High Lymphocyte Infiltration Research Articles

Epstein-Barr Virus-Associated Pulmonary Carcinoma: Proposing an Alternative Term and Expanding the Histologic Spectrum of Lymphoepithelioma-like Carcinoma of the Lung.

Lymphoepithelioma-like carcinoma (LELC) of the lung is a rare Epstein-Barr virus (EBV)-associated carcinoma. It is histologically characterized by a syncytial growth pattern with marked lymphocytic infiltration that is indistinguishable from the histology observed in undifferentiated nasopharyngeal carcinomas. However, it has been noted that LELC can display nonclassic morphology and lack significant lymphocytic infiltration. In this study, we conducted a comprehensive clinicopathologic analysis of 61 patients with pulmonary LELC and performed automatic quantification of the lymphocytic infiltrate using the IHC Profiler software. We demonstrated that pulmonary LELCs have a morphologically continuous spectrum, ranging from classic poorly differentiated tumors with intense lymphocytic infiltration to nonclassic morphology with little lymphocytic infiltration. These EBV-associated tumors represent a distinct entity and usually occur in female and nonsmoking patients. Tumors with low lymphocytic infiltration can closely resemble nonkeratinizing squamous cell carcinoma and tend to be larger in size, have higher maximum standardized uptake values on radiography, and exhibit shorter times to recurrence than those with high lymphocytic infiltration. Through detailed pathologic examination, we observed several distinct morphologic features in pulmonary LELCs, including granulomatous inflammation, focal keratinization, spread through alveolar spaces, and lepidic spreading pattern. We also found that patients with tumors exhibiting granulomatous inflammation have favorable outcomes; however, spread through alveolar spaces did not significantly correlate with prognosis. As many of these "LELCs" do not resemble undifferentiated nasopharyngeal carcinoma or lymphoepithelioma, we propose using an alternative term, EBV-associated pulmonary carcinoma, to encompass the entire morphologic spectrum of this distinct disease entity.

Abstract 3141: Immune gene expression and prognosis in localized clear cell (cc) renal cell carcinoma (RCC)

Abstract Background: We previously reported that increased infiltration of immune cells (lymphocytes, plasma cells, macrophages, neutrophils) in localized ccRCC is associated with recurrence. In these tumors we now study the immune gene expression to identify a signature to predict recurrence. Methods: We identified pts with ccRCC with >T1b tumor who underwent nephrectomy for whom we had annotation for objective tumor recurrence and a minimum follow-up of 2 years. All histologic slides were examined and tumor section with the highest amount of intratumoral immune infiltration were selected from each tumor for gene expression profiling. Total mRNA isolated from the macrodissected FFPE tumor underwent gene expression profiling for 770 genes in the Immune Profile Panel by NanoString Technologies. Digital raw counts of mRNA abundance were normalized using positive controls as well as 16 housekeeping genes. The mean signals were used to calculate fold change in gene expression between recurrers and non-recurrers, and a p-value <0.05 using empirical Bayes methods was considered significant. Cox regression was also conducted to correlate gene expression with time to recurrence. Results: Of 132 patients (pts), 24 (18%) had recurrence and 108 did not. The median age of pts was 59 years and 57 were female. The median time to recurrence was 25.7 months. Fifty pts had low immune infiltration score and 82 pts had high infiltration based on previously conducted morphologic assessment. Only 4 genes (IL8, NCAM1, ARG2, PPBP) had >1.5 times increased mean expression in recurrers vs non-recurrers (p<0.05). Only 5 genes (CX3CL1, HLA-G, VCAM1, IL17RB, CXCL14) had >1.5 times decreased mean expression in recurrers vs non-recurrers (p<0.05). Overexpression of the following immunoregulatory genes was associated with a shorter time to recurrence: TGFB1, STAT3, NFATC4, B7-H3, ADORA2A, OX-40L, IL10, RUNX1, LILRA4, MARCO, CR1, FCER2, IL2RA (HR 1.5-3.6, p<0.05). When compared to pts with low lymphocyte infiltration, pts with high lymphocyte infiltration overexpressed T cell (CD3E, CD5), cytotoxic T cell (CD8B, SLAMF6, IL2RB, GZMK, TCF7, CTSW, EBI3), Th1 (CD38, CXCR3, CXCR6, IL16), Th2 (ITK) and Tregs (IL2RB, IRF4, TIGIT) genes (FDR p<0.01). Conclusions: Specific immune related somatic genes were differentially expressed based on recurrence and time to recurrence in patients with localized ccRCC undergoing surgery. With further validation, these genes warrant functional validation since they may represent therapeutic targets in the perioperative setting. The results also highlight the heterogeneity of immune cell infiltration. Citation Format: Pooja Ghatalia, Karthik Devarajan, Jennifer Gordetsky, Essel Dulaimi, Sejong Bae, Gurudatta Naik, Guru Sonpavde, Elizabeth Plimack. Immune gene expression and prognosis in localized clear cell (cc) renal cell carcinoma (RCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3141.

