Expansion of tumor-infiltrating lymphocytes from head and neck squamous cell carcinoma to assess the potential of adoptive cell therapy.

BACKGROUND: Adoptive transfer of in vitro expanded tumor-infiltrating lymphocytes (TILs) has been effective in regressing several types of malignant tumors. This study assessed the yield and factors influencing the successful expansion of tumor-infiltrating lymphocytes (TILs) from head and neck squamous cell carcinoma (HNSCC), along with their immune phenotypes. METHODS: TILs were expanded from 47 surgically resected HNSCC specimens and their metastasized lymph nodes. The cancer tissues were cut into small pieces (1-2 mm) and underwent initial expansion for 2 weeks. Tumor location, smoking history, stromal TIL percentage, human papillomavirus infection, and programmed death-ligand 1 score were examined for their impact on successful expansion of TILs. Expanded TILs were evaluated by flow cytometry using fluorescence-activated cell sorting. A second round of TIL expansion following the rapid expansion protocol was performed on a subset of samples with successful TIL expansion. RESULTS: TILs were successfully expanded from 36.2% samples. Failure was due to contamination (27.6%) or insufficient expansion (36.2%). Only the stromal TIL percentage was significantly associated with successful TIL expansion (p = 0.032). The stromal TIL percentage also displayed a correlation with the expanded TILs per fragment (r = 0.341, p = 0.048). On flow cytometry analysis using 13 samples with successful TIL expansion, CD4 + T cell dominancy was seen in 69.2% of cases. Effector memory T cells were the major phenotype of expanded CD4 + and CD8 + T cells in all cases. CONCLUSION: We could expand TILs from approximately one-third of HNSCC samples. TIL expansion could be applicable in HNSCC samples with diverse clinicopathological characteristics.

Factors associated with engraftment success of patient-derived xenografts of breast cancer.

BACKGROUND: Patient-derived xenograft (PDX) models serve as a valuable tool for the preclinical evaluation of novel therapies. They closely replicate the genetic, phenotypic, and histopathological characteristics of primary breast tumors. Despite their promise, the rate of successful PDX engraftment is various in the literature. This study aimed to identify the key factors associated with successful PDX engraftment of primary breast cancer. METHODS: We integrated clinicopathological data with morphological attributes quantified using a trained artificial intelligence (AI) model to identify the principal factors affecting PDX engraftment. RESULTS: Multivariate logistic regression analyses demonstrated that several factors, including a high Ki-67 labeling index (Ki-67LI) (p < 0.001), younger age at diagnosis (p = 0.032), post neoadjuvant chemotherapy (NAC) (p = 0.006), higher histologic grade (p = 0.039), larger tumor size (p = 0.029), and AI-assessed higher intratumoral necrosis (p = 0.027) and intratumoral invasive carcinoma (p = 0.040) proportions, were significant factors for successful PDX engraftment (area under the curve [AUC] 0.905). In the NAC group, a higher Ki-67LI (p < 0.001), lower Miller-Payne grade (p < 0.001), and reduced proportion of intratumoral normal breast glands as assessed by AI (p = 0.06) collectively provided excellent prediction accuracy for successful PDX engraftment (AUC 0.89). CONCLUSIONS: We found that high Ki-67LI, younger age, post-NAC status, higher histologic grade, larger tumor size, and specific morphological attributes were significant factors for predicting successful PDX engraftment of primary breast cancer.

