Neoplasia-related and treatment-induced lymphopenia: impact on the outcome of chemoradiotherapy in laryngeal cancer.

INTRODUCTION: The role of the immune system in the efficacy of radiotherapy (RT) has been well established. We examined the role of neoplasia-related and treatment-induced lymphopenia in the outcome of RT or chemoradiotherapy (CRT) in squamous cell laryngeal cancer. MATERIALS AND METHODS: We retrospectively analyzed a series of 135 laryngeal carcinomas treated with radical or postoperative RT/CRT. Six lymphocyte-related variables were defined and examined: i. lymphocyte counts (LCs) before a brief course of induction chemotherapy, ii. pre-RT LCs, iii. post-RT LCs, iv. pre-RT neutrophil/lymphocyte ratio (N/L), v. pre-RT monocyte/lymphocyte ratio (M/L), and vi. pre-RT platelet/lymphocyte ratio (Pt/L). RESULTS: RT and CRT resulted in a significant decrease of LCs at the end of therapy, and this was significantly more prominent in patients treated with radical intent and neck irradiation (median LC nadir 810/µl vs. 1250/µl; p = .0003). Induction chemotherapy did not intensify the lymphotoxic effect of RT. LCs lower than the 33rd percentile before RT (<1718/µl) and after RT (<720/µl) were significantly linked to poor locoregional progression-free survival (LRFS; p = .02 and p = .08, respectively) and disease-specific overall survival (OS; p = .02 and p = .03, respectively). This was also confirmed multivariate analysis (LRFS: p = .006/HR = 2.41 and p = .08/HR = 1.76, respectively; OS: p = .001/HR = 3.06 and p = .02/HR = 2.07, respectively). High pre-RT N/L, M/L, and Pt/L ratios were also of ominous prognostic relevance. CONCLUSIONS: Both neoplasia-related and RT-induced lymphopenia define the outcome of RT in terms of locoregional failure, incidence of metastasis, and, finally, disease-specific survival of patients with laryngeal cancer. Restoration of pre-RT lymphopenia and protection of peripheral lymphocytes during RT emerge as critical issues that demand therapeutic interventions to maximize the efficacy of RT/CRT in patients with laryngeal cancer.

Cytokine Plasma Levels in Breast Cancer Patients, Before and After Surgery.

Studying the levels of cytokines in the plasma of patients could be valuable in guiding immunotherapy policies. We assessed the plasma levels of 4 major cytokines [interferon (IFN)-ß, interleukin-2 (IL-2), tumor necrosis factor alpha (TNF-α), transforming growth factor beta (TGF-ß)] collected from 19 patients with ductal breast cancer (BCa), before surgery (BS) and 5 days after surgery (AS). The ratio AS/BS was also calculated and correlated with histopathological variables and tumor-infiltrating lymphocyte (TIL) density. The IFN-ß and TNF-α levels were significantly higher in BCa patients, BS and AS, than healthy controls (P < 0.02). High IL-2 levels BS were linked with node involvement (P = 0.02), and marginally with HER2 expression (P = 0.08), while high TNF-α levels were linked with high PgR expression (P = 0.02). Increasing IFN-ß, IL-2, and TNF-α levels were noted AS, which was more evident in patients with larger tumors. The TGF-ß levels were significantly lower in BCa patients (P < 0.007). Linear regression analysis showed a direct association of IFN-ß levels AS (P = 0.02, r = 0.52) and of TNF-α AS/BS-ratio (P = 0.001, r = 0.72) with TIL-density. It is suggested that although effector immune response is evident in the majority of early stage BCa patients, removal of the primary tumor further unblocks such responses.

HIF1α-dependent and independent pathways regulate the expression of PD-L1 in prostate cancer.

PD-L1/PD-1 pathway is a major pathway exploited by human cancer types, which is a target for current immunotherapy. We investigated tumor microenvironmental factors involved in PD-L1 induction in prostate cancer (PC). We studied the expression of PD-L1 in a series of 66 PCs, in parallel with the expression of hypoxia- and acidity-related immunohistochemical markers (Hypoxia-inducible factor HIF1α, and lactate dehydrogenase LDHA) and tumor-infiltrating lymphocyte TIL density. Experiments with three PC cell lines, the 22Rv1, DU145, and PC3 were conducted focusing on the inducibility of PD-L1 by hypoxia, acidity, lymphocyte interactions, and radiation. In tissues, PD-L1 expression by cancer cells was directly related to PD-L1 expression by TILs and macrophages (p < 0.05), and the overexpression of HIF1α and LDH5 (p < 0.05). TIL density was inversely related to ΗΙF1α (p = 0.02). Exposure of PC cell lines to hypoxia strongly induced PD-L1 and protein and mRNA levels, directly controlled by HIF1α function (p < 0.001). Irradiation with 20 Gy had no apparent effect on PD-L1 expression. Culturing PC cell lines with culture medium (CM) from PBMCs strongly induced PD-L1 at protein and mRNA levels, independently from HIF1α, which was also confirmed when cells were incubated with Interferon-γ (p < 0.001). It is concluded that the combination of anti-PD-L1/PD-1 immunotherapy with hypoxia/HIF-targeting may be important in the treatment of specific subgroups of PC patients.