Molecular signatures in hepatocellular carcinoma: A step toward rationally designed cancer therapy

Molecular characterization of hepatocellular carcinoma (HCC) has greatly improved our understanding of disease pathogenesis. Mutational analysis, RNA and microRNA expression profiling, and epigenetic characterization have revealed common aberrations in oncogenes and tumor suppressors that correlate with disease biology and serve as a guide for the rational design of targeted therapies. These approaches have also led to the discovery of novel targets, including mutations in isocitrate dehydrogenase and chromatin remodeling enzymes. With the advent of immunotherapy, RNA expression profiling of the tumor microenvironment has identified a subset of HCC with high lymphocyte infiltration that may benefit from checkpoint inhibitor therapy. Molecular signatures thus capture the biology of a tumor, providing a supplement to current staging schema, which are based on tumor size and number, for more accurate prognostication of recurrence risk and survival. Molecular signatures may also be used to guide interventional therapy by defining those most suitable for transplantation or locoregional therapy rather than surgical resection. Finally, a multiomics approach involves the aggregation and analysis of multiple signatures for a more comprehensive characterization of pathogenic mechanisms. This broader approach attempts to address issues with signaling pathway cross-talk and redundancy, which have greatly limited the potential value of targeted therapies to date. Cancer 2018. © 2018 American Cancer Society.

PD-L1 and PD-1 and characterization of tumor-infiltrating lymphocytes in high grade sarcomas of soft tissue – prognostic implications and rationale for immunotherapy

ABSTRACTTherapies targeting programmed death 1-(PD-1) or its ligand (PD-L1), promoting antitumor T-cell activity have been successfully introduced into clinical practice. Clinical response correlates with PD-L1 expression by tumor cells or immune cells within the tumor microenvironment. The PD-L1/PD-1 axis and tumor microenvironment has been rarely studied in high-grade sarcomas of soft tissue (hSTS), a group of rare, genetically heterogenous and clinically aggressive tumors. We examined PD-L1 protein and CD274/PD-L1 gene copy number variations in 128 primary resected, therapy-naive hSTS using immunohistochemistry and fluorescence-in-situ hybridization. Frequency of tumoral PD-L1 expression varied widely in different disease subentities, with highest rates of positivity (40%) seen in undifferentiated pleomorphic sarcomas (UPS) and rare positivity detected in synovial sarcomas (6%). Amplification of the CD274/PD-L1 gene occurred in 14% of UPS and was rare in other subtypes. PD-L1 protein expression was significantly more frequent in CD274/PD-L1 amplified cases (p = 0.015). The subgroup of UPS was further characterized regarding the interaction between PD-L1 and the immunologic tumor microenvironment. High density of CD3+ and CD8+ tumor infiltrating lymphocytes (TILs) was significantly correlated with the presence of PD-L1 expression and seen more frequently in tumors with lower TNM stage (p = 0.024). Both, PD-L1 expression and high density lymphocytic infiltration were independent prognostic factors for a favorable overall (p = 0.001, HR 6.105 (2.041–8.258)), disease-specific (p = 0.003, HR 10.536 (2.186–50.774)) and disease-free survival (p = 0.020, HR 3.317 (1.209–9.106); values for CD8) in this particular subgroup of hSTS, whereas PD-L1 expression in TILs or CD274/PD-L1 gene amplification were not associated with outcome. These findings represent novel insights into the immune landscape of soft tissue sarcomas, in particular UPS and strengthen the rationale for immunotherapy, including targeting the PD-1/PD-L1 axis in these tumors.

Survival impact of lymphocyte infiltration into the tumor of hepatocellular carcinoma in hepatitis B virus-positive or non-B non-C patients who underwent curative resection.