Mental health of South Korean adolescents in the COVID-19 Era: Web-based survey focused on suicide-related factors.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic has negatively impacted many aspects of life. Measures for preventing the spread of COVID-19 (e.g., school lockdowns, remote and hybrid classes, group and outdoor activity restrictions, and social distancing in the classroom and meal time) could have led to adolescents to experience anxiety and depressive symptoms. Such mental health impacts could increase the risk of suicidal ideation in this population. Moreover, according to a report by the Organization for Economic Co-operation and Development, although the total number of suicide deaths in South Korea decreased in 2021, the suicide rate of those aged 10-29 years increased. One factor affecting the result is adolescent mental health by COVID-19. This study examines the mental health status of South Korean adolescents amid the prolonged COVID-19 pandemic, and identifies and analyzes predictors of suicidal ideation, suicide planning, and suicide attempts. METHODS: The study used data from 54,948 adolescents who participated in the 2020 Korea Youth Risk Behavior Web-based Survey. Based on their responses to suicide-related questions, the sample was divided into a healthy group, suicide-ideation group, suicide-planning group, and suicide-attempt group. The descriptive statistics of these groups were then analyzed. An analysis of covariance, post-hoc tests, and multiple logistic regression analysis were performed on the four groups. RESULTS: Overall, 6.9% of the participants reported suicidal ideation, 2.2% reported planning suicide, and 1.9% reported attempting suicide in the previous 12 months. CONCLUSIONS: During the prolonged COVID-19 pandemic, there is a strong need for various individualized programs that identify and intervene to support adolescents at risk of suicide by accurately assessing their mental health risk factors, such as stress, sadness and despair, loneliness, and generalized anxiety disorder. Accordingly, it is necessary to develop and distribute a mental health and well-being curriculum, strengthen suicide prevention programs and support services, expand mental health diagnostic tests, and school-based mental health programs.

Establishing Patient-Derived Cancer Cell Cultures and Xenografts in Biliary Tract Cancer.

PURPOSE: Biliary tract cancers (BTCs) are rare and show a dismal prognosis with limited treatment options. To improve our understanding of these heterogeneous tumors and develop effective therapeutic agents, suitable preclinical models reflecting diverse tumor characteristics are needed. We established and characterized new patient-derived cancer cell cultures and patient-derived xenograft (PDX) models using malignant ascites from five patients with BTC. MATERIALS AND METHODS: Five patient-derived cancer cell cultures and three PDX models derived from malignant ascites of five patients with BTC, AMCBTC-01, -02, -03, -04, and -05, were established. To characterize the models histogenetically and confirm whether characteristics of the primary tumor were maintained, targeted sequencing and histopathological comparison between primary tissue and xenograft tumors were performed. RESULTS: From malignant ascites of five BTC patients, five patient-derived cancer cell cultures (100% success rate), and three PDXs (60% success rate) were established. The morphological characteristics of three primary xenograft tumors were compared with those of matched primary tumors, and they displayed a similar morphology. The mutated genes in samples (models, primary tumor tissue, or both) from more than one patient were TP53 (n=2), KRAS (n=2), and STK11 (n=2). Overall, the pattern of commonly mutated genes in BTC cell cultures was different from that in commercially available BTC cell lines. CONCLUSION: We successfully established the patient-derived cancer cell cultures and xenograft models derived from malignant ascites in BTC patients. These models accompanied by different genetic characteristics from commercially available models will help better understand BTC biology.

Phenotypic Differences of CD103+ Tissue-Resident Memory T Cells Associated with Various Cancers.

BACKGROUND/AIMS: The presence and clinical importance of tissue-resident memory T (TRM) cells have been recently described in association with various cancer types. However, the frequency and the traditional naïve-effector-memory phenotypic characteristics of TRM cells are largely unknown. METHODS: We analyzed single-cell populations of colorectal cancer (CC, n = 18), stomach cancer (SC, n = 13), renal cell carcinoma (RCC, n = 19), and breast cancer (BC, n = 16) by dissociation of tumor tissue with collagenase/hyaluronidase. We investigated populations of naïve, effector, and memory T and TRM cells by flow cytometry. RESULTS: Among CD8- cells, CC was associated with a significantly higher proportion of CD103+ T cells than other tumor types (p < 0.001). Among CD8+ cells, CC and SC were associated with higher CD103+ T-cell proportions than RCC and BC (p < 0.001). Significantly more CD8+ than CD8- cells expressed CD103 (p < 0.001). In association with SC, RCC, and BC, CD8+ T cells had a similar T-cell phenotype composition pattern: fewer effector T cells and more memory-type T cells among CD103+ cells compared with CD103- cells (p < 0.05). Tumors with higher proportion of CD103+ cells had no specific clinicopathologic characteristics than those with lower proportion of CD103+ cells. CONCLUSION: TRM cell abundance and phenotypes varied among CC, SC, RCC, and BC. Further studies regarding the functional differences of TRM associated with various tumors are warranted.

