Characteristics Of Tumor-infiltrating Lymphocytes Research Articles

Abstract P6-15-04: Characterization of tumor infiltrating lymphocytes and its association response in breast cancer

Abstract Background: The magnitude of tumor infiltrating lymphocytes (TIL) is a predictor of good prognosis in the context of neoadyuvant chemotherapy (NAC) in breast cancer, and is expressed in the capacity to achieve a pathological complete response (pCR) and therefore to obtain a major survival. The characteristics of TIL are influenced of different clinical and pathological features as well as its predictive value. The goal of this study is to define the different associations between level of stromal (sTIL) and interstitial (iTIL) tumor infiltrating lymphocytes and pathological and clinical feature of breast cancer. Methods: The level of TIL was analyzed in 483 patients diagnosed with breast cancer of the Instituto Nacional de Enfermedades Neoplasicas from 2003 to 2014. We evaluate patient characteristics such as age, clinical stage, histological subtype and grade, clinical T, N and M, presence of estrogen (ER) and progesterone (PgR) receptors, HER-2 expression and molecular subtype as well as pathological response. Results: We found a positive association between sTIL and iTIL proportion and histological grade (G1-2 vs G3, p<0.05 and p<0.05 respectively), hormone receptor (ER- vs ER+PgR- vs ER+PgR+, p<0.05 and p=0.001 respectively), molecular subtype (Luminal A vs Luminal B vs HER2 vs Triple Negative (TN), p<0.05 and p=0.002 respectively), clinical N (positive vs negative, p=0.006 and p<0.05 respectively). Also we found association between sTIL with lymph nodes after surgery (negative vs positive, p=0.028) and pathological response (pCR vs <pCR, p=0.018), there was no association between sTIL and iTIL with age, histological subtype, clinical T and M. Conclusions: The level of tumor infiltrating lymphocytes are affected by different clinical and pathological features, they are present in both lobular and ductal breast cancer without significant difference; higher level of TIL are associated with high histological grade, ER-, TNBC and high clinical N to diagnosis; and specially sTIL are associated with to achieve a negative lymph node after NAC and obtain a pathological complete response. Citation Format: Galvez M, Castaneda C, Villegas V. Characterization of tumor infiltrating lymphocytes and its association response in breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P6-15-04.

Distinct dominant T-cell receptors with a tissue resident memory phenotype in individual melanoma metastases.

3 Background: Expression of inhibitory immune checkpoints (ICPs) within tumors has emerged as an important barrier for effective anti-tumor immunity. Antibody-mediated blockade of ICPs can lead to durable responses in patients. Interestingly, only a small subset of tumor infiltrating lymphocytes (TILs) express these checkpoints and there is a need to better understand the characteristics of this subset. We undertook this study to understand characteristics of TILs within melanoma. Methods: We used single cell mass cytometry, gene expression profiling of purified T cell subsets, T cell receptor (TCR) sequencing as well as functional studies to understand the characteristics of TILs in melanoma patients (n=50). We also performed exome sequencing of tumor cells in some patients. Results: We find that TILs are functionally and phenotypically distinct from circulating T cells. They express higher levels of inhibitory ICPs (PD-1, TIM-3) and secrete less IL2, IFNg and TNFa than T cells in circulation. Expression of vascular endothelial growth factor within tumors correlated with reduced T cell infiltration. Expression of ICPs (PD-1, TIM-3, PD-L1) were enriched in T cells with a phenotype and expression profile of tissue resident memory T (TRM) cells with most cells expressing multiple checkpoints. Within the myeloid compartment, ICPs were predominantly expressed on CD14+CD16+ subset. TCR sequencing revealed that individual melanoma metastases revealed that the top clones within each of the lesions have distinct TCRs. Concurrent TCR and tumor exome sequencing of individual metastases in the same patient revealed that inter-lesional diversity of TCRs exceeded differences in mutation/neoantigen load in tumor cells. Conclusions: Our findings suggest that TRM cells and CD16+ myeloid cells may be the major target of ICP blockade within tumors. The ability to activate, and retain TRM cells may be an important determinant of the T cell content of the tumor microenvironment and should be a goal for future vaccines. Importantly, our study illustrates inter-lesional diversity of TCRs within individual metastases which may differentially impact the outcome of immune therapy at each site.

