Aaron S. Mansfield

Detection and localization of surgically resectable cancers with a multi-analyte blood test

SEEK and you may find cancer earlier Many cancers can be cured by surgery and/or systemic therapies when detected before they have metastasized. This clinical reality, coupled with the growing appreciation that cancers rapid genetic evolution limits its response to drugs, have fueled interest in methodologies for earlier detection of the disease. Cohen et al. developed a noninvasive blood test, called CancerSEEK that can detect eight common human cancer types (see the Perspective by Kalinich and Haber). The test assesses eight circulating protein biomarkers and tumor-specific mutations in circulating DNA. In a study of 1000 patients previously diagnosed with cancer and 850 healthy control individuals, CancerSEEK detected cancer with a sensitivity of 69 to 98% (depending on cancer type) and 99% specificity. Science, this issue p. 926; see also p. 866 A blood test that combines protein and DNA markers may allow earlier detection of eight common cancer types. Earlier detection is key to reducing cancer deaths. Here, we describe a blood test that can detect eight common cancer types through assessment of the levels of circulating proteins and mutations in cell-free DNA. We applied this test, called CancerSEEK, to 1005 patients with nonmetastatic, clinically detected cancers of the ovary, liver, stomach, pancreas, esophagus, colorectum, lung, or breast. CancerSEEK tests were positive in a median of 70% of the eight cancer types. The sensitivities ranged from 69 to 98% for the detection of five cancer types (ovary, liver, stomach, pancreas, and esophagus) for which there are no screening tests available for average-risk individuals. The specificity of CancerSEEK was greater than 99%: only 7 of 812 healthy controls scored positive. In addition, CancerSEEK localized the cancer to a small number of anatomic sites in a median of 83% of the patients.

PD-1 Restrains Radiotherapy-Induced Abscopal Effect

Park, Dong, and colleagues show in mouse models of melanoma and renal cell carcinoma that stereotactic ablative radiotherapy synergized with PD-1 blockade to induce near-complete regression of the irradiated tumors, and a tumor-specific 66% reduction in the nonirradiated tumors outside the radiation field. We investigated the influence of PD-1 expression on the systemic antitumor response (abscopal effect) induced by stereotactic ablative radiotherapy (SABR) in preclinical melanoma and renal cell carcinoma models. We compared the SABR-induced antitumor response in PD-1–expressing wild-type (WT) and PD-1–deficient knockout (KO) mice and found that PD-1 expression compromises the survival of tumor-bearing mice treated with SABR. None of the PD-1 WT mice survived beyond 25 days, whereas 20% of the PD-1 KO mice survived beyond 40 days. Similarly, PD-1–blocking antibody in WT mice was able to recapitulate SABR-induced antitumor responses observed in PD-1 KO mice and led to increased survival. The combination of SABR plus PD-1 blockade induced near complete regression of the irradiated primary tumor (synergistic effect), as opposed to SABR alone or SABR plus control antibody. The combination of SABR plus PD-1 blockade therapy elicited a 66% reduction in size of nonirradiated, secondary tumors outside the SABR radiation field (abscopal effect). The observed abscopal effect was tumor specific and was not dependent on tumor histology or host genetic background. The CD11ahigh CD8+ T-cell phenotype identifies a tumor-reactive population, which was associated in frequency and function with a SABR-induced antitumor immune response in PD-1 KO mice. We conclude that SABR induces an abscopal tumor-specific immune response in both the irradiated and nonirradiated tumors, which is potentiated by PD-1 blockade. The combination of SABR and PD-1 blockade has the potential to translate into a potent immunotherapy strategy in the management of patients with metastatic cancer. Cancer Immunol Res; 3(6); 610–9. ©2015 AACR.

B7-H1 Expression in Malignant Pleural Mesothelioma is Associated with Sarcomatoid Histology and Poor Prognosis

Introduction: B7 homolog 1 (B7-H1; aka programmed cell death 1 ligand 1) is a negative costimulatory molecule that is associated with poor prognosis in many tumor types. Given the poor prognosis and the limited treatments available for mesothelioma, we decided to examine B7-H1 expression and its association with survival in patients with mesothelioma. Methods: Expression of B7-H1 was determined in 106 patients using a mouse monoclonal antihuman B7-H1 (clone 5H1-A3) antibody with immunohistochemistry. Positive expression was defined as ≥5% positively stained cells. Clinicopathologic features and survival were compared between B7-H1-positive and B7-H1-negative groups. Results: Malignant mesotheliomas of 42 patients (40%) expressed B7-H1. Patients with B7-H1-postive tumors were less likely to be offered or undergo therapeutic surgery (p = 0.03). All sarcomatoid mesotheliomas except one desmoplastic subtype expressed B7-H1. Survival was significantly decreased for patients whose tumors expressed B7-H1 (5 months median, 2–9.5 months interquartile range) compared with those whose tumors did not (14.5 months, 9.25–19 months; p < 0.0001). In a multivariate model, B7-H1 expression and sarcomatoid mesothelioma remained significantly associated with worse survival (risk ratio 1.71, 95% confidence interval 1.03–2.78 [p = 0.04] and risk ratio 2.18, 1.08–4.23 [p = 0.03], respectively). Conclusions: B7-H1 is expressed in a substantial proportion of malignant pleural mesotheliomas and is associated with poor survival. Almost all malignant pleural mesotheliomas with sarcomatoid differentiation expressed B7-H1. The expression of B7-H1 may have important therapeutic implications for the management of malignant pleural mesothelioma.

