IFN-Type-I Response and Systemic Immunity in Rectal Adenocarcinoma Patients Treated with Conventional or Hypofractionated Neoadjuvant Radiotherapy.

The IFN-type-I pathway is involved in radiotherapy (RT)-mediated immune responses. Large RT fractions have been suggested to potently induce this pathway. Neoadjuvant hypofractionated short-course (scRT) and conventional long-course (lcRT) RT applied for the treatment of locally advanced rectal adenocarcinoma patients provides a unique model to address the immuno-stimulatory properties of RT on a systemic level. We prospectively analyzed the IFNß plasma levels and lymphocyte counts (LCs) of rectal adenocarcinoma patients before and after treatment with scRT (n = 22) and lcRT (n = 40). Flow cytometry was conducted to assess the effects on lymphocytic subpopulations in a subset of 20 patients. A statistically significant increase in the post-RT IFNß plasma levels was noted in patients undergoing scRT (p = 0.004). Improved pathological tumor regression was associated with elevated post-RT IFNß levels (p = 0.003). Although all patients experienced substantial lymphopenia after treatment, the post-RT LC of patients treated with scRT were significantly higher compared to lcRT (p = 0.001). Patients undergoing scRT displayed significantly lower percentages of regulatory CD4+/CD25+ T-cells after therapy (p = 0.02). scRT enables effective stimulation of the IFN-type-I pathway on a systemic level and confers decreased lymphocytic cytotoxicity and limited regulatory T-cell activation compared to lcRT, supporting its increasing role in immuno-RT trials.

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.

Next-Generation Sequencing Analysis of Mutations in Circulating Tumor DNA from the Plasma of Patients with Head-Neck Cancer Undergoing Chemo-Radiotherapy Using a Pan-Cancer Cell-Free Assay.

Using next-generation sequencing (NGS), we investigated DNA mutations in the plasma tumor cell-free circulating DNA (ctDNA) of 38 patients with inoperable squamous cell head neck cancer (SCHNC) before and after the completion of chemoradiotherapy (CRT). Baseline mutations of the TP53 were recorded in 10/38 (26.3%) and persisted in 4/10 patients after CRT. ΤP53 mutations were further detected post CRT in 7/38 additional patients with undetectable mutations at baseline (overall rate 44.7%). Furthermore, 4/38 patients exhibited baseline mutations of the EGFR, AR, FGFR3, and FBXW3, and four new gene mutations were detected after CRT (MTOR, EGFR3, ALK, and SF3B1). Τ4 stage was related with a significantly higher rate of mutations (TP53 and overall). Mutations were observed in 8/30 (26.6%) responders (complete/partial response) vs. in 6/8 (75%) of the rest of the patients (p = 0.03). Significant poorer LRFS was noted for patients with mutations detected before and after CRT (p = 0.02). Patients who had detectable mutations either before or after CRT had significantly worse DMFS (p = 0.04 overall, and p = 0.02 for TP53 mutations). It was concluded that assessment of mutations before and after the end of CRT is essential to characterize patients with a high risk of locoregional recurrence or metastatic progression.

Preoperative chemoradiotherapy induces multiple pathways related to anti-tumour immunity in rectal cancer.

BACKGROUND: Rectal cancer treated with preoperative radiotherapy (RT) provides an interesting model to study changes induced on cancer cell immuno-phenotype that could be exploited by immunotherapy interventions to improve prognosis. MATERIALS AND METHODS: We assessed the expression of HLA-class-I, ß2-microglobulin, TAP1, PD-L1 and STING/IFNß in preoperative biopsies and respective post-RT surgical specimens from patients with rectal cancer (n = 27). The effect of radiation was further investigated in colorectal adenocarcinoma cell lines HT-29 and Caco-2. RESULTS: Rectal carcinomas exhibited extensive loss of expression of HLA-Class-I related molecules, which was restored in post-irradiation surgical specimens (P < 0.0001). RT induced the expression of IFNß and STING in cancer cells and tumour-infiltrating lymphocytes (P < 0.0001). In in vitro experiments, irradiation with 4 Gy or 10 Gy induced the expression of HLA-class-I protein (P < 0.001). PD-L1 levels were transiently induced for two days (P < 0.001). cGAS, STING, IFNß and the downstream genes (MX1, MX2, UBE2L6v2, IFI6v2 and IFI44) mRNA levels significantly increased after 3 × 8 Gy or 1 × 20 Gy irradiation (P < 0.001). TREX1 mRNA levels remained unaltered. CONCLUSIONS: RT induces the IFN-type-I pathway and the expression of HLA-class-I molecules on rectal carcinoma. The transient induction of PD-L1 expression suggests that long-course daily RT may sustain increased PD-L1 levels. Anti-PD-L1/PD-1 immunotherapy could block this immunosuppressive pathway.

