Tumor-infiltrating Lymphocytes Counts Research Articles

Obesity is a risk factor for intrahepatic cholangiocarcinoma progression associated with alterations of metabolic activity and immune status

Body mass index (BMI) is well known to be associated with poor prognosis in several cancers. The relationship between BMI and the long-term outcomes of patients with intrahepatic cholangiocarcinoma (ICC) is incompletely understood. This study investigated the relationships of BMI with clinicopathological characteristics and patient outcomes, focusing on metabolic activity and immune status. The relationship between BMI and the maximum standardized uptake value (SUVmax) on fluorine-18 fluorodeoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET/CT) was analyzed. In addition, immunohistochemistry was performed for programmed cell death-ligand 1 (PD-L1), cluster of differentiation 8 (CD8), and forkhead box protein P3 (Foxp3). Seventy-four patients with ICC were classified into normal weight (BMI < 25.0 kg/m2, n = 48) and obesity groups (BMI ≥ 25.0 kg/m2, n = 26), respectively. Serum carbohydrate antigen 19–9 levels were higher in the obesity group than in the normal weight group. Tumor size and the intrahepatic metastasis rate were significantly larger in the obesity group. Patients in the obesity group had significantly worse prognoses than those in the normal weight group. Moreover, BMI displayed a positive correlation with SUVmax on 18F-FDG PET/CT (n = 46, r = 0.5152). Patients with high 18F-FDG uptake had a significantly higher rate of PD-L1 expression, lower CD8 + tumor-infiltrating lymphocyte (TIL) counts, and higher Foxp3 + TIL counts. The elevated BMI might predict the outcomes of patients with ICC. Obesity might be associated with ICC progression, possibly through alterations in metabolic activity and the immune status.

Abstract PS6-45: Prognostic significance of tumor-infiltrating lymphocytes and neutrophil-to-lymphocyte ratio in patients with breast cancer receivingneoadjuvant chemotherapy

Abstract Introduction and objectives: Tumor infiltrating lymphocytes (TILs) and neutrophil-to-lymphocyte ratio (NLR) play a prognostic role in early stage breast cancer (BC). There is no evidence about the combined effect of both factors. Our objective was to evaluate the integrated clinical significance of TILs and NLR in patients with early BC treated with neoadjuvant therapy. Materials and methods: Retrospective, single-center analysis of a cohort of patients with early BC treated with neoadjuvant chemotherapy between 2001-2010. Pre-treatment TILs (CD3+-TIL count) was evaluated using a tumor tissue microarray. NLR was calculated within one month of cancer diagnosis. TILs (logarithmic transformed) and NLR were analyzed as continuous variables. Survival analysis was performed using multivariable Cox regression models. Results:A total of 121 patients were included. Median age: 56 years. Cancer stage at diagnosis: 16% IIA, 28% IIB, 33% IIIA, 7% IIIB and 16% IIIC. Molecular subtype: 64% hormone receptor(HR)-positive (12% HER2-positive), 11% HER2-positive HR-negative and 22% triple-negative. Pathological complete response (pCR): 16.5%. Median follow-up: 12 years. Pre-treatment TIL analysis was available in 71 patients (59%) and NLR in 101 (83%). There was no correlation between both variables (Spearman's Rho: 0.03, p = 0.98). In the univariate analysis, the NLR showed a negative prognostic value for overall survival (OS) (HR 1.23, 95%CI 1.11-1.36; p &amp;lt;0.001, C-index: 0.64 95%CI 0.52-0.77, p = 0.69). The effect was opposite for TILs (HR: 0.76 95%CI 0.61-0.95, p = 0.02; C-index: 0.69 95%CI 0.57-0.81, p = 0.69). The linear approximation was adequate, and there was no suspicion of non-proportionality of the hazards. In the multivariate analysis, including or not cancer staging after neoadjuvant therapy, NLR remained as an independent variable (HR 1.18, 95%CI 1.04-1.33; p = 0.01) and a statistic trend for TILs was also observed (HR 0.83, 95%CI 0.65-1.07; p = 0.16). Given the limited sample size, the multivariate analysis did not provide clear evidence of an additive effect. Nevertheless, the combined analysis of both parameters showed a better fit with respect to the two variables separately (Akaike Information Criterion for the combined model, for TILs and for NLR: 106, 133 and 214, respectively). Conclusion:The integrated characterization of TILs and NLR identifies different prognostic subgroups in early BC patients receiving neoadjuvant chemotherapy. Future validation of these findings in large, multicenter cohorts might allow treatment optimization by means of new strategies such as immunotherapy. Citation Format: Esmeralda García-Torralba, Francisco Ayala de la Peña, Beatriz Alvarez-Abril, Pilar de la Morena Barrio, Alejandra Ivars Rubio, Elisa Garcia Garre, Gema Marin Zafra, Alberto Carmona-Bayonas, Elena Garcia Martinez. Prognostic significance of tumor-infiltrating lymphocytes and neutrophil-to-lymphocyte ratio in patients with breast cancer receivingneoadjuvant chemotherapy [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS6-45.

