Outcome and Immune Correlates of a Phase II Trial of High-Dose Interleukin-2 and Stereotactic Ablative Radiotherapy for Metastatic Renal Cell Carcinoma.

PURPOSE: This phase II clinical trial evaluated whether the addition of stereotactic ablative radiotherapy (SAbR), which may promote tumor antigen presentation, improves the overall response rate (ORR) to high-dose IL2 (HD IL2) in metastatic renal cell carcinoma (mRCC). PATIENTS AND METHODS: Patients with pathologic evidence of clear cell renal cell carcinoma (RCC) and radiographic evidence of metastasis were enrolled in this single-arm trial and were treated with SAbR, followed by HD IL2. ORR was assessed based on nonirradiated metastases. Secondary endpoints included overall survival (OS), progression-free survival (PFS), toxicity, and treatment-related tumor-specific immune response. Correlative studies involved whole-exome and transcriptome sequencing, T-cell receptor sequencing, cytokine analysis, and mass cytometry on patient samples. RESULTS: Thirty ethnically diverse mRCC patients were enrolled. A median of two metastases were treated with SAbR. Among 25 patients evaluable by RECIST v1.1, ORR was 16% with 8% complete responses. Median OS was 37 months. Treatment-related adverse events (AE) included 22 grade ≥3 events that were not dissimilar from HD IL2 alone. There were no grade 5 AEs. A correlation was observed between SAbR to lung metastases and improved PFS (P = 0.0165). Clinical benefit correlated with frameshift mutational load, mast cell tumor infiltration, decreased circulating tumor-associated T-cell clones, and T-cell clonal expansion. Higher regulatory/CD8+ T-cell ratios at baseline in the tumor and periphery correlated with no clinical benefit. CONCLUSIONS: Adding SAbR did not improve the response rate to HD IL2 in patients with mRCC in this study. Tissue analyses suggest a possible correlation between frameshift mutation load as well as tumor immune infiltrates and clinical outcomes.

Rationale and evidence to combine radiation therapy and immunotherapy for cancer treatment.

Cancer immunotherapy exploits the immune system's ability to differentiate between tumor target cells and host cells. Except for limited success against a few tumor types, most immunotherapies have not achieved the desired clinical efficacy until recently. The field of cancer immunotherapy has flourished with a variety of new agents for clinical use, and remarkable progress has been made in the design of effective immunotherapeutic regimens. Furthermore, the therapeutic outcome of these novel agents is enhanced when combined with conventional cancer treatment modalities including radiotherapy (RT). An increasing number of studies have demonstrated the abscopal effect, an immunologic response occurring in cancer sites distant from irradiated areas. The present work reviews studies on the combination between RT and immunotherapy to induce synergistic and abscopal effects involved in cancer immunomodulation. Further insight into the complex interactions between the immune system and cancer cells in the tumor microenvironment, and their modulation by RT, may reveal the abscopal effect as a clinically relevant and reproducible event leading to improved cancer outcome.

Initial experience with oropharynx-targeted radiation therapy for metastatic squamous cell carcinoma of unknown primary of the head and neck.

AIM: Metastasis of unknown primary (MUP) is commonly treated with radiation therapy (RT) to the entire mucosal surfaces and bilateral neck nodes (LN). We report outcomes of oropharynx-targeted RT, retropharyngeal nodes (RPN) and bilateral LN in this context. PATIENTS AND METHODS: Single-Institution retrospective study of 68 patients. Forty percent were treated with intensity-modulated radiation therapy (IMRT). Fifty-six percent received concurrent chemoradiotherapy (CCRT). The median age was 58 years, 82% were Caucasian, and 75% males. Stage III disease was present in 9%, stage IVA in 75% and IVB in 16%. RESULTS: At a median follow-up of 3.5 years, the actuarial locoregional control was 95.5%. The emergence of primary developed in 1patient (1.5%) and 2patients (3%) failed in the neck. The median time-to-locoregional failure (LRF) was 18 months. Actuarial long-term RT toxicity was grade 1 xerostomia (68%), dysphagia (35%), neck stiffness (15%) and trismus (6%). CONCLUSION: RT to the oropharynx, RPN, and bilateral neck provides excellent oncological and functional outcomes in MUP in non-Asian patients. Sparing the mucosal surfaces of the nasopharynx, hypopharynx, and larynx seems reasonable without impacting on survival and locoregional control.

Long-term outcome of seropositive HIV patients with head and neck squamous cell carcinoma treated with radiation therapy and chemotherapy.