The Association of Estrogen Receptor Activity, Interferon Signaling, and MHC Class I Expression in Breast Cancer.

PURPOSE: The expression of major histocompatibility complex class I (MHC I) has previously been reported to be negatively associated with estrogen receptor (ER) expression. Furthermore, MHC I expression, level of tumor-infiltrating lymphocytes (TILs), and expression of interferon (IFN) mediator MxA are positively associated with one another in human breast cancers. This study aimed to investigate the mechanisms of association of MHC I with ER and IFN signaling. MATERIALS AND METHODS: The human leukocyte antigen (HLA)-ABC protein expression was analyzed in breast cancer cell lines. The expressions of HLA-A and MxA mRNAs were analyzed in MCF-7 cells in Gene Expression Omnibus (GEO) data. ER and HLA-ABC expressions, Ki-67 labeling index and TIL levels in tumor tissue were also analyzed in ER+/ human epidermal growth factor receptor 2 (HER2)- breast cancer patients who randomly received either neoadjuvant chemotherapy or estrogen modulator treatment followed by resection. RESULTS: HLA-ABC protein expression was decreased after ß-estradiol treatment or hESR-GFP transfection and increased after fulvestrant or IFN-γ treatment in cell lines. In GEO data, HLA-A and MxA expression was increased after ESR1 shRNA transfection. In patients, ER Allred score was significantly lower and the HLA-ABC expression, TIL levels, and Ki-67 were significantly higher in the estrogen modulator treated group than the chemotherapy treated group. CONCLUSION: MHC I expression and TIL levels might be affected by ER pathway modulation and IFN treatment. Further studies elucidating the mechanism of MHC I regulation could suggest a way to boost TIL influx in cancer in a clinical setting.

Relationship between the Stereocomplex Crystallization Behavior and Mechanical Properties of PLLA/PDLA Blends.

Poly (l-lactic acid) (PLLA) is a promising biomedical polymer material with a wide range of applications. The diverse enantiomeric forms of PLLA provide great opportunities for thermal and mechanical enhancement through stereocomplex formation. The addition of poly (d-lactic acid) (PDLA) as a nucleation agent and the formation of stereocomplex crystallization (SC) have been proven to be an effective method to improve the crystallization and mechanical properties of the PLLA. In this study, PLLA was blended with different amounts of PDLA through a melt blending process and their properties were calculated. The effect of the PDLA on the crystallization behavior, thermal, and mechanical properties of PLLA were investigated systematically by thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), X-ray diffraction (XRD), polarized optical microscopy (POM), dynamic mechanical analysis (DMA), and tensile test. Based on our findings, SC formed easily when PDLA content was increased, and acts as nucleation sites. Both SC and homo crystals (HC) were observed in the PLLA/PDLA blends. As the content of PDLA increased, the degree of crystallization increased, and the mechanical strength also increased.

Anion Exchange Membrane Based on Sulfonated Poly (Styrene-Ethylene-Butylene-Styrene) Copolymers.