Abstract PR03: CD8+ T-cell distribution and immunomodulator expression in BRAF-mutant melanoma affect the response to BRAF inhibitor and chemotherapy

Abstract Although intense effort has been made in studying immune cell dynamics in tumors after treatment with targeted therapies and to recharacterize tumor stages based on their immune cell components, less is known about how baseline immune cell characteristics in tumors affect response to approved therapies. Here we describe baseline expression of immune regulators and CD8+ T-cell distribution in BRAF-mutated metastatic melanoma and their relationship with progression-free survival (PFS) on vemurafenib and chemotherapy. To our knowledge, we are the first to describe the baseline characteristics of tumor-infiltrating lymphocytes and their impact on outcomes in a large randomized, controlled trial in melanoma. Archival or baseline samples were collected from patients in 2 clinical trials (BRIM2 and BRIM3) and analyzed. A total of 252 RNA samples were prepared from formalin-fixed, paraffin-embedded (FFPE) tissue samples and profiled for expression using a panel of 96 immune genes on the Fluidigm platform (Fluidigm Corp). Additionally, for 277 patients from the BRIM3 trial, CD8+ T cells were detected by immunohistochemistry (IHC) and quantified using Definiens (Definiens AG). Patients were defined as high or low expressers based on a median cutoff, and hazard ratios (HRs) were determined by cox proportional hazards modeling of PFS. HRs refer to the comparison of high and low expressing groups, where an HR<1 is indicative of a better outcome for the high-expressing group. A discovery (BRIM2) – validation (BRIM3) scheme was applied to assess the prognostic value of the expression of immune-related genes. Samples from the BRIM3 dacarbazine and vemurafenib arms were compared to assess the predictive value of the markers on the treatment effect of vemurafenib. Of the genes tested, 26 met our discovery criteria in the BRIM2 trial and 2, IL7 and IL12A, were validated in BRIM3. In the vemurafenib arm of BRIM3, patients with high expression of IL7 had improved PFS compared with patients with low IL7, with an HR of 0.63 (95% confidence interval [CI]: 0.41-0.97; P=0.03); high expression of IL12A had an HR of 0.58 (0.38-0.88; P=0.01). Neither gene was associated with PFS in the dacarbazine-treated arm; however, high and low expressers of both genes benefited from vemurafenib treatment. Because immune contexture is known to be associated with outcomes, slides were stained for CD8+ T-cell content in 3 marker areas: center, peripheral, and invasive margin. In the dacarbazine arm, increased PFS was seen in patients with higher CD8+ T-cell content in the tumor center or periphery, with HRs of 0.58 (0.43-0.94; P=0.02) and 0.39 (0.25-0.59; P<0.01), respectively. CD8+ T-cell content did not show a significant effect in vemurafenib-treated patients, and all groups benefited from vemurafenib treatment. Based on these characterizations, tumor immune cell components seem to play important but varying roles in response to dacarbazine or vemurafenib treatment in BRAF-mutant melanoma. This abstract is also being presented as Poster B22. Citation Format: Matthew Wongchenko, Christina Rabe, Jeffrey Sosman, Grant McArthur, Yuanyuan Xiao, Houston Gilbert, Luc Andries, Mark Kockx, Hartmut Koeppen, Priti Hegde, Lukas Amler, Yibing Yan, Antoni Ribas. CD8+ T-cell distribution and immunomodulator expression in BRAF-mutant melanoma affect the response to BRAF inhibitor and chemotherapy. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr PR03.

Clinicopathological characteristics of tumor-infiltrating lymphocytes in colorectal cancer

Clinicopathological characteristics of tumor-infiltrating lymphocytes in colorectal cancer

Evidence for existence of oligoclonal tumor-infiltrating lymphocytes and predominant production of T helper 1/T cytotoxic 1 type cytokines in gastric and colorectal tumors

Tumor-infiltrating lymphocytes (TIL) play a central role in cellular immunity against tumor. We have revealed the characteristics of TILs in terms of T-cell receptor (TCR) repertoire, T-cell clonality, and cytokine production. TCR repertoire analyses and CDR3 size spectratyping were performed using peripheral blood mononuclear cells (PBMCs) and tissue specimens of gastric or colorectal cancers surgically resected from 11 patients. The cytokine expression was measured by real-time quantitative polymerase chain reaction. TCR repertoires were similar among multiple tissue specimens from different sites of the same tumor. Similar peak patterns of CDR3 size spectratyping were observed among these tumor specimens, but not in normal tissues or PBMCs. In addition, identical peaks were detected in multiple specimens of the same tumor. The ratio of the levels of IFN-gamma to that of IL-4 is significantly higher for tumor lesions compared with PBMCs. These results suggested that a limited number of TILs locally expand in response to tumor antigens exiting within gastric or colorectal cancers and local predominant production of the T helper 1/T cytotoxic 1 type cytokine may affect the anti-tumor immune response of TILs.