AIM: To report the outcome of radiation therapy (RT) +/- chemotherapy in HIV-seropositive patients with Head and Neck Squamous Cell Carcinoma (HNSCC). PATIENTS AND METHODS: This is the largest single-Institution retrospective study to date, consisting of 73 HIV patients with HNSCC treated from January 1997-2010. The median age at RT, HIV diagnosis and the duration of patients being HIV seropositive were 51, 34, and 11 years, respectively. Seventy patients had SCC and one had submandibular salivary duct carcinoma. Stages I-II, III and IVA/B were: 22%, 27% and 51%, respectively. Primary cancer sites comprised the larynx (37%), oropharynx (32%), oral cavity (13%), hypopharynx (7%), nasopharynx (4%), unknown primary (MUP) (4%), nasal cavity (3%), and submandibular salivary duct (1%). All patients had an ECOG performance scale of ≤1 and were treated with RT +/- chemotherapy. Fifty patients (70%) were on highly active anti-retroviral therapy (HAART) during treatment, and the median CD4 count was 290 (range: 203-1142). Median dose of 70, 63, and 54 Gy were delivered to the gross disease, high-risk neck, and low-risk neck respectively. Median duration of treatment was 52 (range: 49-64) days. Twelve patients (17%) underwent neck dissection for N3 disease. RESULTS: After a median follow-up of 47 months (range: 7-140), the 4-year locoregional control (LRC) and overall survival (OS) were 69% and 55% respectively. Seven patients (10%) developed second primary sites within the first 5 years of completing RT (2 anal SCCs and 5 HNSCCs). The LRC for Stages III/IV larynx and oropharynx SCC (which represent the majority of the cohort) were 76% and 70%, respectively. Chemo/RT-related late toxicities were dysphagia of grade≤2, 3, and 4 found in 74%, 15% and 11% of patients, respectively. Hoarseness (grade 1) was reported in 10% of patients; no patient experienced grade ≥2. Xerostomia grade ≤2, and 3 was found in 77% and 23% of patients, respectively. A Chi-square test and univariate analysis showed statistically significant relationships between LRC and duration of RT (p<0.001), as well as positive trends for weight loss (<10%) and absence of second malignancy. CONCLUSION: Definitive RT +/- chemotherapy for HIV-seropositive patients with HNSCC appears to be less effective compared to the observed rates of LRC and OS of other HNSCC without HIV. Due to advances in the HAART which prolongs HIV patients' survival, it is extremely important to establish better treatment strategies to improve therapeutic ratio in this growing patient population.

Wiskott-Aldrich syndrome protein regulates leukocyte-dependent breast cancer metastasis.

A paracrine interaction between epidermal growth factor (EGF)-secreting tumor-associated macrophages (TAMs) and colony-stimulating factor 1 (CSF-1)-secreting breast carcinoma cells promotes invasion and metastasis. Here, we show that mice deficient in the hematopoietic-cell-specific Wiskott-Aldrich syndrome protein (WASp) are unable to support TAM-dependent carcinoma cell invasion and metastasis in both orthotopic and transgenic models of mammary tumorigenesis. Motility and invasion defects of tumor cells were recapitulated ex vivo upon coculture with WASp(-/-) macrophages. Mechanistically, WASp is required for macrophages to migrate toward CSF-1-producing carcinoma cells, as well as for the release of EGF through metalloprotease-dependent shedding of EGF from the cell surface of macrophages. Our findings suggest that WASp acts to support both the migration of TAMs and the production of EGF, which in concert promote breast tumor metastasis.

Tolerance and toxicity of primary radiation therapy in the management of seropositive HIV patients with squamous cell carcinoma of the head and neck.

OBJECTIVES/HYPOTHESIS: To report tolerance and toxicity of radiotherapy (RT) with or without chemotherapy in HIV seropositive patients with squamous cell carcinoma of the head and neck (SCCHN). METHODS: This is a single institution retrospective study of 73 HIV seropositive patients with SCCHN treated from January 1997 through 2010. Stages I, II, III, and IV were 8%, 10%, 24%, and 58%, respectively. The median age at RT, HIV diagnosis. and the duration of HIV seropositive were 51 (32-72), 34 (25-50), and 11 (6-20) years, respectively. Patients were treated definitively with RT alone (35%) or concurrent chemo-RT (65%). Median dose of 70 Gy (66-70) was delivered to the gross disease. Median duration of treatment was 52 (49-64) days. Fifty patients (70%) were on HAART. RESULTS: RT± chemotherapy induced acute toxicity was: median weight loss 20 pounds (6-40), 100% developed dysgeusia and xerostomia (grades 1-3). Acute mucositis and dysphagia/odynophagia grades ≤ 2 and 3 were 83% and 17%, respectively. Treatment breaks in excess of 10, 7, and 3 days were found in 5%, 13%, and 15% of patients, respectively. With a median follow-up of 4 years (2-12) the RT ±chemotherapy induced late dysphagia and xerostomia grades >2 were 26% and 23% of patients, respectively. CONCLUSION: Our data show that primary RT ±chemotherapy for HIV seropositive SCCHN is less tolerated compared to the historical data for SCCHN without HIV. LEVEL OF EVIDENCE: 2b.

The impact of computed tomography on early glottic cancer outcomes.