Abstract PS5-08: Comparison of PD-L1 protein expression between primary tumors and metastatic lesions in triple negative breast cancers

Abstract Background: Several recent studies that compared small cohorts of metastatic and primary lesions, suggested substantial heterogeneity in tumor infiltrating lymphocyte count, immune gene expression and PD-L1 protein expression across different metastatic sites and between primary breast cancers and metastasis. Understanding the frequency of PD-L1 positivity rates across different tissue sites can indicate differences in the immune microenvironment and may also guide biopsy site selection. We compared PD-L1 positivity on immune cells and tumor cells in primary and metastatic triple negative breast cancer tumors (TNBC).Methods: A retrospective data analysis of the Foundation Medicine PD-L1 IHC database was conducted on 340 cases of TNBC. PD-L1 positivity was determined by IHC using SP142CDx. Results are reported as percent of PD-L1 stained immune cells (IC) in the tumor area. A tumor was considered PD-L1 positive if ≥ 1% IC stained positive with PD-L1. As an exploratory analysis, PD-L1 positivity of tumor cells (TC) was also assessed. PD-L1 percent positive staining results are reported as means with 95% CI. The proportion of PD-L1 positive and negative IC and TC in primary tumors vs metastatic sites was compared using Chi-Square test. Prism 8 was used for all data analysis.Results: All patients were female, with median age 56 years (range 26-89); 179 samples were from primary tumors and 161 from metastatic lesions, representing 15 different tissue sites. Overall, PD-L1 expression on immune cells was statistically significantly more frequent in primary tumors compared to metastatic sites (63.7% [n=114] vs 42.9% [n=69]), p&amp;lt;0.0001). This was driven by the lower PD-L1 positivity rates in skin (23.8%, 95% CI: 8.22% - 47.2%), liver (17.4%, 95%CI: 5.00% - 38.8%) and bone (16.7%, 95%CI: 2.10% - 48.4%) metastases. Lung (68.8%, 95% CI: 41.3 - 90.0), soft tissues (65.2%, 95% CI: 42.7 - 83.6) and lymph nodes 51.1%, 95% CI (35.8 - 66.3) had PD-L1 % positivity rates similar to primary tumors. PD-L1 expression was rare on tumor cells in both the breast and metastatic sites (8.3% vs 4.3%, p=0.13). Conclusion: We observed substantial heterogeneity in PD-L1 positivity rates across metastatic sites. Lung, soft tissues and lymph node metastases had PD-L1 % positivity rates that were similar to that of primary tumors whereas skin, liver and bone metastases had significantly lower PD-L1 % positivity rates. These results raise the possibility that response to immune therapy could depend on the location and the PD-L1 positivity of the metastatic site. Limited current experience in breast cancer is not sufficient to correlate tumor response with PD-L1 expression in metastases, but as more patients receive treatment, this could be examined in the future. Table 1: Sample Characteristics and % PD-L1 positivity on immune cellsSample typeTotal N (%)N PD-L1 positive (%, 95% CI)Primary Tumor179 (52.6)114 (63.7%, 56.2% - 70.7%)Metastatic Lesion161 (47.4)69 (42.9%, 35.1% - 50.9%)Sites of MetastasesN (% of metastatic samples)N PD-L1 positive (%, 95% CI)Lung16 (10.0)11 (68.8%, 41.3% - 90.0%)Soft Tissues23 (14.3)15 (65.2%, 42.7% - 83.6%)Lymph Nodes45 (28.0)23 (51.1%, 35.8% - 66.3%)Skin21 (13.0)5 (23.8%, 8.22% - 47.2%)Liver23 (14.3)4 (17.4%, 5.00% - 38.8%)Bone12 (7.5)2 (16.7%, 2.10% - 48.4%)Brain9 (5.6)5Mediastinum4 (2.5)1Pleura2 (1.2)0Muscle1 (&amp;lt;1)0Omentum1 (&amp;lt;1)1Ovary1 (&amp;lt;1)0Pelvis1 (&amp;lt;1)0Retroperitoneum1 (&amp;lt;1)0Adrenal Gland1 (&amp;lt;1)1 Table 2: Comparison of PD-L1 positivity in primary versus metastatic sitesTissuePDL1+ Immune CellPDL1- Immune CellP valuePDL1+ Tumor CellPDL- Tumor CellP valuePrimary114650.0001151640.1313Metastasis69927154 Citation Format: Mariya Rozenblit, Richard Huang, Natalie Danziger, Brian Alexander, Shakti Ramkissoon, Kim Blenman, Jeffrey Ross, David Rimm, Lajos Pusztai. Comparison of PD-L1 protein expression between primary tumors and metastatic lesions in triple negative breast cancers [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS5-08.