Sulfonated poly(styrene-ethylene-butylene-styrene) copolymer (S-SEBS) was prepared as an anion exchange membrane using the casting method. The prepared S-SEBS was further modified with sulfonic acid groups and grafted with maleic anhydride (MA) to improve the ionic conducting properties. The prepared MA-grafted S-SEBS (S-SEBS-g-MA) membranes were characterized by Fourier transform infrared red (FT-IR) spectroscopy and dynamic modulus analysis (DMA). The morphology of the S-SEBS and S-SEBS-g-MA was investigated using atomic force microscopy (AFM) analysis. The modified membranes formed ionic channels by means of association with the sulfonate group and carboxyl group in the SEBS. The electrochemical properties of the modified SEBS membranes, such as water uptake capability, impedance spectroscopy, ionic conductivity, and ionic exchange capacity (IEC), were also measured. The electrochemical analysis revealed that the S-SEBS-g-MA anion exchange membrane showed ionic conductivity of 0.25 S/cm at 100% relative humidity, with 72.5% water uptake capacity. Interestingly, we did not observe any changes in their mechanical and chemical properties, which revealed the robustness of the modified SEBS membrane.

Evaluation of silver nanowires (AgNWs) toxicity on reproductive success of Daphnia magna over two generations and their teratogenic effect on embryonic development.

This study assessed the reproductive toxicity of silver nanowires (AgNWs) in Daphnia magna over two consecutive generations. An acute immobilization test was conducted according to the ΟECD 202 guidelines. To perform reproductive toxicity tests in both F0 and F1 generations, the animals were exposed to different concentrations of AgNWs (0, 0.4, 2, 10, and 50 µg L-1) and pyriproxyfen (0.4 µg L-1), as a positive control, based on the ΟECD 211 principles. Overall, AgNWs were acutely toxic to D. magna with EC50 value of 0.063 mg L-1. Compared to the control groups, AgNWs disrupted reproductive performances of D. magna through increasing the egg development time and time to production of first brood as well as decreasing the total offspring production and molting frequency in both F0 and F1 generations. After exposure to AgNWs, the number of male neonates and non-reproductive females increased in the F0 generation, whereas just male neonates raised in the F1 generation. Moreover, AgNWs caused several congenital anomalies including underdeveloped antennae, 2nd antennae, malpighian tube, rostrum, sensory bristles, tail spine, and malformed eyes. Together, AgNWs could disrupt reproductive health of D. magna, and these types of bioperturbations could dramatically change the good health state of aquatic ecosystems.

Clinicopathological factors associated with tumor-infiltrating lymphocyte reactivity in breast cancer.

BACKGROUND: The clinical significance of adoptive tumor-infiltrating lymphocyte (TIL) therapy has been demonstrated in many clinical trials. We analyzed the in vitro reactivity of cultured TILs against autologous breast cancer cells. METHODS: TILs and cancer cells were cultured from 31 breast tumor tissues. Reactivity of TILs against cancer cells was determined by measuring secreted interferon-gamma. Expression levels of epithelial markers, major histocompatibility complex molecules, and programmed death-ligand 1 (PD-L1) in cancer cells, and T cell markers (memory, T cell activation and exhaustion, and regulatory T cell markers) in expanded TILs were analyzed and compared between the reactive and non-reactive groups. RESULTS: In seven cases, TILs showed reactivity to autologous cancer cells. Six of these cases were associated with triple-negative breast cancer (TNBC). All reactive TNBCs were derived from surgical specimens after neoadjuvant chemotherapy (NAC). Higher expression of Ki67 in tumor tissues and lower expression of PD-L1 in cultured cancer cells were associated with reactivity. Proliferation of reactive TILs was high. High proportions of T cells and PD-1+CD4+ and PD1+CD8+ T cells were associated with reactivity in TNBC cases, while other activation or exhaustion markers were not. CONCLUSION: TILs from approximately half the TNBC cases with NAC showed reactivity against autologous cancer cells. The proportion of PD-1+ T cells was higher in the reactive group. Adoptive TIL therapy combined with PD-1 inhibitors might be promising for TNBC patients with residual tumors after NAC.

Comparative toxicity of silver nanoparticles (AgNPs) and silver nanowires (AgNWs) on saltwater microcrustacean, Artemia salina.