Prostate Cancer Stem Cells: Biology and Treatment Implications.

Stem cells differentiate into mature organ/tissue-specific cells at a steady pace under normal conditions, but their growth can be accelerated during the process of tissue healing or in the context of certain diseases. It is postulated that the proliferation and growth of carcinomas are sustained by the presence of a vital cellular compartment resembling stem cells residing in normal tissues: 'stem-like cancer cells' or cancer stem cells (CSCs). Mutations in prostate stem cells can lead to the formation of prostate cancer. Prostate CSCs (PCSCs) have been identified and partially characterized. These express surface markers include CD44, CD133, integrin α2ß1, and pluripotency factors like OCT4, NANOG, and SOX2. Several signaling pathways are also over-activated, including Notch, PTEN/Akt/PI3K, RAS-RAF-MEK-ERK and HH. Moreover, PCSCs appear to induce resistance to radiotherapy and chemotherapy, while their presence has been linked to aggressive cancer behavior and higher relapse rates. The development of treatment policies to target PCSCs in tumors is appealing as radiotherapy and chemotherapy, through cancer cell killing, trigger tumor repopulation via activated stem cells. Thus, blocking this reactive stem cell mobilization may facilitate a positive outcome through cytotoxic treatment.

Ultra-Hypofractionated Re-Irradiation with Anti-PD-1 Immunotherapy for Locoregionally Recurrent (after Radical Chemo-Radiotherapy) Non-Small Cell Lung Cancer.

Large fractions of radiotherapy of 8 Gy (ultra-hypofractionated RT, ultra-hypoRT) promote anti-tumor immune responses that have been clinically substantiated in combination trials with immune checkpoint inhibitors (ICIs). In the current study, we postulated that ultra-hypoRT in combination with ICIs may enhance tumor clearance in NSCLC patients with locoregional relapse after radical chemo-RT. Between 2019 and 2021, eleven patients received re-irradiation with one or two fractions of 8 Gy concurrently with anti-PD1 immunotherapy (nivolumab or pembrolizumab). RT-related toxicities were negligible, while immune-related adverse events enforced immunotherapy interruption in 36% of patients. The overall response rate was 81.8%. Tumor reduction between 80 and 100% was noted in 63.5% of patients. Within a median follow-up of 22 months, the locoregional relapse-free rate was 54.5%, while the projected 2-year disease-specific overall survival was 62%. The results were independent of PD-L1 status. The current report provides encouraging evidence that a relatively low biological dose of RT delivered with 8 Gy fractions is feasible and can be safely combined with anti-PD-1 immunotherapy. Despite the low number of patients, the significant tumor regression achieved and the long-lasting locoregional control and overall progression-free intervals provide a basis to pursue immuno-RT trials with U-hypoRT schemes in this group of NSCLC patients of poor prognosis.

Anti-PD-1 immunotherapy with dose-adjusted ultra-hypofractionated re-irradiation in patients with locoregionally recurrent head and neck cancer

Introduction Patients with recurrent inoperable squamous-cell head-neck cancer (HNSCC) after chemo-radiotherapy have an ominous prognosis. Re-irradiation can be applied with some efficacy and high toxicity rates. Anti-PD-1 immunotherapy is effective in 25% of patients. Immunogenic death produced by large radiotherapy (RT) fractions may enhance immune response. Materials and methods We evaluated the efficacy and tolerance of ultra-hypofractionated immuno-radiotherapy (uhypo-IRT) in 17 patients with recurrent HNSCC and 1 with melanoma. Four of HNSCC patients also had oligometastatic disease. Using a dose/time/toxicity-based algorithm, 7, 7 and 4 patients received 1, 2 and 3 fractions of 8 Gy to the tumor, respectively. Nivolumab anti-PD-1 immunotherapy was administered concurrently with RT and continued for 24 cycles, or until disease progression or manifestation of immune-related adverse events (irAEs). Results Early and late RT toxicities were minimal. Three patients developed irAEs (16%). After the 12th cycle, 7/17 (41.2%) and 5/17 (29.4%) patients with HNSCC showed complete (CR) and partial response (PR), respectively. CR was also achieved in the melanoma patient. The objective response rates in HNSCC patients were 57%, 86% and 66%, after 1, 2 and 3 fractions, respectively (overall response rate 70.6%). Most responders experienced an increase in peripheral lymphocyte counts. The median time to progression was 10 months. The 3-year projected locoregional progression-free survival was 35%, while the 3-year disease-specific overall survival was 50%. Conclusions Anti-PD1 uhypo-IRT is safe and effective in patients with recurrent HNSCC. The high objective response rates and the long survival without evidence of disease support further trials on uhypo-IRT (AU)