The prognostic value of lymphocyte infiltration into hepatocellular carcinoma (HCC) is still controversial, and it has not been reported in hepatitis B virus (HBV)-positive or non-B non-C (NBNC) HCC. The aim of this study is to assess the prognostic significance of lymphocyte infiltrate in tumor for HBV-positive and NBNC HCC patients. This study investigated 145 HBV-positive or NBNC patients who underwent hepatectomy for HCC between January 2001 and May 2009. Cumulative recurrence rate, overall survival (OS), and clinicopathological parameters were analyzed according to lymphocyte infiltration in tumor. In patients with low lymphocyte infiltration, the 5-year recurrence rate was higher and OS was poor (86.4 and 44.1%, respectively) than that of the patients with high lymphocyte infiltration (55.3 and 83.7%, respectively). Multivariate analyses revealed that independent risk factors for recurrence were low albumin value (hazard ratio [HR] 2.33, P = 0.009), high American Joint Committee on Cancer (AJCC) T stage (HR 2.31, P < 0.0001), high α-fetoprotein (AFP) value (HR 2.06, P = 0.005), and low lymphocyte infiltration (HR 2.50, P = 0.0001). The independent risk factors for OS were low albumin value (HR 3.69, P = 0.003), high AJCC T stage (HR 2.10, P = 0.049), high AFP value (HR 3.98, P < 0.001), and low lymphocyte infiltration (HR 3.47, P = 0.001). Lymphocyte infiltrate in tumor is significantly associated high recurrence rate and poor overall survival. Evaluation of the infiltrating lymphocyte could improve the prediction of prognosis in HCC patients after curative resection.

Abstract 588: Advanced molecular characterization of severe autoimmune toxicities associated with checkpoint inhibitor therapies

Abstract Immune checkpoint inhibitors (ICIs) have made a profound impact on the treatment of a variety of cancers. However, as with any systemic treatment, toxicities are inevitable. With most classes of cancer therapies, toxicities are relatively predictable based on clinical trial safety data and therefore can be handled with prophylactic or supportive care measures. However, ICIs are unique in their ability to cause rare but severe auto-immune toxicities. The molecular underpinnings of these toxicities, as well as unique features of the patient, tumor, or affected tissue, have not been extensively explored. We recently reported a small case series of two patients with myocarditis resulting in death arising following combination ICI therapy (Johnson et al, N Engl J Med, 2016). High lymphocytic infiltration, coupled with PD-L1 expression was present in the affected myocardium and skeletal muscle. Common T cell clones were identified between the affected tissue and tumor, and abnormal expression of muscle-specific transcripts was identified in the associated tumor, suggesting release of peripheral tolerance to tumor-expressed self-antigens.To expand upon our reported study, we collected healthy and afflicted tissue from a series of cancer patients with immune-related colitis, myocarditis (MC), and encephalopathy following ICI treatment. We hypothesize that molecular analysis of these tissues will identify causal factors in the etiology of these toxicities, and how to better predict, prevent, and treat them. Thus, we performed molecular characterization of the immune infiltrate and diseased tissue microenvironment. A total of 20 affected (colon, cardiac, brain) and non-diseased control specimens were examined by spatial digital profiling (nanoString). This process generates a spatial heatmap of digital counts of 20 selected immunology and cellular markers and proteins across each specimen. Using this technology, the landscape of inflammation in ICI-affected organs can be resolved for insights into the mechanism whereby ICI-mediated auto-immunity occurs. Targeted RNAseq for selected immuno-oncology mRNA targets was also performed. In initial RNA sequencing analyses of MC cases, affected myocardium, skeletal muscle, and patient-matched tumors all demonstrated expression of immune activation markers (e.g. interferon-gamma and granzyme B), expression of PD-L1, and muscle-specific genes. In the expanded population, including colitis, digital spatial profiling analyses and targeted NGS (RNAseq) are underway. Although data analyses are incomplete at the time of this abstract, this work will be the largest and most comprehensive analysis of the molecular underpinnings of ICI-mediated auto-immune toxicity reported to date. These data should offer clarity in the mechanisms and features of these adverse events, how to prevent or predict them with precision medicine, and how to treat them when they do occur. Citation Format: Justin M. Balko, Daniel Y. Wang, Yu Wang, Rami Al-Rohil, Margaret Compton, Jeffery A. Sosman, Igor Puzanov, Bret Mobley, Robert D. Hoffman, Yaomin Xu, Javid J. Moslehi, Chanjuan Shi, Douglas B. Johnson. Advanced molecular characterization of severe autoimmune toxicities associated with checkpoint inhibitor therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 588. doi:10.1158/1538-7445.AM2017-588

Predictive value of the combination of SMAD4 expression and lymphocyte infiltration in malignant transformation of oral leukoplakia