This study evaluated the potential toxic effects of silver nanoparticles (AgNPs) and silver nanowires (AgNWs) on saltwater microcrustacean Artemia salina nauplii under ISO TS 20787 guideline. To investigate the acute toxicity of these nanomaterials, the nauplii were exposed to different concentrations of 0 (control), 0.39, 1.56, 6.25, 25 and 100 mg/L AgNPs and concentrations of 0 (control), 0.01, 0.1, 1, 10, 50 and 100 mg/L AgNWs for 72 h. Immobilization rate of A. salina exposed to both AgNPs and AgNWs for 72 h increased significantly in a concentration-dependent manner (P < 0.05). The 72 h EC10 and EC50 were found to be 1.48 ±â€¯0.6 and 10.70 ±â€¯1.3 mg/L for AgNPs, respectively, and 0.03 ±â€¯0.02 and 0.43 ±â€¯0.04 mg/L for AgNWs, respectively. Based on the EC10 and EC50 values, the toxicity of AgNWs was significantly higher than AgNPs (P < 0.05). Oxidative stress resulted from 48 h exposure to both AgNPs and AgNWs in A. salina was assessed by measuring reactive oxygen species (ROS) production and superoxide dismutase (SOD) activity. The results revealed that both AgNPs and AgNWs could induce ROS production. The SOD activity decreased significantly with the increase of exposure concentration (P < 0.05). In conclusion, the present results show that both nanomaterials have toxic effects on A. salina nauplii and thus, more effort should be made to prevent their release into saltwater ecosystems and trophic transfer in the aquatic food chain.

Presence of tertiary lymphoid structures determines the level of tumor-infiltrating lymphocytes in primary breast cancer and metastasis.

The level of tumor-infiltrating lymphocytes and presence of tertiary lymphoid structures are significant prognostic and predictive factors in primary breast cancer. However, the understanding about differences in tumor-infiltrating lymphocytes and tertiary lymphoid structures at various metastatic sites or between primary breast tumors and metastatic sites is limited. A total of 335 cases of metastatic breast cancer from four metastatic sites (lung, liver, brain, and ovary) were included. We analyzed the percentages of tumor-infiltrating lymphocytes and presence of tertiary lymphoid structures in the primary and metastatic sites. The mean level of tumor-infiltrating lymphocytes in the lung metastases was higher than in the liver, brain, ovary, and matched primary tumors, while metastatic tumors of the liver and brain showed lower levels of tumor-infiltrating lymphocytes than primary tumors. Tertiary lymphoid structures were only found in the lung and liver, and in cases of brain metastases the change of tertiary lymphoid structures from present to absent significantly affected the level of tumor-infiltrating lymphocytes in metastases compared with that in matched primary tumors. Patients with a lower histological grade, hormone receptor positivity in primary tumors and metastases, a lower level of tumor-infiltrating lymphocytes and absence of tertiary lymphoid structures in primary tumors, a higher level of tumor-infiltrating lymphocytes and presence of tertiary lymphoid structures in metastases, and lung metastases showed significantly better overall survival. Our results showed that metastatic breast tumors in the lung had more tumor-infiltrating lymphocytes than did tumors at other sites and matched primary tumors. In addition, the presence of tertiary lymphoid structures in metastatic sites is a critical factor for the level of tumor-infiltrating lymphocytes.

Expression of Immunoproteasome Subunit LMP7 in Breast Cancer and Its Association with Immune-Related Markers.