Circulating Plasma Cell-free DNA (cfDNA) as a Predictive Biomarker for Radiotherapy: Results from a Prospective Trial in Head and Neck Cancer.

Background/Aim: The plasma levels of cell-free DNA (cfDNA) in cancer patients increase due to rapid cancer cell proliferation and death. Therefore, cfDNA can be used to study specific tumor-DNA features. In addition, the non-specific cfDNA concentration may be an important biomarker of cancer prognosis. Patients and Methods: We prospectively examined the predictive role of cfDNA levels and the kinetics in the outcome of chemo-radiotherapy (CRT) in a cohort of 47 patients with locally advanced squamous cell head-neck cancer (SCHNC) treated with definitive chemo-radiotherapy. Results: Increased cfDNA levels after therapy completion (after/before treatment ratio; A/B-ratio >1) were found in 26/47 patients (55.3%). Locally advanced T4-stage was significantly associated with higher cfDNA levels after CRT (3.3 ng/µl in T4-stage vs. 1.3 ng/µl in T1-3 stages, p=0.007). Patients who responded to CRT (partial/complete response) had significantly lower cfDNA levels before therapy (mean values 1.2 ng/µl vs. 2.7 ng/µl, p=0.03). A significantly worse locoregional progression-free survival in patients with an A/B-ratio >1 was documented (p=0.01; hazard ratio 3.5, 95%CI=1.2-9.7). This was also confirmed in multivariate analysis, where the A/B-ratio was an independent predictive variable of locoregional relapse (p=0.03, hazard ratio 3.9, 95%CI=1.2-13). Conclusion: High post-CRT cfDNA levels could be an early biomarker for the immediate recruitment of patients with SCHNC in consolidation chemo-immunotherapy protocols.

Using Liquid Biopsy to Predict Relapse After Radiotherapy in Squamous Cell Head-Neck and Esophageal Cancer.

Circulating cell-free DNA (cfDNA) in the blood of cancer patients contains tumor-specific mutated genes and viral genome that can be identified and quantified as 'tumor-specific cfDNA' (circulating tumor DNA, ctDNA). Various technologies are available that offer reliable detection of ctDNA at a low concentration. Quantitative and qualitative analysis of ctDNA may be of prognostic and predictive value in oncology. Here, we present concisely the experience on the assessment of ctDNA levels and kinetics during therapy in the outcome of radiotherapy (RT) and chemo-radiotherapy (CRT) in squamous cell head-neck cancer and esophageal squamous cell cancer patients. The levels of circulating viral (human papilloma virus or Epstein-Barr) ctDNA, and levels of total, mutated or methylated ctDNA at diagnosis are linked with tumor burden and clinical aggressiveness, and may be of prognostic or even predictive value of RT/CRT efficacy. Persistent ctDNA levels after therapy seem to predict high rates of tumor relapse several months before radiological documentation. This can prove of value for the identification of subgroups of patients who could benefit from RT dose-escalation or consolidation chemotherapy and immunotherapy, a hypothesis that should be tested in clinical trials.

Irradiation-induced IFN-type-I pathway activation in prostate cancer cell lines.