Oral leukoplakia (OL) is a common, potentially malignant disorder of the oral cavity. SMAD4 was initially identified as a tumor suppressor and central mediator of transforming growth factor (TGF)‐β signaling. In this study, we aimed to determine the expression patterns of SMAD4 in OL, its relationship with the degree of inflammation, and its clinical implications as a biomarker for OL malignant transformation. A total of 150 patients with OL were enrolled in this study. Paraffin‐embedded sections obtained from biopsy or resection specimens were subjected to immunohistochemical analysis. Associations among the status of epithelial SMAD4 expression, stromal lymphocyte infiltration, and malignant transformation of OL were examined. Malignant transformation was significantly associated with the status of SMAD4 expression (P = 0.0017) and lymphocyte infiltration status (P = 0.0054). Cox regression analysis, based on the event‐free survival (EFS), revealed that a low SMAD4 expression was a significant prognostic factor in OL patients (hazard ratio, 2.632; P = 0.043). In addition, a low SMAD4 expression was closely correlated with high lymphocyte infiltration (P = 0.00035), resulting in a significant correlation between the combination of low SMAD4 expression and high lymphocyte infiltration with malignant transformation of OL (P = 0.00027). The combination of the status of epithelial SMAD4 expression and stromal lymphocyte infiltration may be a useful biomarker for predicting malignant transformation in OL patients. These results suggest that not only epithelial SMAD4 loss, but also stromal features, may regulate the risk of malignant transformation of OL.

Folic acid protects against lead acetate-induced hepatotoxicity by decreasing NF-κB, IL-1β production and lipid peroxidation mediataed cell injury

Folic acid plays an important role in cellular metabolic activities. The present study was designed to investigate the protective effect of folic acid against lead acetate-induced hepatotoxicity. Twenty four male Wistar albino rats were randomly divided into four groups, six animals each. Negative control group received the vehicle, positive control group received 1mg/kg folic acid for five consecutive days/week for 4 weeks orally, lead-exposed group received 10mg/kg lead acetate intraperitoneally (IP) for five consecutive days/week for 4 weeks, and lead-treated group received 10mg/kg lead acetate IP and 1mg/kg folic acid orally for five consecutive days/week for 4 weeks concurrently. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and γ- glutamyltransferase (GGT) were measured. Hepatic total peroxide and interleukin-1β (IL-1β) were also investigated. Histopathological studies using hematoxylin-eosin (H&E) and periodic acid shiff's (PAS) were carried out. The expression of nuclear factor kappa B (NF-κB) was evaluated using immunohistochemistry. Serum AST, ALT and GGT and hepatic total peroxide and IL-1β were significantly increased in lead-exposed group and were positively correlated with hepatic lead level. Moreover, lead-exposed rats showed hydropic degeneration, nuclear vesiculation, high lymphocytic infiltration, depletion of glycogen content and NF-κB expression. Concomitant folic acid administration resulted in a significant alleviation of biochemical and structural alteration-induced by lead. This was associated with reduction of hepatic total peroxide and IL-1β and reduction of NF-κB expression. In conclusion, folic acid protects against lead acetate-induced hepatotoxicity by decreasing NF-κB, IL-1β production and lipid peroxidation mediataed cell injury.

Identification of BRCA1-like triple-negative breast cancers by quantitative multiplex-ligation-dependent probe amplification (MLPA) analysis of BRCA1-associated chromosomal regions: a validation study.

BackgroundTriple-negative breast cancer (TNBC) with a BRCA1-like molecular signature has been demonstrated to remarkably respond to platinum-based chemotherapy and might be suited for a future treatment with poly(ADP-ribose)polymerase (PARP) inhibitors. In order to rapidly assess this signature we have previously developed a multiplex-ligation-dependent probe amplification (MLPA)-based assay. Here we present an independent validation of this assay to confirm its important clinical impact.MethodsOne-hundred-forty-four TNBC tumor specimens were analysed by the MLPA-based “BRCA1-like” test. Classification into BRCA1-like vs. non-BRCA1-like samples was performed by our formerly established nearest shrunken centroids classifier. Data were subsequently compared with the BRCA1-mutation/methylation status of the samples. T-lymphocyte infiltration and expression of the main target of PARP inhibitors, PARP1, were assessed on a subset of samples by immunohistochemistry. Data acquisition and interpretation was performed in a blinded manner.ResultsIn the studied TNBC cohort, 63 out of 144 (44 %) tumors were classified into the BRCA1-like category. Among these, the MLPA test correctly predicted 15 out of 18 (83 %) samples with a pathogenic BRCA1-mutation and 20 of 22 (91 %) samples exhibiting BRCA1-promoter methylation. Five false-negative samples were observed. We identified high lymphocyte infiltration as one possible basis for misclassification. However, two falsely classified BRCA1-mutated tumors were also characterized by rather non-BRCA1-associated histopathological features such as borderline ER expression. The BRCA1-like vs. non-BRCA1-like signature was specifically enriched in high-grade (G3) cancers (90 % vs. 58 %, p = 0.0004) and was also frequent in tumors with strong (3+) nuclear PARP1 expression (37 % vs. 16 %; p = 0.087).ConclusionsThis validation study confirmed the good performance of the initial MLPA assay which might thus serve as a valuable tool to select patients for platinum-based chemotherapy regimens. Moreover, frequent PARP1 upregulation in BRCA1-like tumors may also point to susceptibility to treatment with PARP inhibitors. Limitations are the requirement of high tumor content and high-quality DNA.