Abstract SP122: Novel biomarkers in immune therapy response

Abstract Four randomized trials demonstrated statistically significant increase in pCR rates when an immune checkpoint inhibitor (ICI) was added to neoadjuvant chemotherapy in triple negative breast cancer (TNBC), these include the KN-522, Impassion-031, I-SPY2 pembrolizumab, and I-SPY2 durvalumab/oleparib arms. One trial, GeparNuevo, showed a non-significant trend for higher pCR rate, and one trial (NeoTrip-aPDL1) showed no effect. In each of these randomized trials, high tumor infiltrating lymphocyte count, high expression of PD-L1 protein and a broad range of immune-related genes and immune gene signatures predicted for higher pCR rate in both chemotherapy alone and chemotherapy plus ICI treatment arms. Unexpectedly, the positive trials also consistently demonstrated that pCR rates significantly improved with ICI therapy even in PDL-1 negative TNBC. For example, in KN-522 the pCR rates were 30% vs 45% in the PDL-1 negative and 55% vs 69% in the PD-L1 positive populations. There is less data on the efficacy of neoadjuvant immune checkpoint therapy in estrogen receptor positive (ER+) cancers. However, both the pembrolizumab and durvalumab + olaparib arms of the I-SPY2 trail demonstrated improvement in pCR rates in ER+ cancers, and these improvements were also independent of PDL-1 mRNA expression or other immune markers. On the other hand, the ER+ MammaPrint ultrahigh (MP2) subset that is characterized by high proliferation, and low ER-related gene expression showed selective benefit from ICI therapy, while no benefit was seen in the MP1 group. The same MP2 group also benefited from adding carboplatin and veliparib to paclitaxel in an earlier arm of I-SPY2 suggesting that among ER+ cancers, the MP2 tumors are more chemotherapy sensitive and this sensitivity might be further enhanced by adding an immune checkpoint inhibitor.A very different biomarker picture emerged from the randomized first line trials in metastatic TNBC (mTNBC). In this clinical setting, the efficacy of immune checkpoint inhibitors (ICI) is proportional to the level of PD-L1 protein expression by immunohistochemistry, or by immune infiltration that can be quantified by tumor infiltrating lymphocyte count. In PDL-1 negative, or immune low, mTNBC no benefit is seen from ICI therapy. In immune rich metastatic TNBC (defined as CPS&amp;gt;20 with 22C3 Assay), close to 30% response rate was seen with single agent pembrolizumab in the KN-199 trial. Two large randomized first line trials, IMpassion-130 and KN-355, demonstrated improved response rates and increased progression free survival (and overall survival in Impassion-130) when ICI was added to nab-paclitaxel (Impassion-130), or taxol or nab-paclitaxel or gemcitabine/carboplatin (KN-355) chemotherapies, respectivel. Impassion-130 also demonstrated that different immunohistochemistry assays for PDL-1 determination have different accuracy to define the atezolizumab sensitive population. The SP142 Ventana assay that defines approximately 40% of the mTNBC population as PDL-1 positive had the highest overall accuracy. A third, and smallest, randomized trial, IMPassion-131, failed to show significant benefit (HR:0.82, p=0.2) from adding atezolizumab to paclitaxel. This failure is likely due to differences in patient composition and limited sample size and power, rather than choice of chemotherapy. Pembrolizumab is also approved in the US for metastatic solid tumors, including breast cancer regardless of receptor status, that have either microsatellite instability, or high tumor mutation burden (&amp;gt;10 mutation /MB). Few trials examined the role of ICI in ER+ metastatic breast cancer, and these studies showed low single agent activity. Due to the paucity of data, the relationship between immune therapy efficacy and PDL-1 expression, or recurrence score, or luminal A versus B subtype is unknown in ER+ metastatic disease. Citation Format: L Pusztai. Novel biomarkers in immune therapy response [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr SP122.

Abstract PS12-09: Pre-operative pembrolizumab (Pembro) with radiation therapy (RT) in patients with operable triple-negative breast cancer (TNBC)

Abstract Background: Pembrolizumab (pembro)-mediated checkpoint blockade has shown only modest efficacy in triple negative breast cancer (TNBC). Pathologic complete response (pCR) rates in TNBC are 34-55% with neoadjuvant chemotherapy and 26% with neoadjuvant stereotactic radiation therapy (RT) alone. However, because RT induces immune-mediated cell death that can generate a rich supply of tumor antigens and trigger anti-tumor immunity, the addition of pembro to RT can generate robust anti-tumor immune responses as demonstrated in metastatic TNBC. If administered in the pre-operative setting, immune therapy plus RT could induce long-term tumor-specific memory, and ultimately, improve cure rates. This study aimed to establish the safety of pre-operative pembro plus RT in 20 TNBC patients for whom lumpectomy and adjuvant RT were planned and to explore predictive biomarkers (NCT03366844). Methods: Women planning breast conserving surgery for operable, stage II/III, TNBC were enrolled in this single-institution pilot study. Study treatment consisted of pre-operative pembro (200mg IV) followed 3 weeks later by pembro (200mg IV) plus RT (24 Gy/3 fractions) to the primary breast tumor followed 3-5 weeks later by standard-of-care (SOC) neoadjuvant chemotherapy. Adjuvant RT was administered per SOC after surgery, but without a boost dose. Research blood and tumor biopsies were obtained at baseline, at week 4 prior to pembro/RT, and at week 6-8 prior to chemotherapy initiation. Co-primary endpoints were: safety/tolerability (defined by the number of patients who do not necessitate a delay in SOC chemotherapy or surgery) and change in tumor infiltrating lymphocyte (TIL) score. Secondary endpoints include safety/toxicity up to 19 weeks after study enrollment and pathologic complete response (pCR) rate. Results: 20 patients were enrolled and completed all treatment (pembro, RT, chemotherapy and surgery) as of July 2020. The median age is 55y (range 33-70y). The stage distribution is IIA (11), IIB (6), IIIA (2) and IIIC (1) with biopsy proven nodal involvement in 55%. All patients received a taxane containing regimen, 12/20 (60%) received carboplatin and 15/20 (75%) received an anthracycline. Three patients did not complete the planned course of chemotherapy, two of whom had residual disease. None of the patients experienced significant delay (&amp;gt;2 weeks) in receiving SOC treatment. There were no observed grade 3 or 4 toxicities observed during the pembro +/- RT treatment. Grade 4 colitis in 2/20 was reported during SOC chemotherapy and was attributed to pembro. The most frequent grade 1/2 toxicities were nausea (4/20), arthralgia (15/20), fatigue (16/20), maculopapular rash (1/20), diarrhea (1/20) and mucositis (1/20). At the time of surgery, 12/20 (60%) were ypT0N0 and 13/20 (65%) were ypT0/Tis. 15/20 (75%) achieved an RCB 0/1, 4/20 (20%) were RCB 2 and 1/20 (5%) was RCB 3. Of the 11 patients with node-positive disease at diagnosis, 9/11 (82%) became ypN0, 1/11 (9%) became ypN1mic and 1/11 (9%) became ypN1a. No patients progressed during treatment. Change in TILs after pembro or pembro-RT did not correlate with treatment response, however baseline TIL count ≥10% in the initial biopsy was associated with pCR (p = 0.005). Further profiling of the immune cells in the biopsies revealed that RCB 0/1 patients were strongly associated with increased numbers of CD8+ T cells and lower numbers of CD11b+ myeloid cells following the combination of pembro and RT. Conclusions: Neoadjuvant pembro plus RT prior to SOC chemotherapy is safe/feasible and may increase pCR rates and clearance of nodal metastases beyond chemotherapy alone. A phase 2 study of checkpoint blockade + RT expansion of this trial (n=50) is currently accruing. Citation Format: Heather McArthur, Stephen Shiao, Scott Karlan, Reva Basho, Farin Amersi, Brittany Arnold, Michele Burnison, Alice Chung, Cathie Chung, Catherine Dang, Armando Giuliano, Nimmi Kapoor, Negin Habibi Khameneh, Simon Knott, Cynthia Martin, Philomena McAndrew, Monica Mita, Dorothy Park, Christina DiLauro Abaya, Alice Ho. Pre-operative pembrolizumab (Pembro) with radiation therapy (RT) in patients with operable triple-negative breast cancer (TNBC) [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS12-09.