The Interferon (ΙFN) Type-I pathway has an important role in the activation of an anti-tumor immune response. We investigated the effects of two different dose fractionations of radiation (3 daily 8 Gy fractions vs. one fraction of 20 Gy) on the activation of the Type-I IFN-pathway in three hormone-dependent (22Rv1) and independent (DU145, PC3), prostate cancer (PC) cell lines. Regardless of the dose schedules, radiation-induced the expression of IFN-stimulated genes in all PC cell lines, with a strong up-regulation of the IFI6v2 and IFI44 genes. In addition, strong up-regulation of the MX1 and MX2 genes was noted in the PC3 cell line. This effect was independent of the expression of IFNß, cGAS, or TREX1 levels. It is suggested that the RT-induced IFN type-I response could be exploited for the development of immuno-RT policies for localized and metastatic PC.

Anti-Tumor Immunity and Preoperative Radiovaccination: Emerging New Concepts in the Treatment of Breast Cancer.

Neoadjuvant chemotherapy (NACT) for certain breast cancer (BC) subtypes confers significant tumor regression rates and a survival benefit for patients with a complete pathologic response. Clinical and preclinical studies have demonstrated that immune-related factors are responsible for better treatment outcomes, and thus, neoadjuvant immunotherapy (IO) has emerged as a means to further improve patient survival rates. Innate immunological "coldness", however, of specific BC subtypes, especially of the luminal ones, due to their immunosuppressive tumor microenvironment, hinders the efficacy of immune checkpoint inhibitors. Treatment policies aiming to reverse this immunological inertia are, therefore, needed. Moreover, radiotherapy (RT) has been proven to have a significant interplay with the immune system and promote anti-tumor immunity. This "radiovaccination" effect could be exploited in the neoadjuvant setting of BC and significantly enhance the effects of the already established clinical practice. Modern stereotactic irradiation techniques directed to the primary tumor and involved lymph nodes may prove important for the RT-NACT-IO combination. In this review, we provide an overview and critically discuss the biological rationale, clinical experience, and ongoing research underlying the interplay between neoadjuvant chemotherapy, anti-tumor immune response, and the emerging role of RT as a preoperative adjunct with immunological therapeutic implications in BC.

Immune Response and Immune Checkpoint Molecules in Patients with Rectal Cancer Undergoing Neoadjuvant Chemoradiotherapy: A Review.

It is well-established that tumor antigens and molecules expressed and secreted by cancer cells trigger innate and adaptive immune responses. These two types of anti-tumor immunity lead to the infiltration of the tumor's microenvironment by immune cells with either regulatory or cytotoxic properties. Whether this response is associated with tumor eradication after radiotherapy and chemotherapy or regrowth has been a matter of extensive research through the years, mainly focusing on tumor-infiltrating lymphocytes and monocytes and their subtypes, and the expression of immune checkpoint and other immune-related molecules by both immune and cancer cells in the tumor microenvironment. A literature search has been conducted on studies dealing with the immune response in patients with rectal cancer treated with neoadjuvant radiotherapy or chemoradiotherapy, assessing its impact on locoregional control and survival and underlying the potential role of immunotherapy in the treatment of this cancer subtype. Here, we provide an overview of the interactions between local/systemic anti-tumor immunity, cancer-related immune checkpoint, and other immunological pathways and radiotherapy, and how these affect the prognosis of rectal cancer patients. Chemoradiotherapy induces critical immunological changes in the tumor microenvironment and cancer cells that can be exploited for therapeutic interventions in rectal cancer.

Current status of locally advanced rectal cancer therapy and future prospects.

Rectal cancer treatment has been evolving ever since the beginning of the 20th century. Surgery was originally the only available method regardless of the extent of tumor invasion or nodal involvement status. Total mesorectal excision was established as the standard procedure in the early 1990 s. Advances in the utilization of radiation for rectal cancer led to the addition of radiotherapy (RT) combined with chemotherapy to the postoperative treatment algorithm. The promising results of the Swedish short-course preoperative RT set the basis for a number of large randomized trials investigating the efficacy of neoadjuvant RT or chemoradiotherapy (CRT) for advanced rectal cancer. Both short-course RT and long-course preoperative CRT compared favorably to adjuvant treatment and became the standard of choice for patients with extramural invasion or lymph node involvement. Recently, the focus of clinical research has been shifted towards total neoadjuvant therapy (TNT), delivering the whole course of RT and chemotherapy before surgery, and showing good tolerance and encouraging efficacy. Although targeted therapies haven't displayed a benefit in the neoadjuvant setting, preliminary evidence suggests impressive efficacy of immunotherapy in rectal carcinomas with mismatch-repair deficiency. In this review, we provide an in-depth critical overview of all significant randomized trials that have shaped the current treatment guidelines for locally advanced rectal cancer and discuss future trends for the treatment of this common malignancy.