Abstract 1424: Predicting response to immune checkpoint therapy using an mutation burden threshold

Abstract Treatment with antibodies to PD-1 or CTLA-4 leads to prolonged response in some cancer patients. Patients whose tumors have a high mutational burden have a better response to anti-CTLA-4 treatment with ipilimumab in melanoma5, and to anti-PD-1 treatment with pembrolizumab in non-small cell lung6 and colorectal cancer7. These results suggest that a sufficiently high non-synonymous somatic mutation burden in the tumor may lead to protein alterations that serve as neo-antigens to stimulate CD8 T-cell immune response. This immune response may be blocked by the tumor by engaging the immune checkpoint pathway, making such tumors especially vulnerable to immune checkpoint disruption. However, the specific criteria to identify patients likely to respond to immune checkpoint therapy have remained elusive so far. Using somatic mutation and RNA-Seq data from TCGA, we propose that there exists a mutational threshold, which we call the “Activated Immune Mutational” (AIM) Threshold, which can identify patients likely to respond to immune checkpoint therapy. Compared to AIM- patients (potential non-responders), AIM+ patients (potential responders) have tumors that meet four criteria: (1) a high non-synonymous mutation burden; (2) a high level of immune infiltration in the tumor; (3) a high CD8 T cell fraction in the leukocyte component of the immune infiltrate plus high expression of the T cell marker CD8A; and (4) a high expression of immune checkpoint genes PD-1, PD-L1, PD-L2, CTLA-4. We find that in melanoma, endometrial, colon, cervical and breast cancer, a clear AIM threshold satisfying all four criteria exists. The distribution of mutations in AIM- versus AIM+ tumors for these cancers is very different. In the former, there are high frequency somatic mutations in only a few genes, while in the latter, high frequency somatic mutations are found in many genes distributed over the whole genome. Pathological analysis of high-resolution images from 150 TCGA tumors (30 from each of the five tumor types) validated the presence of significantly higher lymphocytic infiltration in the AIM+ tumors compared to AIM- tumors. We show that AIM+ tumors can be identified using sequencing assays which are currently in clinical use. Finally, survival analysis in melanoma patients from TCGA treated with immunotherapy will be presented. Citation Format: Anshuman Panda, Anil Betigeri, Kalyanasundaram Subramanian, Kim Hirshfield, Lorna Rodriguez, Shridar Ganesan, Gyan Bhanot. Predicting response to immune checkpoint therapy using an mutation burden threshold. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1424.

KEYNOTE-177: First-line, open-label, randomized, phase III study of pembrolizumab (MK-3475) versus investigator-choice chemotherapy for mismatch repair deficient or microsatellite instability-high metastatic colorectal carcinoma.

TPS789 Background: Mismatch repair deficient (dMMR) tumors are characterized by high mutational load and lymphocyte infiltration and may be good candidates for immune checkpoint blockade. Pembrolizumab is a monoclonal antibody against PD-1 that is designed to block its interaction with PD-L1 and PD-L2 and thus allow an antitumor immune response. In the KEYNOTE-016 proof-of-concept study, pembrolizumab showed promising antitumor activity against dMMR tumors in patients (pts) with treatment-refractory metastatic colorectal carcinoma (mCRC). KEYNOTE-177 is an international, randomized trial designed to evaluate the efficacy and safety of pembrolizumab compared with standard-of-care (SOC) chemotherapy in the first-line setting for dMMR or microsatellite instability-high (MSI-H) mCRC. Methods: Key eligibility criteria include age ≥ 18 y, confirmed MSI-H or dMMR mCRC, ECOG PS 0-1, no active autoimmune disease or brain metastases, and no prior therapy for metastatic disease. Pts will be randomized 1:1 to receive either pembrolizumab 200 mg Q3W or investigator’s choice of SOC chemotherapy. Chemotherapy must be chosen prior to randomization; options include mFOLFOX6 or FOLFIRI alone or in combination with bevacizumab or cetuximab. Treatment will continue until PD, unacceptable toxicity, pt/investigator decision, or completion of 35 cycles (pembrolizumab only). Response will be evaluated every 9 wk per RECIST v1.1 by central review and per RECIST adapted for immunotherapy response patterns. Eligible pts may continue pembrolizumab beyond initial RECIST-defined progression. Pts in the SOC arm who have PD and meet crossover criteria may be eligible to receive pembrolizumab for up to 17 treatment cycles. AEs will be assessed throughout treatment and for 30 d thereafter (up to 90 d for serious AEs) and graded per NCI CTCAE v4.0. Pts will be followed for survival every 9 wk. PFS per RECIST v1.1 is the primary end point; OS and ORR are key secondary end points. Other end points include duration of response and health-related quality of life. Planned enrollment in KEYNOTE-177 is 270 pts.