Tumor Microenvironment and Immune Response in Lip Cancer.

Tumor-infiltrating lymphocytes (TILs) play a significant role in cancer progression and prognosis of patients. The tumor microenvironment (TME) may affect the anti-tumor immune response. We examined the TIL and tertiary lymphoid structure (TLS) density in the invading front and inner tumor stroma, and the lymphocyte subpopulation (CD8, CD4, FOXP3) density in 60 squamous cell carcinomas of the lip. Analysis was performed in parallel with markers of hypoxia (hypoxia-inducible factor (HIF1α), lactate dehydrogenase (LDHA)) and angiogenesis. Low TIL density in the invading tumor front was related with larger tumor size (p = 0.05), deep invasion (p = 0.01), high smooth-muscle actin (SMA) expression (p = 0.01), and high HIF1α and LDH5 expression (p = 0.04). FOXP3+ TILs infiltration and FOXP3+/CD8+ ratios were higher in inner tumor areas, linked with LDH5 expression, and higher MIB1 proliferation index (p = 0.03) and SMA expression (p = 0.001). Dense CD4+ lymphocytic infiltration in the invading front is related to high tumor-budding (TB) (p = 0.04) and angiogenesis (p = 0.04 and p = 0.006, respectively). Low CD8+ TIL density, high CD20+ B-cell density, high FOXP3+/CD8+ ratio and high CD68+ macrophage presence characterized tumors with local invasion (p = 0.02, 0.01, 0.02 and 0.006, respectively). High angiogenic activity was linked with high CD4+, FOXP3+, and low CD8+ TIL density (p = 0.05, 0.01 and 0.01, respectively), as well as high CD68+ macrophage presence (p = 0.003). LDH5 expression was linked with high CD4+ and FOXP3+ TIL density (p = 0.05 and 0.01, respectively). Further research is needed to explore the prognostic and therapeutic value of TME/TIL interactions.

Cancer Microenvironment Defines Tumor-Infiltrating Lymphocyte Density and Tertiary Lymphoid Structure Formation in Laryngeal Cancer.

BACKGROUND: The presence and activity of tumor-infiltrating lymphocytes (TILs) is a key parameter related to the antitumor immune response. A large number of studies reveal TIL density as a prognostic marker and predictor of response to radiotherapy, chemotherapy, and immunotherapy. METHODS: We examined the TIL and tertiary lymphoid structure TLS density in the invading front and inner tumor stroma, in a 33 squamous cell laryngeal carcinomas (LSCC) treated with laryngectomy. TIL and TLS densities were in parallel examined with markers of anaerobic metabolism, vascular density (VD), vascular survival ability (VSA), and histopathological parameters. RESULTS: TIL and TLS densities significantly decreased in inner tumor areas (p < 0.0001). TIL density in the invading tumor front was inversely related with lymph node involvement (p = 0.03), HIF1α expression (p = 0.008), vessel density (p = 0.02), and MIB1 (p = 0.006). TIL density in inner stroma was inversely linked to local invasion (marginal p = 0.05), tumor budding (TB) (p = 0.005), MIB1 (p = 0.02), and HIF1α expression (p = 0.02). Low-TLS density in the invading front and in inner tumor areas was related to high TB (p = 0.02 and 0.002, respectively), HIF1α (p = 0.003 and 0.01, respectively), and LDH5 expression (p = 0.003 and 0.007, respectively). CD4+, FOXP3+ TIL density, and FOXP3+/CD8+ ratio were directly associated with VSA (p = 0.008, 0.02, and 0.05, respectively). CONCLUSION: Poor immune response is related to hypoxic background and anaerobic metabolism, as well as increased invasive and metastatic ability. Regulatory TIL markers are linked with increased angiogenic potential. The prognostic, predictive, and therapy-guiding value of TILs in clinical practice demands thorough investigation.

Tumor-infiltrating lymphocytes, PD-L1, and MMR-deficiency combined characterization may identify subgroups of rectal cancer patients who would benefit from immunotherapy.