Anti-PD-1 immunotherapy with dose-adjusted ultra-hypofractionated re-irradiation in patients with locoregionally recurrent head and neck cancer.

INTRODUCTION: Patients with recurrent inoperable squamous-cell head-neck cancer (HNSCC) after chemo-radiotherapy have an ominous prognosis. Re-irradiation can be applied with some efficacy and high toxicity rates. Anti-PD-1 immunotherapy is effective in 25% of patients. Immunogenic death produced by large radiotherapy (RT) fractions may enhance immune response. MATERIALS AND METHODS: We evaluated the efficacy and tolerance of ultra-hypofractionated immuno-radiotherapy (uhypo-IRT) in 17 patients with recurrent HNSCC and 1 with melanoma. Four of HNSCC patients also had oligometastatic disease. Using a dose/time/toxicity-based algorithm, 7, 7 and 4 patients received 1, 2 and 3 fractions of 8 Gy to the tumor, respectively. Nivolumab anti-PD-1 immunotherapy was administered concurrently with RT and continued for 24 cycles, or until disease progression or manifestation of immune-related adverse events (irAEs). RESULTS: Early and late RT toxicities were minimal. Three patients developed irAEs (16%). After the 12th cycle, 7/17 (41.2%) and 5/17 (29.4%) patients with HNSCC showed complete (CR) and partial response (PR), respectively. CR was also achieved in the melanoma patient. The objective response rates in HNSCC patients were 57%, 86% and 66%, after 1, 2 and 3 fractions, respectively (overall response rate 70.6%). Most responders experienced an increase in peripheral lymphocyte counts. The median time to progression was 10 months. The 3-year projected locoregional progression-free survival was 35%, while the 3-year disease-specific overall survival was 50%. CONCLUSIONS: Anti-PD1 uhypo-IRT is safe and effective in patients with recurrent HNSCC. The high objective response rates and the long survival without evidence of disease support further trials on uhypo-IRT.

Combining the past and present to advance immuno-radiotherapy of cancer.

Since its first clinical application, 120 years ago, radiotherapy evolved into a major anti-cancer treatment modality, offering high cure rates in many human malignancies. During the past ten years, the establishment of immune checkpoint inhibitors (ICIs) in cancer therapeutics has vigorously reintroduced the immune system's role in the outcome of radiotherapy and, conversely, the role of radio-vaccination in the efficacy of immunotherapy. The knowledge and clinical experience that founded the current era of immuno-radiotherapy started alongside with the birth of radiotherapy, and evolved through exhaustive experimental work, clinical trials on active specific immunotherapy, frustrating attempts to validate the importance of cytokine administration with radiotherapy, and, finally, the encouraging ICI-based clinical trials that opened the door to a far more encouraging perspective; radio-vaccination, through its old and new methods, is rising as a research field that promises to cure, previously incurable, disease. In this critical review, we focus on the scientific knowledge gathered through more than a century of research on radiotherapy interactions with the immune system. Understanding the origins of this promising therapeutic approach will substantially contribute to developing new immuno-radiotherapy policies in the fight against cancer.

The molecular basis of immuno-radiotherapy.

PURPOSE: Radiotherapy (RT) and immunotherapy are powerful anti-tumor treatment modalities. Experimental research has demonstrated an important interplay between the cytotoxic effects of RT and the immune system. This systematic review provides an overview of the basics of anti-tumor immunity and focuses on the mechanisms underlying the interplay between RT and immune anti-tumor response that set the molecular basis of immuno-RT. CONCLUSIONS: An 'immunity acquired equilibrium' mimicking tumor dormancy can be achieved post-irradiation treatment, with the balance shifted toward tumor eradication or regrowth when immune cells' cytotoxic effects or cancer proliferation rate prevail, respectively. RT has both immunosuppressive and immune-enhancing properties. The latter effect is also known as radio-vaccination. Its mechanisms involve up- or down-regulation of membrane molecules, such as PD-L1, HLA-class-I, CD80/86, CD47, and Fas/CD95, that play a vital role in immune checkpoint pathways and increased cytokine expression (e.g. INFα,ß,γ, IL1,2, and TNFα) by cancer or immune cells. Moreover, the interactions of radiation with the tumor microenvironment (fibroblasts, tumor-infiltrating lymphocytes, monocytes, and dendritic cells are also an important component of radio-vaccination. Thus, RT may have anti-tumor vaccine properties, whose sequels can be exploited by immunotherapy agents to treat different cancer subtypes effectively.