Abstract PS18-06: Proteomic analysis of breast cancer formalin-fixed paraffin-embedded clinical specimens identifies biologically-important subtypes with distinct clinical outcomes

Abstract Background: Genomic classification of breast cancer has advanced breast cancer diagnosis and outcomes. However, extensive heterogeneity still exists beyond their DNA or RNA profiles. Newer classifications based on protein profiling are being developed to investigate the molecular oncology of breast cancers at the level where most drugs act. Using a recently-developed technology, we performed global proteomic profiling of 300 breast cancer specimens linked to outcome data. Methods: Sections of 75 samples from each PAM50 intrinsic subtype (Luminal A, Luminal B, Her2-enriched, Basal-like; n = 300) were macrodissected and analyzed using the Single-Pot Solid-Phase enhanced Sample Preparation Clinical Tissue Proteomics, a highly sensitive 11-sample multiplex massspectrometry protocol applicable to formalin-fixed, paraffin embedded (FFPE) specimens. This methodology enables comprehensive quantification of protein expression for classifier and biomarker discovery. Patients were diagnosed during 2008-2013 (n = 178, dataset I) and 1986-1992 (n = 122, dataset II). Results: In-depth proteomic analysis measured 9088 proteins in total, including 4214 proteins quantified in every sample. Consensus clustering of 174 evaluable cases in dataset I identified four distinct groups based on expression values for 1054 highly variant proteins. Cluster 3 (n = 47, mostly basal-like with HER2-Enriched) displayed the most favorable recurrence free survival (RFS) when compared to other clusters (HR = 0.22, 95%CI [0.08-0.63], p = 0.005). This cluster was enriched for immune related pathways including antigen processing and presentation and type I &amp; II interferon signaling, and displayed high tumor infiltrating lymphocyte counts, characterizing this cluster as “immune hot”. In contrast, cluster 2 (n = 50, mostly basal-like) exhibited the poorest RFS (HR = 2.88, 95%CI [1.45-5.70], p = 0.002) and was enriched for proteins related to stromal and extracellular matrix with few immune related peptides. Cluster 1 (n = 34, luminal B and HER2-Enriched) was associated with lipid metabolism, whereas cluster 4 (n = 43, mostly HER2-Enriched with luminal A and luminal B) had a profile enriched for extracellular matrix, blood coagulation and complement activation. Conclusions: Global proteomic analysis on FFPE specimens can characterize the heterogeneity of breast cancer in a reliable and clinically-applicable high throughput manner. Our methodology identifies protein candidates that potentially serve as therapeutic targets and could be adapted to archived clinical specimens from other tumors. Citation Format: Karama Asleh, Gian Luca Negri, Sandra E. Spencer Miko, Shane Colborne, Christopher S. Hughes, Xiu Q. Wang, Dongxia Gao, C. Blake Gilks, Stephen K.L. Chia, Torsten O. Nielsen, Gregg B. Morin. Proteomic analysis of breast cancer formalin-fixed paraffin-embedded clinical specimens identifies biologically-important subtypes with distinct clinical outcomes [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS18-06.

Molecular analyses of triple-negative breast cancer in the young and elderly

BackgroundBreast cancer in young adults has been implicated with a worse outcome. Analyses of genomic traits associated with age have been heterogenous, likely because of an incomplete accounting for underlying molecular subtypes. We aimed to resolve whether triple-negative breast cancer (TNBC) in younger versus older patients represent similar or different molecular diseases in the context of genetic and transcriptional subtypes and immune cell infiltration.Patients and methodsIn total, 237 patients from a reported population-based south Swedish TNBC cohort profiled by RNA sequencing and whole-genome sequencing (WGS) were included. Patients were binned in 10-year intervals. Complimentary PD-L1 and CD20 immunohistochemistry and estimation of tumor-infiltrating lymphocytes (TILs) were performed. Cases were analyzed for differences in patient outcome, genomic, transcriptional, and immune landscape features versus age at diagnosis. Additionally, 560 public WGS breast cancer profiles were used for validation.ResultsMedian age at diagnosis was 62 years (range 26–91). Age was not associated with invasive disease-free survival or overall survival after adjuvant chemotherapy. Among the BRCA1-deficient cases (82/237), 90% were diagnosed before the age of 70 and were predominantly of the basal-like subtype. In the full TNBC cohort, reported associations of patient age with changes in Ki67 expression, PIK3CA mutations, and a luminal androgen receptor subtype were confirmed. Within DNA repair deficiency or gene expression defined molecular subgroups, age-related alterations in, e.g., overall gene expression, immune cell marker gene expression, genetic mutational and rearrangement signatures, amount of copy number alterations, and tumor mutational burden did, however, not appear distinct. Similar non-significant associations for genetic alterations with age were obtained for other breast cancer subgroups in public WGS data. Consistent with age-related immunosenescence, TIL counts decreased linearly with patient age across different genetic TNBC subtypes.ConclusionsAge-related alterations in TNBC, as well as breast cancer in general, need to be viewed in the context of underlying genomic phenotypes. Based on this notion, age at diagnosis alone does not appear to provide an additional layer of biological complexity above that of proposed genetic and transcriptional phenotypes of TNBC. Consequently, treatment decisions should be less influenced by age and more driven by tumor biology.