Integrative analysis of breast cancer reveals prognostic haematopoietic activity and patient-specific immune response profiles.

Transcriptional programmes active in haematopoietic cells enable a variety of functions including dedifferentiation, innate immunity and adaptive immunity. Understanding how these programmes function in the context of cancer can provide valuable insights into host immune response, cancer severity and potential therapy response. Here we present a method that uses the transcriptomes of over 200 murine haematopoietic cells, to infer the lineage-specific haematopoietic activity present in human breast tumours. Correlating this activity with patient survival and tumour purity reveals that the transcriptional programmes of many cell types influence patient prognosis and are found in environments of high lymphocytic infiltration. Collectively, these results allow for a detailed and personalized assessment of the patient immune response to a tumour. When combined with routinely collected patient biopsy genomic data, this method can enable a richer understanding of the complex interplay between the host immune system and cancer.

Abstract B109: In-depth tissue analysis using multiplexed immunohistochemical consecutive staining on single slide

Abstract Recent successes of cancer immunotherapy, in particular immune checkpoint blockade, have been largely attributed to factors in the tumor microenvironment (TME). In addition, many studies have found that high lymphocyte infiltration in tumors is prognostic of progression-free or overall survival, while in some cases, myeloid cells rather than lymphocytes are associated with favorable clinical outcome. Importantly, assessment of immune markers usually requires not only quantifying their presence in tumors but also assessing their spatial localization. To assess spatial distribution of tumor-infiltrating immune cells and suppressive molecules, multidimensional immunohistochemical analyses are poised to become the new standard in pathology. A major limitation for such high-dimensional analyses however is tumor tissue availability and new multiplexed imaging approaches are desperately needed. Here, we will describe the development of a new multiplexed immunohistochemistry (IHC) method independent of proprietary reagents or equipment to assess a large panel of phenotypical markers that could easily be integrated in routine clinical pathology. This new technique named Multiplexed Immunohistochemical Consecutive Staining on Single Slide (MICSSS) can be performed in any laboratory already conducting traditional IHC with limited cost and no additional instrumentation. The MICSSS method is performed on formalin-fixed paraffin-embedded tissue using iterative cycles of staining, revelation, scanning, and bleaching of chromogenic substrate. It should easily be adaptable to most existing staining protocols with primary antibody staining and secondary antibody detection, thus retaining previously established specificity and sensitivity parameters. The MICSSS method can characterize a large panel of parameters, including marker colocalization on cells, using a single slide while preserving tissue antigenicity and architecture. It is not limited by antibody cross-reactivity, photo-bleaching, or autofluorescence, while still allowing prolonged slide storage for future reuse as new markers become available. Here, we will describe the MICSSS method workflow and show potential applications of multiplex immunostaining including its use to characterize the tumor immune environment and identify prognostic factors or predictive biomarkers of response to CTLA-4 blockade. In conclusion, the MICSSS method will provide new powerful ways to map the tumor microenvironment with precision, in a sample-sparing manner, to monitor the immune response at the tumor site during therapy and discover new prognostic and predictive markers that might be of major interest for the clinical management of patients with various diseases. Citation Format: Romain Remark, Taha Merghoub, Diane Damotte, Jedd D. Wolchok, Miriam Merad, Sacha Gnjatic. In-depth tissue analysis using multiplexed immunohistochemical consecutive staining on single slide. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B109.

Abstract IA20: Cancer subtypes and their immune microenvironments

Abstract In the vast majority of cancer types, as demonstrated and exemplified in colorectal cancer (CRC) and non-small cell lung cancer (NSCLC), a high density of CD8+ T cells associated with high densities of tertiary lymphoid structures (TLS) in the tumor microenvironment correlates with a good prognosis for the patient. However, an opposite association has been reported in primary clear cell renal cell carcinoma (RCC) and several other cancers. Our team studied the immune infiltrates of pulmonary metastases from CRC and RCC. As in the primary tumors, a high density of CD8+ T cells correlated with good prognosis for CRC metastases, while it correlated with a bad prognosis for RCC metastases. These results suggested that the identity of the tumor cells, rather than the organ where they grow, is critical for shaping the immune contexture of a given tumor. Transcriptomic analyses of large cohorts of human CRC and RCC allow to define molecular subgroups associated with distinct risks of tumor progression. We therefore characterized the immune microenvironments of the molecular subgroups of CRC and RCC. In CRC, the MSI-enriched subgroup identified patients with high cytotoxic T cells infiltration and favorable prognosis. In CRC and RCC, we confirmed that patients with low tumoral T and B cell infiltrations presented with high risk of death; In addition, in both cancer types, we identified subgroups of poor-prognosis patients with high tumoral lymphocyte infiltration, in the context of high expression of genes related to inflammation, immunosuppression, and angiogenesis. Integration of molecular and immune tumor phenotypes refines prognostic cancer groups. Moreover, these classifications allow to select immunotherapies appropriate to specific, potentially responding, groups of patients. Citation Format: Etienne Becht, Nicolas A. Giraldo, Aurelien De Reynies, Pierre Laurent-Puig, Benoit Beuselinck, Jessica Zucman-Rossi, Marie Caroline Dieu-Nosjean, Catherine Sautes-Fridman, Wolf H. Fridman. Cancer subtypes and their immune microenvironments. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr IA20.