Is anti-PD-1 immunotherapy a means for post-irradiation tumor clearance in head and neck cancer?

Chemo-radiotherapy is the standard treatment for locally advanced head-neck cancer (LA-HNC). However, about 30% of tumors do not respond or even progress shortly after the completion of radiotherapy. We investigated whether anti-PD1 immunotherapy can eradicate the irradiated tumor and reverse the ominous prognosis of these patients. We retrospectively analyzed a small series of 9 patients with LA-HNC who did not respond (6/9) or showed local disease progression (3/9) during chemo-radiotherapy and were treated with nivolumab anti-PD1 immunotherapy. Immunotherapy started 1.5 months after the end of radiotherapy. Out of 9 patients, 3 (33.3%) had a complete response and 3 (33.3%) partial response at 6 months after the onset of immunotherapy. Two patients are alive with no evidence of disease at 36 months. One more patient with partial response and without disease progression survived 16 months after therapy when he died from intercurrent disease. Immunotherapy showed an excellent tolerance profile. One patient developed an extensive skin rash on the 16th cycle. Anti-PD-1 immunotherapy after radiotherapy can lead to clearance of the remnant tumor and ameliorate the prognosis of patients. Randomized trials are necessary to establish post-irradiation immunotherapy as a standard of care in this ill-fated subgroup of HNC 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.

Expression of CD47 and SIRPα Macrophage Immune-Checkpoint Pathway in Non-Small-Cell Lung Cancer.

BACKGROUND: Cancer cells escape macrophage phagocytosis by expressing the CD47 integrin-associated protein that binds to the SIRPα ligand (signal regulatory protein alpha) expressed by macrophages. Immunotherapy targeting this pathway is under clinical development. METHODS: We investigated the expression of CD47/SIRPα molecules in a series of 98 NSCLCs, in parallel with the infiltration of tumor stroma by CD68+ macrophages, tumor-infiltrating lymphocytes (TILs), and PD-L1/PD-1 molecules. RESULTS: Extensive membranous CD47 expression by cancer cells characterized 29/98 cases. SIRPα and CD68 were expressed, to a varying extent, by tumor-associated macrophages (Μφ, TAMs). A high CD68Mφ-score in inner tumor areas was linked with improved overall survival (p = 0.005); and this was independent of the stage (p = 0.02, hazard ratio 0.4). In contrast, high SIRPα expression by CD68+ TAMs (SIRPα/CD68-ratio) was linked with CD47 expression by cancer cells, low TIL-score, and poor prognosis (p = 0.02). A direct association of CD47 expression by cancer cells and the % FOXP3+ TILs (p = 0.01, r = 0.25) was also noted. CONCLUSIONS: TAMs play an important role in the prognosis of operable NSCLC. As SIRPα+ macrophages adversely affect prognosis, it is suggested that the CD47/SIRPα axis is a sound target for adjuvant immunotherapy policies, aiming to improve the cure rates in operable NSCLC.

Loss of HLA-class-I expression in non-small-cell lung cancer: Association with prognosis and anaerobic metabolism.

Cancer immuno-editing frequently leads to loss of HLA-class-I molecule (HLA) expression and impaired immune surveillance. We investigated the expression of HLAs in a series of operable non-small-cell lung carcinomas (NSCLCs). Complete loss and extensive loss of expression was noted in 41.5% and 23.4% of cases, respectively. Low CD8 + and FOXP3 + TIL-density was significantly associated with loss of HLAs (p < 0.05 and p = 0.003, respectively). High PD-L1 expression was linked with sustained expression of HLAs. A significant association of loss of HLA-expression with overexpression of LDH5 (p = 0.01) and marginally with HIF1α was recorded. Cell line experiments confirmed that hypoxia and acidity down-regulate the expression of HLAs. A direct association between HLAs and Beclin-1 expression was also noted (p = 0.01). Loss of HLA-expression was linked with poorer survival (p < 0.01), independent of stage. It is concluded that loss of HLA-class-I molecules is frequent in NSCLC and directly linked to micro-environmental hypoxia and acidity.