Infiltrating immune cells in benign breast disease and risk of subsequent invasive breast cancer

BackgroundIt is well established that tumors are antigenic and can induce an immune response by the host, entailing lymphocytic infiltration of the tumor and surrounding stroma. The extent and composition of the immune response to the tumor, assessed through evaluation of tumor-infiltrating lymphocyte counts, has been shown in many studies to have prognostic and predictive value for invasive breast cancer, but currently, there is little evidence regarding the association between infiltrating immune cell counts (IICCs) in women with benign breast disease (BBD) and risk of subsequent invasive breast cancer.MethodsUsing a cohort of 15,395 women biopsied for BBD at Kaiser Permanente Northwest, we conducted a nested case-control study in which cases were women who developed a subsequent invasive breast cancer during follow-up and controls were individually matched to cases on age at BBD diagnosis. We assessed IICCs in normal tissue and in the BBD lesions, and we used unconditional logistic regression to estimate the multivariable odds ratios (OR) and 95% confidence intervals (CI) for the associations between IICCs and breast cancer risk.ResultsThere was no association between the IICC in normal tissue (multivariable OR per 5% increase in IICC = 1.05, 95% CI = 0.96–1.16) or in the BBD lesion (OR per 5% increase in IICC = 1.06, 95% CI = 0.96–1.18) and risk of subsequent invasive breast cancer. Also, there were no associations within subgroups defined by menopausal status, BBD histology, BMI, and history of smoking.ConclusionThe results of this study suggest that IICCs in BBD tissue are not associated with altered risk of subsequent invasive breast cancer.

Expression of immune response biomarkers (PD‑L1, p16, CD3+ and CD8+ TILs) in recurrent head and neck squamous cell carcinoma within previously irradiated areas.

The immune landscape of head and neck squamous cell carcinoma in pretreated areas remains poorly documented. We aimed to assess the tumor microenvironment for biomarkers of antitumor immune responses in tumors in previously irradiated areas compared with denovo tumors. This retrospective monocentric study analyzed 100 paraffin‑embedded surgical samples of invasive head and neck squamous cell carcinoma (oral cavity, oropharynx, larynx, hypopharynx) from patients who underwent surgery between January2010 and November2017. We compared the immune microenvironment in 50denovo tumors and 50tumors recurring within irradiated areas. We used immunohistochemistry to assess p16 status, CD3+/CD8+ tumor‑infiltrating lymphocytes (TILs), and programmed death‑ligand1 (PD‑L1) expression on tumor and immune cells in stromal and intratumoral components. CD3+ TIL counts were significantly lower in intratumoral and stromal components (P=0.003 and P=0.020, respectively) in the irradiated area cohort; there was no significant difference between CD8+TIL counts in the two cohorts. The percentage of tumors with PD‑L1+ tumor cells (tumor proportion score≥1%) was significantly lower within the irradiated area cohort than the denovo cohort (56.0%vs.86.0%, P<0.001). There were also significantly fewer tumors with PD‑L1+ immune cells in the irradiated area cohort. Predominantly, tumors from the irradiated area cohort had microenvironments classified as 'adaptive immune resistance'. There was persistence of cytotoxic cells in tumors in the irradiated areas but lower PD‑L1 expression and CD3+ TIL counts than in the denovo tumors. This offers an initial hypothesis to explain why these lesions are less responsive to immunotherapy, even though they may still have antitumor capacities. Assessment of immune response biomarkers in patients treated with immunotherapy in randomized trials is required.

Programmed Death-Ligand 1 and Programmed Death-Ligand 2 Expression Can Affect Prognosis in Extramammary Paget's Disease.

Extramammary Paget's disease (EMPD) is a type of carcinoma that usually progresses slowly but may cause metastasis and subsequent death of patients. We investigated the relationship between the expression of programmed death-ligand 1 (PD-L1)/programmed death-ligand 2 (PD-L2) and stromal CD8+ tumor-infiltrating lymphocytes (TILs) in EMPD and clinicopathological findings, including prognosis. We examined 47 cases of EMPD and performed immunohistochemical staining of formalin-fixed paraffin-embedded full-face sections. PD-L1 expression in tumor cells was observed in 13 cases (27.7%) while PD-L2 expression was observed in 21 cases (44.7%). The cumulative postoperative recurrence-free rate in the group with positivity for PD-L1 and/or PD-L2 with a low CD8+ TIL count was significantly lower than that of the corresponding group with a high CD8+ TIL count and of the PD-L1- and PD-L2-negative group (p=0.026). The expression of PD-L1/PD-L2 in tumor cells was shown to be a factor for poor prognosis.