The Influence of Host Factors on the Prognosis of Breast Cancer: Stroma and Immune Cell Components as Cancer Biomarkers.

In this paper, we will review the data on stromal components and immunological parameters in the cancer microenvironment as prognostic and predictive markers in breast cancer. Host immunological response to cancer has gained importance because of recent breakthroughs in immunotherapy. Currently, molecular and clinical subtyping of breast cancer is solely based on the molecular features of the cancer cells without considering the importance of stromal components. There is now clear evidence that infiltrating immune and inflammatory cells influence the biology and clinical course of breast cancer. However, the prognostic and predictive function of immune cells differs between breast cancer subtypes. Immune parameters are established and validated prognostic and predictive markers in triple negative and for HER2 positive breast cancers and may be ready to be used as stratification parameters in clinical trials and as adjunct variables when making clinical decisions. On the other hand, the prognostic and predictive impact is minimal in low grade, luminal A type breast cancers. The strong association between higher lymphocytic infiltration and better outcome (including greater chemotherapy sensitivity) in TNBC and HER2 positive cancers also raises novel therapeutic options that target immune cells to increase their activity. Immune markers also carry the potential to serve as predictive markers to select patients for immunotherapeutic regimens (e.g. checkpoint inhibitors).

Tumor-infiltrating immune cell profiles and their change after neoadjuvant chemotherapy predict response and prognosis of breast cancer.

IntroductionTumor microenvironment immunity is associated with breast cancer outcome. A high lymphocytic infiltration has been associated with response to neoadjuvant chemotherapy, but the contribution to response and prognosis of immune cell subpopulations profiles in both pre-treated and post-treatment residual tumor is still unclear.MethodsWe analyzed pre- and post-treatment tumor-infiltrating immune cells (CD3, CD4, CD8, CD20, CD68, Foxp3) by immunohistochemistry in a series of 121 breast cancer patients homogeneously treated with neoadjuvant chemotherapy. Immune cell profiles were analyzed and correlated with response and survival.ResultsWe identified three tumor-infiltrating immune cell profiles, which were able to predict pathological complete response (pCR) to neoadjuvant chemotherapy (cluster B: 58%, versus clusters A and C: 7%). A higher infiltration by CD4 lymphocytes was the main factor explaining the occurrence of pCR, and this association was validated in six public genomic datasets. A higher chemotherapy effect on lymphocytic infiltration, including an inversion of CD4/CD8 ratio, was associated with pCR and with better prognosis. Analysis of the immune infiltrate in post-chemotherapy residual tumor identified a profile (cluster Y), mainly characterized by high CD3 and CD68 infiltration, with a worse disease free survival.ConclusionsBreast cancer immune cell subpopulation profiles, determined by immunohistochemistry-based computerized analysis, identify groups of patients characterized by high response (in the pre-treatment setting) and poor prognosis (in the post-treatment setting). Further understanding of the mechanisms underlying the distribution of immune cells and their changes after chemotherapy may contribute to the development of new immune-targeted therapies for breast cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0488-5) contains supplementary material, which is available to authorized users.

Systematic analysis of human oncogenic viruses in colon cancer revealed EBV latency in lymphoid infiltrates.

BackgroundEnvironmental factors may play a role in colon cancer. In this view, several studies investigated tumor samples for the presence of various viral DNA with conflicting results.FindingsWe undertook a systematic DNA analysis of 44 consecutive, prospectively collected primary tumor samples by real time and qualitative PCR for viruses of known or potential oncogenic role in humans, including polyomavirus (JCV, BKV, Merkel cell polyomavirus), HPV, HTLV, HHV-8 and EBV. Negative controls consisted of surgical resection margins. No evidence of genomic DNA fragments from tested virus were detected, except for EBV, which was found in a significant portion of tumors (23/44, 52%). Real-time PCR showed that EBV DNA was present at a highly variable content (median 258 copies in 105 cells, range 15–4837). Presence of EBV DNA had a trend to be associated with high lymphocyte infiltration (p = 0.06, χ2 test), and in situ hybridization with EBER1-2 probes revealed latency in a fraction of these lymphoid cells, with just a few scattered plasma cells positive for BZLF-1, an immediate early protein expressed during lytic replication. LMP-1 expression was undetectable by immunohistochemistry.ConclusionsThese results argue against a significant involvement of the tested oncogenic viruses in established colon cancer.