PURPOSE: In the presence of interferon, proteasome subunits are replaced by their inducible counterparts to form an immunoproteasome (IP) plays a key role in generation of antigenic peptides presented by MHC class I molecules, leading to elicitation of a T cell‒mediated immune response. Although the roles of IP in other cancers, and inflammatory diseases have been extensively studied, its significance in breast cancer is unclear. MATERIALS AND METHODS: We investigated the expression of LMP7, an IP subunit, and its relationship with immune system components in two breast cancer cohorts. RESULTS: In 668 consecutive breast cancer cohort, 40% of tumors showed high level of LMP7 expression, and tumors with high expression of LMP7 had more tumor-infiltrating lymphocytes (TILs) in each subtype of breast cancer. In another cohort of 681 triple-negative breast cancer patients cohort, the expression of LMP7 in tumor cells was significantly correlated with the amount of TILs and the expression of interferon-associated molecules (MxA [p < 0.001] and PKR [p < 0.001]), endoplasmic reticulum stress-associated molecules (PERK [p=0.012], p-eIF2a [p=0.001], and XBP1 [p < 0.001]), and damage-associated molecular patterns (HMGN1 [p < 0.001] and HMGB1 [p < 0.001]). Patients with higher LMP7 expression had better disease-free survival outcomes than those with no or low expression in the positive lymph node metastasis group (p=0.041). CONCLUSION: Close association between the TIL levels and LMP7 expression in breast cancer indicates that better antigen presentation through greater LMP7 expression might be associated with more TILs.

Retained or altered expression of major histocompatibility complex class I in patient-derived xenograft models in breast cancer.

The expression of major histocompatibility complex class I (MHC I) in tumor cells is regulated by interferon signaling, and it is an important factor in the efficacy of cytotoxic T cell-dependent immunotherapy. To determine the impact of immune cells in MHC I expression on tumor cells, we compared the expression of MHC I in tumor cells derived from primary breast cancers and patient-derived xenograft (PDX) models. MHC I and myxovirus resistance gene A (MxA) expression were analyzed using immunohistochemistry in 23 cases of tumor tissue and corresponding primary and secondary PDXs. The median H score of MHC I was 210 (0-300) in patient tumor tissues, 197.5 (0-300) in primary PDX tumors, and 157.5 (5-300) in secondary PDX tumors. Cases were divided into four groups based on the difference in MHC I expression between the patient tumor tissues and secondary PDXs. Eleven cases constituted the high MHC I group, four constituted the low MHC I group, six comprised the decreased MHC I group, and two comprised the increased MHC I group. MHC I and MxA expressions in each tumor were weakly correlated within patients' tumors, while strongly correlated within PDX models. Retained or altered expression of MHC I in breast cancer PDXs reveals the presence of intrinsic and extrinsic interferon signaling pathways in tumor cells. Thus, considering MHC I expression in PDX is important when using PDX models to evaluate the efficacy of cancer immunotherapy in a preclinical setting.

Short-term inhalation study of graphene oxide nanoplates.

Graphene oxides possess unique physicochemical properties with important potential applications in electronics, pharmaceuticals, and medicine. However, the toxicity following inhalation exposure to graphene oxide has not yet been clarified. Therefore, this study conducted a short-term graphene oxide inhalation toxicity analysis using a nose-only inhalation exposure system and male Sprague-Dawley rats. A total of four groups (15 rats per group) were exposed: (1) control (fresh air), (2) low concentration (0.76 ± 0.16 mg/m3), (3) moderate concentration (2.60 ± 0.19 mg/m3), and (4) high concentration (9.78 ± 0.29 mg/m3). The rats were exposed to graphene oxide for 6 h/day for 5 days, followed by recovery for 1, 3, and 21 days. No significant body or organ weight changes were noted after the short-term exposure or during the recovery period. Similarly, no significant systemic effects of toxicological importance were noted in the hematological assays, bronchoalveolar lavage fluid (BAL) inflammatory markers, BAL fluid cytokines, or blood biochemical assays following the graphene oxide exposure or during the post-exposure observation period. Moreover, no significant differences were observed in the BAL cell differentials, such as lymphocytes, macrophages, or polymorphonuclear cells. Graphene oxide-ingested alveolar macrophages as a spontaneous clearance reaction were observed in the lungs of all the concentration groups from post 1 day to post 21 days. Histopathological examination of the liver and kidneys did not reveal any significant test-article-relevant histopathological lesions. Importantly, similar to previously reported graphene inhalation data, this short-term nose-only inhalation study found only minimal or unnoticeable graphene oxide toxicity in the lungs and other organs.