Temporal and spatial discordance of programmed cell death-ligand 1 expression and lymphocyte tumor infiltration between paired primary lesions and brain metastases in lung cancer

The tumor microenvironments of paired primary lung cancers and brain metastases are significantly different, such that many of the metastases lose PD-L1 expression, lymphocyte infiltration or both with greater discrepancies over time. The spatial and temporal heterogeneity of PD-L1 expression may limit its use as a tissue-based predictive biomarker in lung cancer.

Heterogeneity of Programmed Cell Death Ligand 1 Expression in Multifocal Lung Cancer

Purpose: The expression of programmed cell death ligand 1 (PD-L1) provides limited predictive value in identifying patients most likely to respond to immunotherapy. As the heterogeneity of PD-L1 expression may lead to sampling error and the misclassification of PD-L1 status, we assessed the distribution of PD-L1 expression in paired, resected multifocal lung cancers. Experimental Design: PD-L1 was assessed by IHC. Paired lesions were defined as independent primaries or related lesions using mate pair next-generation sequencing. Agreement statistics were used for analysis. Results: Sixty-seven multifocal lung cancers from 32 patients were sequenced and stained for PD-L1. There was agreement of PD-L1 expression by the tumor cells in paired lesions of 20 patients and disagreement of PD-L1 expression by the tumor cells in paired lesions of 12 patients (κ = 0.01). Sequencing identified that 23 patients had independent primary lung cancers and that 9 patients had related cancers. In paired lesions of patients with independent cancers, there was agreement of PD-L1 expression by the tumor cells in 12 patients and disagreement in 11 patients (κ = 0.31). In paired lesions of patients with related lung cancers, there was agreement of PD-L1 expression by the tumor cells in 8 patients and disagreement in 1 patient (κ = 0.73). Conclusions: The expression of PD-L1 is heterogeneous among paired independent lung cancers, but there are high levels of agreement in intrapulmonary metastasis. Clin Cancer Res; 22(9); 2177–82. ©2015 AACR.

Regional immunity in melanoma: immunosuppressive changes precede nodal metastasis

In order to characterize the degree of immunosuppression in regional immunity in patients with melanoma, we used immunohistochemistry to analyze markers of T-cell subtype and polarity, costimulation, dendritic cell maturation, monocytes, lymphatic vasculature, and angiogenesis. Specifically, we analyzed expression of CD4, CD8, CD14, CD40, CD86, CD123, HLA-DR, IL-10, LYVE, VEGFR3, and VEGF-C in lymph nodes. We compared sentinel lymph nodes with and without metastasis from patients with melanoma with both infection inflamed (reactive) and dormant human lymph nodes. There were no differences demonstrated between sentinel lymph nodes with or without metastasis from patients with melanoma in any of the markers that were tested. Both groups of sentinel lymph nodes had fewer CD8+ T cells than either set of control nodes. Whereas the infection inflamed lymph nodes demonstrated Th2 polarity, the dormant lymph nodes demonstrated Th1 polarity. In conclusion, changes in regional immunity appeared to precede metastasis in melanoma. Whether there was tumor present in sentinel lymph nodes or not, these nodes demonstrated a marked decrease in cytotoxic T cells compared with both sets of controls. Furthermore, the control lymph nodes used for comparison can significantly impact interpretation, as the dormant and reactive lymph nodes markedly varied in their immune profiles. These immunologic changes may explain the successful metastasis of melanoma in the midst of the immune environment of the sentinel lymph node, and lend insights into the mechanisms of lymphatic metastases in other solid malignancies.

Immune cell quantitation in normal breast tissue lobules with and without lobulitis

While the immune microenvironment has been investigated in breast cancers, little is known about its role in non-malignant breast tissues. Here we quantify and localize cellular immune components in normal breast tissue lobules, with and without visible immune infiltrates (lobulitis). Up to ten representative lobules each in eleven normal breast tissue samples were assessed for B cells (CD20), cytotoxic T cells (CD8), helper T cells (CD4), dendritic cells (CD11c), leukocytes (CD45), and monocytes/macrophages (CD68). Using digital image analysis, immune cell densities were measured and compared between lobules with/without lobulitis. 109 lobules in 11 normal breast tissue samples were evaluated; 31 with lobulitis and 78 without. Immune cells showed consistent patterns in all normal samples, predominantly localized to lobules rather than stroma. Regardless of lobulitis status, most lobules demonstrated CD8+, CD11c+, CD45+, and CD68+ cells, with lower densities of CD4+ and CD20+ cells. Both CD11c+ and CD8+ cells were consistently and intimately associated with the basal aspect of lobule epithelium. Significantly higher densities of CD4+, CD8+, CD20+, and CD45+ cells were observed in lobules with lobulitis. In contrast, densities of monocytes/macrophages and dendritic cells did not vary with lobulitis. In normal breast tissue, myeloid and lymphoid cells are present and localized to lobules, with cytotoxic T and dendritic cells directly integrated with epithelium. Lobules with lobulitis have significantly more adaptive immune (B and T) cells, but no increase in dendritic cells or monocytes/macrophages. These findings indicate an active and dynamic mucosal immune system in normal breast tissue.