Nuclear adenomatous polyposis coli suppresses colitis-associated tumorigenesis in mice

Mutation of tumor suppressor adenomatous polyposis coli (APC) initiates most colorectal cancers and chronic colitis increases risk. APC is a nucleo-cytoplasmic shuttling protein, best known for antagonizing Wnt signaling by forming a cytoplasmic complex that marks β-catenin for degradation. Using our unique mouse model with compromised nuclear Apc import (Apc(mNLS)), we show that Apc(mNLS/mNLS) mice have increased susceptibility to tumorigenesis induced with azoxymethane (AOM) and dextran sodium sulfate (DSS). The AOM-DSS-induced colon adenoma histopathology, proliferation, apoptosis, stem cell number and β-catenin and Kras mutation spectra were similar in Apc(mNLS/mNLS) and Apc(+/+) mice. However, AOM-DSS-treated Apc(mNLS/mNLS) mice showed more weight loss, more lymphoid follicles and edema, and increased colon shortening than treated Apc(+/+) mice, indicating a colitis predisposition. To test this directly, we induced acute colitis with a 7 day DSS treatment followed by 5 days of recovery. Compared with Apc(+/+) mice, DSS-treated Apc(mNLS/mNLS) mice developed more severe colitis based on clinical grade and histopathology. Apc(mNLS/mNLS) mice also had higher lymphocytic infiltration and reduced expression of stem cell markers, suggesting an increased propensity for chronic inflammation. Moreover, colons from DSS-treated Apc(mNLS/mNLS) mice showed fewer goblet cells and reduced Muc2 expression. Even in untreated Apc(mNLS/mNLS) mice, there were significantly fewer goblet cells in jejuna, and a modest decrease in colonocyte Muc2 expression compared with Apc(+/+) mice. Colonocytes from untreated Apc(mNLS/mNLS) mice also showed increased expression of inflammatory mediators cyclooxygenase-2 (Cox-2) and macrophage inflammatory protein-2 (MIP-2). These findings reveal novel functions for nuclear Apc in goblet cell differentiation and protection against inflammation-induced colon tumorigenesis.

Transplanted terminally differentiated induced pluripotent stem cells are accepted by immune mechanisms similar to self-tolerance.

The exact nature of the immune response elicited by autologous induced pluripotent stem cell (iPSC) progeny is still not well understood. Here we show in murine models that autologous iPSC-derived endothelial cells (iECs) elicit an immune response that resembles the one against a comparable somatic cell, the aortic endothelial cell (AEC). These cells exhibit long-term survival in vivo and prompt a tolerogenic contexture of intra-graft characterized by elevated IL-10 expression. In contrast, undifferentiated iPSCs elicit a very different immune response with high lymphocytic infiltration and elevated IFN-γ, granzyme-B, and perforin intra-graft. Furthermore, the clonal structure of infiltrating T cells from iEC grafts is statistically indistinguishable from that of AECs, but is different from that of undifferentiated iPSC grafts. Taken together, our results indicate that the differentiation of iPSCs results in a loss of immunogenicity and leads to the induction of tolerance, despite expected antigen expression differences between iPSC-derived versus original somatic cells.

Role of Kamut® brand khorasan wheat in the counteraction of non-celiac wheat sensitivity and oxidative damage

It has been suggested that ancient grains show lower immunogenic properties and therefore can be introduced in the diet of non-celiac wheat-sensitive people. In the present study we investigated the possible difference in inflammation caused by feeding ancient Kamut® wheat pasta (KP) compared to modern durum wheat pasta (WP) to rats. The effect of the two experimental diets on the oxidative status was also compared in basal condition and after an exogenous oxidative stress. In rats fed WP the histological evaluation of the duodenum morphology evidenced a flattened mucosa, an unusual shape and shortening of the villi, and a high lymphocyte infiltration, while no modifications were detected in KP fed animals. The fecal metabolite profiling was differently modified by the two diets, suggesting significant changes in the gut microflora. Furthermore, the results confirmed previous data on the antioxidant protection in rats by Kamut® wheat foods. It is conceivable that Kamut® components can act through a hormetic effect, eliciting an adaptive response that protects the organism against both oxidative stress and inflammation.