (p Conclusions PD-L1 expression and RAD 50 foci were quantifiable in both CTCs and CAMLs, and a correlative response to radiotherapy +/- chemotherapy was quantified. This data suggests that sequential tracking of CTCs and immune-related cells from the primary lung tumor is feasible using microfiltration and may potentially serve as a predictive biomarker for cancer therapy sensitivity. Citation Format: Daniel L. Adams, Martin J. Edelman, Penny Fang, Wen Jiang, Jianzhong He, Ting Xu, Hui Gao, James M. Reuben, Yawei Qiao, Steven Hahn, Ritsuko Komaki, Zhongxing Liao, Cha-Mei Tang, Steven H. Lin. Sequential tracking of PD-L1 expression and RAD50 induction in CTCs and circulating stromal cells of lung cancer patients during treatment with radiotherapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4990.

High lymphocyte count during neoadjuvant chemoradiotherapy is associated with improved pathologic complete response in esophageal cancer.

BACKGROUND AND PURPOSE Neoadjuvant chemoradiation (nCRT) can reduce tumor infiltrating lymphocytes. We examined absolute lymphocyte count (ALC) nadir during nCRT for esophageal cancer (EC) and pathologic complete response (pCR). MATERIALS AND METHODS Patients with stage I-IVA EC (n = 313) treated 2007-2013 with nCRT followed by surgery were analyzed. ALC was obtained before, during/weekly, and one month after CRT. pCR was defined as no viable tumor cells at surgery. High ALC was defined as nadir of ≥0.35 × 103/μL (highest tertile). Comparison of continuous and categorical variables by pCR was assessed by ANOVA and Pearsons chi-square. Univariate/multivariate logistic regression was used to assess predictors of pCR and high ALC nadir. RESULTS Eighty-nine patients (27.8%) achieved a complete pathological response (pCR). For patients with pCR, median ALC nadir was significantly higher than those without (0.35 × 103/μL vs 0.29 × 103/μL, p = 0.007). Patients maintaining high ALC nadir had a higher pCR rate (OR1.82, 95%CI 1.08-3.05, p = 0.024). Predictors of high ALC included treatment with proton therapy vs. IMRT (OR4.18, 95%CI 2.34-7.47, p < 0.001), smoking at diagnosis (OR2.80, 95%CI 1.49-5.25, p = 0.001), early stage I-II disease (OR2.33, 95%CI 1.32-4.17, p = 0.005), and SCC histology (OR3.70, 95%CI 1.01-14.29, p = 0.048). Mean body dose (MBD) was inversely related to high ALC nadir (OR0.77 per Gy, 95%CI 0.70-0.84, p < 0.001). CONCLUSION A higher ALC level during nCRT is associated with a higher rate of pCR for esophageal cancer patients undergoing trimodality therapy.

Sequential versus concurrent chemoradiation therapy by surgical margin status in resected non-small cell lung cancer

Background: Postoperative chemoradiotherapy (CRT) for non-small cell lung cancer (NSCLC) can be delivered sequentially (sCRT) or concurrently (cCRT). Without high-volume data, current guidelines recommend either option for patients with negative margins (M-) and cCRT for those with positive margins (M+). In this study, survival was compared between sCRT versus cCRT for M- and M+ disease; survival in patients who underwent sCRT was also assessed with chemotherapy-first versus radiotherapy (RT)-first. Methods: The National Cancer Database was queried for patients with primary NSCLC undergoing surgery followed by CRT. Patients were excluded if they received neoadjuvant chemotherapy or RT. Both M- and M+ (including R1 and R2) subcohorts were evaluated. Multivariable logistic regression ascertained factors associated with cCRT delivery. Kaplan-Meier analysis evaluated overall survival (OS); Cox proportional hazards modeling determined variables associated with OS. Propensity score matching aimed to address group imbalances and indication biases. Results: Of 4,921 total patients, 3,475 (71%) were M-, 1,446 (29%) were M+, 2,271 (46%) received sCRT, and 2,650 (54%) underwent cCRT. Median OS among the sCRT and cCRT groups in patients who were M- was 54.6 versus 39.5 months, respectively (P<.001); differences persisted following propensity score matching (P<.001). In the overall M+ cohort, outcomes for sCRT and cCRT were 36.3 versus 30.5 months (P=.011), but showed equipoise following matching (P=.745). In the R1 and R2 subsets, no differences in OS were seen between cohorts (P=.368 and .553, respectively). When evaluating the sCRT population, there were no OS differences between chemotherapy-first and RT-first after matching (P=.229). Conclusions: Postoperative sCRT was associated with improved survival compared with cCRT in patients with M- disease, with statistical equipoise in those with M+ disease. Differential sequencing of sCRT does not appear to affect survival.

Melanoma brain metastases harboring BRAF V600K or NRAS mutations are associated with an increased local failure rate following conventional therapy

Studies on melanoma brain metastases (MBM) with regard to mutational status are lacking. We investigated the outcomes of MBM in molecularly characterized patients for BRAF and NRAS mutations receiving conventional treatment. We investigated associations between outcomes [competing risk of local and distant brain failure (LF, DF) and overall survival (OS)] and clinical/pathological features of patients with known mutation status following initial treatment of MBM. Competing risk analysis was performed using the methods of Fine and Gray. We identified 235 patients with MBM diagnosed from 2005 to 2011. Mutation prevalence was BRAFnon-V600K 98 (42%), BRAFV600K 34 (14%), NRAS 43 (18%), and wild-type for both genes (WT) 60 (26%) patients. Six month cumulative incidence LF rates were 3% for combined SRS or surgery with adjuvant radiation, 18% for surgery, 18% for SRS, 60% for WBRT, and 67% for systemic therapy. On multivariate analysis, only mutation status and initial treatment type were found to be independent predictors of local control. As compared to WT, NRAS (HR 2.58, 95% CI 1.18–5.67, p = 0.02) and BRAFV600K (HR 2.83, 95% CI 1.23–6.47, p = 0.01) mutational status were statistically significant while BRAFnon-V600K status was not statistically significant (p = 0.23). Mutation status was not associated with DF or OS. BRAFV600K and NRAS mutation status predict increased LF following conventional treatments for MBM. These data can inform the design and interpretation of future MBM trials.