The Role of Vascular Endothelial Growth Factor in the Pathogenesis, Diagnosis and Treatment of Malignant Pleural Effusion

Malignant pleural effusions (MPEs) are a significant source of cancer-related morbidity. Over 150,000 patients in the United States suffer from breathlessness and diminished quality of life due to MPE each year. Current management strategies are of mostly palliative value and focus on symptom control; they do not address the pathobiology of the effusion, nor do they improve survival. Further elucidation of the pathophysiological mechanisms, coupled with the development of novel treatments such as intrapleural chemotherapeutics targeting this process, has the potential to greatly improve the efficacy of our current management options. Vascular endothelial growth factor-A (VEGF-A) has been implicated as a critical cytokine in the formation of malignant pleural effusions. Elevated levels of VEGF produced by tumor cells, mesothelial cells, and infiltrating immune cells result in increased vascular permeability, cancer cell transmigration, and angiogenesis. Therefore antiangiogenic therapies such as Bevacizumab, a monoclonal antibody targeting VEGF-A, may have a potential role in the management of malignant pleural effusions. Herein we review the pathogenesis and potential treatment strategies of malignant pleural effusions, with a focus on angiogenesis and antiangiogenic therapeutics.

The immunomodulatory effects of bevacizumab on systemic immunity in patients with metastatic melanoma.

Metastatic melanoma is near-to-invariably a fatal disease. As novel therapeutic strategies against metastatic melanoma are urgently needed, we have tested a combinatorial regimen consisting of conventional chemotherapy coupled to bevacizumab, a monoclonal antibody that inhibit angiogenesis, demonstrating some clinical benefit. A preliminary assessment of one of our clinical trials points to a previously unrecognized immunomodulatory effect of bevacizumab. Herein, we evaluate the immunomodulatory effect of bevacizumab when administered together with conventional chemotherapy to patients with metastatic melanoma. To this aim, we measured the abundance of various lymphocyte subsets among peripheral blood mononuclear cells (PBMCs) as well as the circulating levels of 42 cytokines, chemokines and growth factors in patients with metastatic melanoma who received albumin-bound paclitaxel plus carboplatin, either as a standalone intervention (AC, 55 subjects) or combined with bevacizumab (ACB, 39 individuals), in the context of clinical trials N057e and N0775, respectively. Relative shifts in PBMC subsets and cytokine levels were calculated (relative to baseline levels) when patients underwent restaging evaluation after two cycles of therapy. The Mann–Whitney U test was used to compare responses between the groups. Bevacizumab failed to affect the TH1/TH2 cell ratio in this patient cohort. However, we observed a significant increase in CD8+ lymphocytes in patients who received ACB (+38%) but not in subjects treated with AC only (−10%) (p = 0.03). Moreover, circulating interleuikin-6 (IL-6) levels were reduced in patients treated with ACB (−42%) but not in individuals receiving AC only (28%) (p = 0.0018). Thus, the addition of bevacizumab to chemotherapy for the treatment of metastatic melanoma exerts immunomodulatory effects.

Phase I dose escalation study of the PKCι inhibitor aurothiomalate for advanced non-small-cell lung cancer, ovarian cancer, and pancreatic cancer.

Protein kinase C iota (PKC&igr;) is overexpressed in non-small-cell lung cancer, ovarian, and pancreatic cancers, where it plays a critical role in oncogenesis. The gold compound aurothiomalate (ATM) has been shown to inhibit PKC&igr; signaling and exerts potent antitumor activity in preclinical models. We sought to determine the maximum tolerated dose (MTD) of ATM. We conducted a phase I dose escalation trial of ATM in patients with non-small-cell lung cancer, ovarian or pancreatic cancer. Patients received ATM intramuscularly weekly for three cycles (cycle duration 4 weeks) at 25, 50, or 75 mg in a 3+3 design. The dose was not escalated for individual patients. Blood samples were analyzed for elemental gold levels. Patients were evaluated every 4 weeks for toxicity and every 8 weeks for response. Fifteen patients were enrolled in this study. Six patients were treated at 25 mg, seven at 50 mg, and two at 75 mg. There was one dose-limiting toxicity at 25 mg (hypokalemia), one at 50 mg (urinary tract infection), and none at 75 mg. There were three grade 3 hematologic toxicities. The recommended MTD of ATM is 50 mg. Patients received treatment for a median of two cycles (range 1–3). There appeared to be a dose-related accumulation of steady-state plasma concentrations of gold consistent with linear pharmacokinetics. In summary, this phase I study was successful in identifying ATM 50 mg intramuscularly weekly as the MTD. Future clinical investigations targeting PKC&igr; are currently in progress.