INTRODUCTION: Mismatch repair deficiency, immunological fertility, and PD-L1 expression status are key histopathological and molecular features defining tumor responsiveness to immunotherapy and, eventually, prognosis. These were investigated in a series of locally advanced rectal cancer patients treated with postoperative chemotherapy and radiotherapy. MATERIALS AND METHODS: Tumor-infiltrating lymphocyte (TIL) density was assessed in hematoxylin-eosin tissue sections. PD-L1 expression and the expression of MMR proteins (MLH1, PSM2, MSH2, and MSH6) were assessed with immunohistochemistry. Their association with histopathological variables (node involvement and tumor budding) and prognosis was assessed. RESULTS: The TIL-density was significantly higher in the invading tumor front and was inversely related to tumor budding and directly with better overall survival (OS) and distant metastasis-free survival (DMFS) (p = 0.02 and 0.02, respectively). Cancer cell PD-L1 expression was related to high TIL-density (p < 0.01) but not to prognosis, although its overexpression defined a trend for poorer OS in patients with high TIL-density. High PD-L1 expression by stroma infiltrating immune cells was linked with better OS and DMFS (p = 0.007 and 0.001, respectively. MMR deficiency was recorded in 26.2 % of cases, and this was linked with higher TIL-density, but not with prognosis. CONCLUSIONS: Dense intratumoral lymphocytic infiltration relates to a better prognosis in rectal cancer, although it is also linked with PD-L1 expression that may adversely modulate the anti-tumor effects of TILs. This latter subgroup of patients (high TIL-density/high cancer cell PD-L1 expression) could be an additional target for anti-PD-1/PD-L1 immunotherapy, along with the established subgroup of MMR deficient patients.

Prognostic and Predictive Relevance of Tumor-Infiltrating Lymphocytes in Squamous Cell Head-Neck Cancer Patients Treated with Radical Radiotherapy/Chemo-Radiotherapy.

Microenvironmental conditions control the entrance and thriving of cytotoxic lymphocytes in tumors, allowing or preventing immune-mediated cancer cell death. We investigated the role of tumor-infiltrating lymphocyte (TIL) density in the outcome of radiotherapy in a series of squamous cell head−neck tumors (HNSCC). Moreover, we assessed the link between markers of hypoxia and TIL density. One-hundred twenty-one patients with HNSCC treated prospectively with radical radiotherapy/chemo-radiotherapy were analyzed. The assessment of TIL density was performed on hematoxylin and eosin biopsy sections before radiotherapy. TIL density ranged from 0.8 to 150 lymphocytes per ×40 optical field (median 27.5). Using the median value, patients were grouped into two categories of low and high TIL density. Early T-stage tumors had a significantly higher TIL density (p < 0.003), but we found no association with N-stage. Overexpression of HIF1α, HIF2α, and CA9 was significantly linked with poor infiltration by TILs (p < 0.03). A significant association of high TIL density with better disease-specific overall survival and improved locoregional relapse-free survival was noted (p = 0.008 and 0.02, respectively), which was also confirmed in multivariate analysis. It is concluded that HNSCC phenotypes that allow for the intratumoral accumulation of lymphocytes have a better outcome following radical radiotherapy/chemo-radiotherapy. Intratumoral-activated HIF- and CA9-related pathways characterize immunologically cold tumors and may be used as targets for therapeutic interventions.

Hypoxia and anaerobic metabolism relate with immunologically cold breast cancer and poor prognosis.

PURPOSE: Hypoxia-Inducible Factor HIF1α and lactate dehydrogenase LDHA drive anaerobic tumor metabolism and define clinical aggressiveness. We investigated their expression in breast cancer and their role in immune response and prognosis of breast cancer. METHODS: Tissue material from 175 breast cancer patients treated in a prospective study were analyzed with immunohistochemistry for HIF1α and LDH5 expression, in parallel with the tumor-infiltrating lymphocyte TIL-density and tertiary lymphoid structure TLS-density. RESULTS: High LDH5 expression was noted in 48/175 tumors, and this was related to HIF1α overexpression (p < 0.0001), triple-negative TNBC histology (p = 0.01), poor disease-specific survival (p < 0.007), metastasis (p < 0.01), and locoregional recurrence (p = 0.03). High HIF1α expression, noted in 39/175 cases, was linked with low steroid receptor expression (p < 0.05), her2 overexpression (p = 0.01), poor survival (p < 0.04), and high metastasis rates (p < 0.004). High TIL-density in the invading tumor front (TILinv) was linked with low LDH5 and HIF expression (p < 0.0001) and better prognosis (p < 0.02). High TIL-density in inner tumor areas (TILinn) was significantly linked with TNBC. Multivariate analysis showed that PgR-status (p = 0.003, HR 2.99, 95% CI 1.4-6.0), TILinv (p = 0.02, HR 2.31, 95% CI 1.1-4.8), LDH5 (p = 0.01, HR 2.43, 95% CI 1.2-5.0), N-stage (p = 0.04, HR 2.42, 95% CI 1.0-5.8), T-stage (p = 0.04, HR 2.31, 95% CI 1.0-5.1), and her2 status (p = 0.05, HR 2.01, 95% CI 1.0-4.2) were independent variables defining death events. CONCLUSION: Overexpression of LDH5, an event directly related to HIF1α overexpression, characterizes a third of breast tumors, which is more frequent in TNBC. Both HIF1α and LDH5 define cold breast cancer microenvironment and poor prognosis. A rational is provided to study further whether metabolic manipulations targeting HIF and LDH5 may enhance the antitumor immune response in breast cancer.