BDNF expression in GISTs predicts poor prognosis when associated with PD-L1 positive tumor-infiltrating lymphocytes

ABSTRACT Substantial evidence indicates that brain-derived neurotrophic factor (BDNF) plays an important role in tumorigenesis, in addition to its primary role in neuronal activity. Gastrointestinal stromal tumors (GISTs), which are the most common mesenchymal neoplasms of the gastrointestinal tract, contain multiple types of tumor-infiltrating lymphocytes (TILs) that express relevant immune checkpoint proteins. However, no data have been reported on the role of BDNF in GISTs. This study aimed to investigate the expression pattern and prognostic value of BDNF in GIST patients with different degrees of risk, as well as the relationship between BDNF expression and immune checkpoints. Immunohistochemistry (IHC) demonstrated that higher BDNF expression was more likely to be present in high-risk patients and suggested a poor prognosis. A similar phenomenon was demonstrated in plasma. Even more interesting was that a positive correlation was present between BDNF and PD-L1+ expression on TILs. Moreover, high BDNF expression levels in combination with a high PD-L1+ TIL count predict extremely poor survival. The combination of BDNF expression and TIL PD-L1+ expression as a single biomarker was a powerful significant independent predictor of prognosis. Taken together, BDNF expression may serve as a significant prognostic factor, as the combination of BDNF expression and the PD-L1+ TIL subset led to superior prediction of GIST prognosis. Furthermore, our research coupled a neurotrophin with immunity, which provides novel evidence of neural and immune regulation in a clinical study of GIST.

CD8+ T-cell lymphocytes infiltration predict clinical outcomes in Wilms' tumor.

The abundance and location of CD8+ tumor-infiltrating lymphocytes demonstrate important facets of the anticancer immune response. CD8-expressing lymphocytes have been used in immunotherapy for multiple cancers. This study aims to determine the association between the abundance and localization of CD8+ tumor-infiltrating lymphocytes and clinical outcomes of Wilms' tumor. This retrospective study employed 42 pediatric patients diagnosed with Wilms' tumor. CD8+ tumor-infiltrating lymphocyte counts were calculated based on the mean percentage of stroma occupied by CD8+ lymphocytes at the center and the invasive border of the tumor using immunohistochemistry. CD8+ tumor-infiltrating lymphocyte counts were significantly higher in the center and the invasive border of the early-stage tumor samples. CD8+ tumor-infiltrating lymphocytes in the invasive border and tumor center positively correlated with tumor invasion, regional lymph node invasion, histological type, metastasis, and stage of the tumor. A high CD8+ tumor-infiltrating lymphocyte scores at the invasive margin of the tumor correlated with low tumor recurrence. Low CD8+ tumor-infiltrating lymphocyte scores in the two tumor regions correlated with poor prognosis and shorter disease-free survival. Overall, these findings show that patients with high CD8+ tumor-infiltrating lymphocytes are associated with better clinical outcomes. Therefore, measuring the abundance of CD8+ tumor-infiltrating lymphocytes may be useful in predicting response to cancer immunotherapies.

A role for the immune system in advanced laryngeal cancer

To investigate the role of the altered activation of the immune system in the prognosis of patients affected by laryngeal squamous cell carcinoma (LSCC). We analyzed 56 patients with advanced LSCC divided into two groups according to their prognosis: the first group relapsed within 24 months after treatment, the second group had no evidence of disease at 2 years. The presence of stromal tumor infiltrating lymphocytes (TILs) at the tumor-host border was investigated. In 43 patients we evaluated the expression of 395 genes related to immune system activation through a next generation sequencing panel. Priority-LASSO models and clustering analyses were integrated with multivariate Cox proportional hazard modeling to identify independent genes associated with relapse and estimate hazard ratios in relation to gene expression and TILs. TILs and the expression of genes related with immune system activation (FCGR1A, IFNA17, FCRLA, NCR3, KREMEN1, CD14, CD3G, CD19, CD20 and CD79A) were significantly associated with prognostic factors or disease specific survival. In patients with lymph node metastases and advanced T stage (pT4), the expression of other genes was altered. Low TILs count was highly associated with relapse within 2 years (p < 0.001). Low TILs and altered expression of specific genes associated with tumor-immune systems interactions emerged as independent risk factors, associated to poor prognosis and relapse within 2 years in advanced LSCC. Evaluation of patients’ immune profile could be useful for prognosis and future therapeutic approaches towards personalized therapy.

Prognostic impact of tumor microvessels in intrahepatic cholangiocarcinoma: association with tumor-infiltrating lymphocytes.

Tumor microvessel density (MVD) is a prognostic factor for patients with intrahepatic cholangiocarcinoma (ICC). Tumor-infiltrating lymphocytes (TILs) are also key components of the tumor microenvironment that play important roles in ICC progression. This study aimed to clarify the relationships between the MVD and immune status and prognosis in patients with ICC. Immunohistochemical staining for cluster of differentiation 34 (CD34), cluster of differentiation 8 (CD8), forkhead box protein P3 (Foxp3), and programmed death-ligand 1 (PD-L1) was performed. The relationships between the MVD and clinicopathological characteristics and outcomes were analyzed. Additionally, the correlations between the MVD, CD8+ and Foxp3+ TIL counts, and PD-L1 expression were evaluated. One hundred ICC patients were classified into high (n = 50) and low (n = 50) MVD groups. The serum platelet and carbohydrate antigen 19-9 levels were higher in the low MVD group than in the high MVD group (P = 0.017 and P = 0.008, respectively). The low MVD group showed a significantly larger tumor size (P = 0.016), more frequent microvascular invasion (P = 0.001), and a higher rate of intrahepatic (P = 0.023) and lymph node (P < 0.001) metastasis than the high MVD group. Moreover, the MVD showed a high positive correlation with CD8+ TILs (r = 0.754, P < 0.001) and a negative correlation with Foxp3+ TILs (r = -0.302, P = 0.003). In contrast, no significant correlation was observed between the MVD and PD-L1 expression in cancer cells (P = 0.817). Patients with low MVDs had a significantly worse prognosis than those with high MVDs. Furthermore, multivariable analyses revealed that a low MVD influenced recurrence-free survival. A decreased intratumoral MVD might predict ICC patient outcomes. Tumor microvessels might be associated with ICC progression, possibly by altering TIL recruitment.