PURPOSE: Aim of this study was to evaluate the impact of computed tomography (CT)-based simulation and planning on early glottic cancer outcomes and toxicity. METHODS: This is a single-institution retrospective study of 253 patients with T1-2 glottic cancer who underwent radiation therapy (RT) from January 1998-2010. Group A (80%) underwent 2-dimensional RT (2DRT) and group B (20%) 3-dimensional RT (3DRT). 76% of patients in group A and 84% in group B had T1 cancer. The median dose and fraction size were 63 Gy and 2.25 Gy, respectively. RESULTS: With a median follow-up of 83, 93, and 30 months for the whole cohort, group A and B, respectively, the loco-regional control (LRC) was 97.6%. The rate of LRC for T1 disease was 99.5% and for T2 disease 91%. According to the RT modality, rates of LRC were 99.4 and 100% in groups A and B for T1, and 89.8 and 100% for T2. Long-term toxicity was negligible in both groups. Kaplan-Meier Curve showed the 5-year cause-specific survival to be 100%. Chi-square and multivariate analysis tests showed a significant relationship between CT simulation (3DRT) and LRC (p < 0.0001). CONCLUSION: CT-based simulation and planning provided better LRC and less acute side effects compared to 2DRT.

The chemotactic defect in wiskott-Aldrich syndrome macrophages is due to the reduced persistence of directional protrusions.

Wiskott-Aldrich syndrome protein (WASp) is an actin nucleation promoting factor that is required for macrophages to directionally migrate towards various chemoattractants. The chemotaxis defect of WASp-deficient cells and its activation by Cdc42 in vivo suggest that WASp plays a role in directional sensing, however, its precise role in macrophage chemotaxis is still unclear. Using shRNA-mediated downregulation of WASp in the murine monocyte/macrophage cell line RAW/LR5 (shWASp), we found that WASp was responsible for the initial wave of actin polymerization in response to global stimulation with CSF-1, which in Dictyostelium discoideum amoebae and carcinoma cells has been correlated with the ability to migrate towards chemoattractants. Real-time monitoring of shWASp cells, as well as WASp⁻/⁻ bone marrow-derived macrophages (BMMs), in response to a CSF-1 gradient revealed that the protrusions from WASp-deficient cells were directional, showing intact directional sensing. However, the protrusions from WASp-deficient cells demonstrated reduced persistence compared to their respective control shRNA and wild-type cells. Further examination showed that tyrosine phosphorylation of WASp was required for both the first wave of actin polymerization following global CSF-1 stimulation and proper directional responses towards CSF-1. Importantly, the PI3K, Rac1 and WAVE2 proteins were incorporated normally in CSF-1 - elicited protrusions in the absence of WASp, suggesting that membrane protrusion driven by the WAVE2 complex signaling is intact. Collectively, these results suggest that WASp and its phosphorylation play critical roles in coordinating the actin cytoskeleton rearrangements necessary for the persistence of protrusions required for directional migration of macrophages towards CSF-1.

Regulation of tyrosine phosphorylation in macrophage phagocytosis and chemotaxis.

Macrophages display a large variety of surface receptors that are critical for their normal cellular functions in host defense, including finding sites of infection (chemotaxis) and removing foreign particles (phagocytosis). However, inappropriate regulation of these processes can lead to human diseases. Many of these receptors utilize tyrosine phosphorylation cascades to initiate and terminate signals leading to cell migration and clearance of infection. Actin remodeling dominates these processes and many regulators have been identified. This review focuses on how tyrosine kinases and phosphatases regulate actin dynamics leading to macrophage chemotaxis and phagocytosis.

N-WASP has the ability to compensate for the loss of WASP in macrophage podosome formation and chemotaxis.

Wiskott-Aldrich syndrome protein (WASP) and its homologue neural-WASP (N-WASP) are nucleation promoting factors that integrate receptor signaling with actin cytoskeleton rearrangement. While hematopoietic cells express both WASP and N-WASP, WASP deficiency results in altered cell morphology, loss of podosomes and defective chemotaxis. It was determined that cells from a mouse derived monocyte/macrophage cell line and primary cells of myeloid lineage expressed approximately 15-fold higher levels of WASP relative to N-WASP. To test whether N-WASP can compensate for the loss of WASP and restore actin cytoskeleton integrity, N-WASP was overexpressed in macrophages, in which endogenous WASP expression was reduced by short hairpin RNA (shWASP cells). Many of the defects associated with the loss of WASP, such as podosome-dependent matrix degradation and chemotaxis were corrected when N-WASP was expressed at equimolar level to that of the wild-type WASP. Furthermore, the ability of N-WASP to partially compensate for the loss of WASP may be physiologically relevant since activated murine WASP-deficient peritoneal macrophages, which show enhanced N-WASP expression, also show an increase in matrix degradation. Our study suggests that expression levels of WASP and N-WASP may influence their roles in actin cytoskeleton rearrangement and shed light to the complex intertwining roles WASP and N-WASP play in macrophages.