Characterization of human tumor-infiltrating lymphocytes expanded in hollow-fiber bioreactors for immunotherapy of cancer.

We attempted to grow tumor-infiltrating lymphocytes (TIL) from 34 fresh tumors of eight different histologies using flasks for the initiation phase and hollow fiber bioreactors to expand TIL to therapeutic numbers. Overall success rate was 76% (26/34) including melanoma (9/14, 64%) and renal cell carcinoma (11/11, 100%). The mean number of days required to reach successful initiation (1 x 10(9) TIL) for all tumor types was 29 +/- 16 days (mean +/- S.D.). Therapeutic doses of TIL required an average of 88 +/- 23 days (initiation plus expansion) with an average TIL number of 3.2 x 10(10) +/- 2.8 x 10(10). TIL phenotype was predominantly CD4+ in 53% (16/30) and CD8+ in 47% (14/30), renal cell carcinoma samples accounted for 12/14 of the predominantly CD8+ TIL. Cells bearing the natural killer (NK) phenotype represented only 0-7% of TIL while LAK phenotype represented 0-68% (mean 11 +/- 15%); LAK was the predominant phenotype in one patient with kidney cancer. Cytotoxicity tests showed consistent NK and LAK activity in addition to cytolysis of autologous tumor. Autologous tumor cell restricted cytolysis was noted for three TIL cultures. The overall success rate and characteristics of TIL were similar to our results with TIL expanded in semi-permeable plastic bags. This work confirms that hollow-fiber bioreactors are a suitable alternative to semi-permeable bags and roller bottle systems for the expansion of human TIL for therapeutic use in cancer patients.

Morphological and functional characteristics of tumor-infiltrating lymphocytes from human colorectal cancers after stimulation with rIL-2.

Tumor-infiltrating lymphocytes (TILs) from colorectal cancers were separated from tumor cells by enzymatic and mechanical tissue disaggregation and discontinuous density gradients. Peripheral blood lymphocytes (PBLs) were isolated using the same procedure. The freshly separated TILs and PBLs were analyzed phenotypically by flow cytometry. The CD4+/CD8+ ratios of the freshly isolated TILs and PBLs were comparable (> 1 in both lymphocyte populations). CD25+, HLA-DR+ and CD56+ cells were significantly more frequent in the TIL than in the PBL population. However, the number of CD45RA+ cells was lower in the TILs as compared to PBLs, while CD29+ accumulated by about 90% in TILs. TILs and autologous PBLs were expanded in vitro with rIL-2. The mean rate of proliferation after 4 weeks was 642-fold in TIL cultures and 335-fold in PBLs. More than 90% of the rIL-2-expanded lymphocytes expressed CD2 and the great majority was CD29+. Stimulation with rIL-2 in vitro induced an outgrowth of CD56+ cells mainly in the TILs. Accordingly the expression of CD3+ and alpha/beta receptor in the TILs was low. Those cells which phenotypically represented lymphokine-activated killer cells displayed a lytic activity against the autologous tumor as well as against allogeneic K562 and Daudi targets. In accordance with the better proliferative response of TILs in long-term cultures with rIL-2, the lytic activity of TILs against autologous and allogeneic tumor targets was significantly higher as compared to PBLs.

Proliferative, phenotypic and functional and molecular characteristics of tumour-infiltrating lymphocytes obtained from unselected patients with malignant melanomas and expanded in vitro in the presence of recombinant interleukin-2

Tumour-infiltrating lymphocytes (TIL) derived from several histotypes, including melanoma, can be expanded in vitro in the presence of recombinant interleukin-2 (rIL-2) and infused as part of experimental adoptive immunotherapy protocols. Several authors have described the isolation and the expansion of TIL, but little is known about the actual proportion of unselected melanoma biopsies that give rise to 'positive' TIL cultures. We evaluated the proliferative, phenotypic, functional and molecular characteristics of cultured TIL obtained from 26 patients with metastatic melanomas, eligible for inclusion in a protocol of adoptive immunotherapy. Sixteen proliferating cultures were obtained. All TIL belonged to the T cell lineage and were characterized by a wide range of cytolytic activity against autologous and, to a lesser extent, allogeneic melanoma cells. Molecular analysis of the constant region of T cell receptor-beta (TCR-beta) showed an oligoclonal population of TIL in the majority of expanded samples, indicating that a selected subset of lymphocytes present in the tumour site could be expanded in vitro. These features, together with the high number of proliferating samples, make these populations of TIL suitable for infusion into patients with advanced disease.