Digital Image Analysis of CD8+ and CD3+ Tumor-Infiltrating Lymphocytes in Tongue Squamous Cell Carcinoma.

PurposeThe presence of CD8+ tumor-infiltrating lymphocytes (TILs) has been reported to be associated with treatment outcomes in many types of solid tumors. However, the results vary due to the various methods of visual estimation and subjective interpretation. The current study is the first to use digital image analyses to evaluate the density of CD8+/CD3+ TILs in tongue squamous cell carcinoma (TSCC).Patients and MethodsFrom 2005 to 2015, a cohort of 258 TSCC patients were consecutively enrolled in this study. After immunohistochemistry on representative sections, the density of CD8+/CD3+ TILs at tumor invasive margins was evaluated by digital image analysis. The subjects were stratified by the median values of CD3+ cell density, CD8+ cell density, CD8/CD3, and scores (0, 1, and 2) to demonstrate the association with various clinicopathological manifestations.ResultsLow CD8+ TIL counts were associated with advanced tumor stages (p=0.034), and low CD8/CD3 ratios were associated with perineural invasion (p=0.043). Both parameters were also associated with increased tumor depths (p=0.034 and 0.004, respectively). Univariate analyses revealed that advanced tumor stages, perineural invasion, extranodal extension, tumor depth, lower CD8 counts, and lower scores (score 0 vs 2) were associated with poorer overall survival, and multivariate analysis further indicated that extranodal extension and low scores (score 0 vs 2) were both independent factors for overall survival (p < 0.0001 and p = 0.0369, respectively).ConclusionThe use of digital image analysis to assess CD8+ TILs at the invasive margins provides an objective indicator of prognoses including overall survival.

Postoperative radiotherapy for laryngeal cancer. The prognostic role of programmed death-ligand 1: An immune microenvironment-based cluster analysis

BackgroundThe prognostic role of programmed death-ligand 1 (PD-L1) expression and the tumor’s immune microenvironment has yet to be investigated in the specific setting of adjuvant postoperative radiotherapy (PORT) for laryngeal carcinoma (LSCC). The main aim of this exploratory study was to investigate, also by cluster analysis, whether PD-L1 expression (in terms of combined positive score [CPS]), tumor-infiltrating lymphocytes (TIL), and tertiary lymphoid structures (TLS) correlated prognostically with response to PORT in a cohort of consecutive LSCC patients. MethodsPD-L1, TIL and TLS were assessed in 24 consecutive patients with LSCC who underwent PORT. Cluster analysis was used to classify cases on the strength of these parameters. ResultsA CPS ≥ 1 was associated with a significantly lower recurrence rate (p = 0.033), and longer disease-free survival (DFS) (p = 0.012) than a CPS < 1. Two clusters of prognostic relevance emerged from our analysis. Cluster 1 was characterized by a mean CPS of 23.0 ± 37.9, a mean TIL count of 68.0 ± 16.4, and the presence of TLS in all cases. Cluster 2 featured a mean CPS of 3.1 ± 7.3, a mean TIL count of 23.9 ± 16.5, and no cases with TLS. Cluster 1 showed a trend towards a lower recurrence rate (p = 0.071) and longer DFS (p = 0.054) than cluster 2. ConclusionsJudging from this preliminary investigation, assessing PD-L1 and immune microenvironment markers seems a promising approach for identifying patients at higher risk of LSCC recurrence after PORT, who might reasonably benefit from adjuvant postoperative chemo-RT, or immunotherapy.

Tumor-infiltrating lymphocytes (TILs) in ER+/HER2- breast cancer.

The prognostic role of tumor-infiltrating lymphocytes (TILs) in ER+/HER2- breast cancer (BC) is debated. We evaluated the association of TILs and clinico-pathological features with distant disease-free survival (DDFS) in patients with ER+/HER2- BC treated at a single institution. A mono-institutional case-cohort series of 987 patients with early ER+/HER2- BC was retrospectively analyzed. TILs were considered both as continuous variable, and dichotomized in low (< 5%) vs high (≥ 5%). The main outcome was DDFS. Median follow-up was 7.5years (0.1-10). Univariate and multivariable Cox proportional hazards regression with inverse sub-cohort sampling probability weighting were used to evaluate the risk across groups. Median TIL count was 2% (Q1-Q3 1-4%). Higher TILs were positively associated with number of lymph nodes involved (p = 0.003), tumor grade (p < 0.0001), peritumoral vascular invasion (p = 0.003), higher Ki-67 (p = 0.0001), luminal B subtype (p < 0.0001), and chemotherapy use (p < 0.00019). In multivariable regression analysis, only higher Ki-67 expression retained significant association with TILs. At univariate Cox regression analysis, TIL expression (≥ 5% vs. < 5%) was not associated with DDFS (HR 1.08, 95% CI 0.80-1.46, p = 0.62). In patients treated with adjuvant chemotherapy, high TILs were associated with better DDFS (HR 0.52, 95%CI 0.33-0.83, p = 0.006), particularly in the group with Ki-67 ≥ 20% (HR 0.50, 95%CI 0.29-0.86, p = 0.01). High TILs in ER+/HER2- BC are significantly associated with clinico-pathological features of dismal outcome. TIL prognostic value seems different in patients treated with or without chemotherapy. Our findings suggest that the high-risk subgroup might be more immunogenic, thus deserving the exploration of immunotherapy approaches.

Prognostic value of tumor-infiltrating lymphocytes (TILs) and their association with PD-L1 expression and DNA repair protein RAD51 in patients with resected non-small cell lung carcinoma

ObjectivesDNA repair proteins have emerged as potential predictors for immunotherapy response alongside PD-L1 expression, tumor-infiltrating lymphocytes (TILs) and tumor mutational burden. We analyzed expression of PD-L1, TILs count and expression of the homologous recombination (HR) protein RAD51, as potential prognostic factors in patients with resected non-small-cell lung carcinoma (NSCLC). Materials and methodsDiscovery set included 96 NSCLC patients from the University Hospital Olomouc (Czech Republic) and a replication set included 1109 NSCLC patients from University Hospital Zurich (Switzerland). Tissue microarrays (TMAs) were stained using the automated staining platform Ventana Benchmark Ultra with antibodies against RAD51,CD3, CD8, CD68 and PD-L1. ResultsLoss of nuclear RAD51 protein was associated with high TILs (r=-0.25, p = 0.01) and PD-L1 status (10.6 vs. 2.4 %, p = 0.012) in patients receiving neoadjuvant chemo-/radiotherapy (CT/RT). In silico analysis from the TCGA data set showed a negative relationship between RAD51 mRNA expression and CD45 (r = ‒0.422, p < 0.0001), CD68 (r = ‒0.326, p < 0.001), CD3 (r = ‒0.266, p < 0.001) and CD8 (r = ‒0.102, p < 0.001). RAD51 low/PD-L1 high patients were clustered as separate entity in the replication set and in TCGA dataset. High TILs status was significantly associated with improved OS in the replication set (unadjusted HR = 0.57, 95 % CI 0.42−0.76, p < 0.001). Similar results have been seen for CD3, CD8 and CD68. ConclusionsIn conclusion, RAD51 nuclear loss is weakly associated with increased TILs and high PD-L1 at the time of surgery in curatively resected NSCLC and after prior exposure to neoadjuvant chemo- or radiotherapy. Both high TILs and RAD51 nuclear loss were confirmed as independent prognostic factors in curatively resected NSCLC.

The relationships between PD-L1 expression, CD8+ TILs and clinico-histomorphological parameters in malignant melanomas

IntroductionMalignant melanomas (MM) are often connected with the expression of PD-L1 protein and the presence of tumor-infiltrating lymphocytes (TILs), however, their impact on prognosis remains controversial. Due to their supposed clinical significance and lack of convincing data, we decided to establish the relationships between CD8 + TIL count, PD-L1 level and certain clinical and histopathological parameters in patients with malignant melanoma, especially those associated with unfavorable prognosis. Materials and methodsWe performed immunohistochemistry for PD-L1 and CD8 on 56 formalin-fixed paraffin-embedded specimens from patients with cutaneous and metastatic malignant melanomas. PD-L1 expression levels were determined by immunohistochemistry (clone 28-8) and subsequently the tumor proportion scores (TPS) were evaluated. CD8 + TIL expressions were classified as either grade 0, 1+, 2+ or 3+, based on the density and distribution of the infiltrating lymphocytes. ResultsThe PD-L1 expression was detected in 20 out of 56 cases (35,71 %). The expression of PD-L1 on tumor cells was significantly increased with higher TILs infiltration in the tumor microenvironment (p = 0,038). Lower TIL score corresponds with poor prognostic clinicopathological parameters such as higher number of mitotic figures (p = 0,005), Clark's level (p = 0,007) and Breslow's depth (p = 0,010). ConclusionsOur results suggest a favorable prognostic value for CD8 + TIL infiltration. Moreover, TIL density was strongly correlated and geographically associated to PD-L1 expression. This analysis provides more insight into the role of TIL count and PD-L1 level in MM and their relationship with each other and association with other prognostic indicators.

Clinical and pathological associations of PTEN expression in ovarian cancer: a multicentre study from the Ovarian Tumour Tissue Analysis Consortium

BackgroundPTEN loss is a putative driver in histotypes of ovarian cancer (high-grade serous (HGSOC), endometrioid (ENOC), clear cell (CCOC), mucinous (MOC), low-grade serous (LGSOC)). We aimed to characterise PTEN expression as a biomarker in epithelial ovarian cancer in a large population-based study.MethodsTumours from 5400 patients from a multicentre observational, prospective cohort study of the Ovarian Tumour Tissue Analysis Consortium were used to evaluate associations between immunohistochemical PTEN patterns and overall survival time, age, stage, grade, residual tumour, CD8+ tumour-infiltrating lymphocytes (TIL) counts, expression of oestrogen receptor (ER), progesterone receptor (PR) and androgen receptor (AR) by means of Cox proportional hazard models and generalised Cochran–Mantel–Haenszel tests.ResultsDownregulation of cytoplasmic PTEN expression was most frequent in ENOC (most frequently in younger patients; p value = 0.0001) and CCOC and was associated with longer overall survival in HGSOC (hazard ratio: 0.78, 95% CI: 0.65–0.94, p value = 0.022). PTEN expression was associated with ER, PR and AR expression (p values: 0.0008, 0.062 and 0.0002, respectively) in HGSOC and with lower CD8 counts in CCOC (p value < 0.0001). Heterogeneous expression of PTEN was more prevalent in advanced HGSOC (p value = 0.019) and associated with higher CD8 counts (p value = 0.0016).ConclusionsPTEN loss is a frequent driver in ovarian carcinoma associating distinctly with expression of hormonal receptors and CD8+ TIL counts in HGSOC and CCOC histotypes.