Acute toxicity, uptake, and elimination of zinc oxide nanoparticles (ZnO NPs) using saltwater microcrustacean, Artemia franciscana.

This study aims to evaluate the potential toxic effects of ZnO nanoparticles on Artemia franciscana nauplii. The ZnO NPs suspension was characterized by TEM, EDS and DLS techniques. Acute toxicity was investigated by exposure of nauplii to concentrations of 1, 5, 7.5, 10, 15, 20, 25 and 30 mg/L of ZnO NPs for 48 h and 96 h. The 96-h EC10 and EC50 values of ZnO NPs were found to be 1.39 mg/L and 4.86 mg/L respectively. The ZnO NPs suspensions did not cause any significant acute toxicity after 48 h of exposure, but the immobilization rate increase significantly compare to control group after 96 h (P < 0.05). The results showed that the uptake, accumulation, and elimination of NPs in nauplii depends on the concentration of NPs and time. The elimination rates of 46.66% and 83.85% were recorded at 1 and 10 mg/L of NPs after 24 h of depuration period, respectively.

ADAR1 expression is associated with tumour-infiltrating lymphocytes in triple-negative breast cancer.

Tumours with a high mutation burden exhibit considerable neoantigens and tumour-infiltrating lymphocytes. RNA editing by ADAR1 is a source of changes in epitope. However, ADAR1 expression in cancer cells and tumour-infiltrating lymphocyte levels in triple-negative breast cancer have not been well evaluated. We immunohistochemically examined ADAR1 expression in 681 triple-negative breast cancer patients and analysed their clinicopathological characteristics. We also analysed basal-like tumours using The Cancer Genome Atlas data. Among the 681 triple-negative breast cancer patients, 45.8% demonstrated high ADAR1 expression. Tumours with high ADAR1 expression exhibited high tumour-infiltrating lymphocyte levels, considerable CD8 + T lymphocyte infiltration, high histological grade and high expression of interferon-related proteins, including HLA-ABC, MxA and PKR. Among patients with lymph node metastasis, those with high tumour-infiltrating lymphocyte levels and low ADAR1 expression demonstrated the best disease-free survival. The Cancer Genome Atlas data analysis of basal-like tumours revealed significant positive correlation between ADAR1 and CD8B expression and positive association of high ADAR1 expression with immune responses and apoptosis pathways. We detected high ADAR1 expression in half of the triple-negative breast cancer patients. In addition to DNA mutations, RNA editing can be related to neoantigens; hence, we need to explore non-synonymous mutations exclusively found using RNA sequencing data to identify clinically relevant neoantigens.

Expression of the MHC class II in triple-negative breast cancer is associated with tumor-infiltrating lymphocytes and interferon signaling.

Tumor-infiltrating lymphocytes (TILs) have been known for their strong prognostic and predictive significance in triple-negative breast cancer (TNBC). Several mechanisms for TIL influx in TNBC have been elucidated. Major histocompatibility complex class II (MHC-II) is an essential component of the adaptive immune system and is generally restricted to the surface of antigen-presenting cells. However, it has been reported that interferon-gamma signaling may induce MHC-II in almost all cell types, including those derived from cancer. We aimed to examine the relationship between MHC-II expression in tumor cells and the amount of TILs in 681 patients with TNBC. Further, the prognostic significance of MHC-II and the association of MHC-II with a couple of molecules involved in the interferon signaling pathway were investigated using immunohistochemical staining. Higher MHC-II expression in tumor cells was associated with the absence of lymphovascular invasion (p = 0.042); larger amounts of TILs (p < 0.001); frequent formations of tertiary lymphoid structures (p < 0.001); higher expression of myxovirus resistance gene A, one of the main mediators of the interferon signaling pathway (p < 0.001); and higher expression of double-stranded RNA-activated protein kinase, which can be induced by interferons (p = 0.008). Moreover, tumors that showed high MHC class I expression and any positivity for MHC-II had larger amounts of CD4- and CD8-positive T lymphocytes (p < 0.001). Positive MHC-II expression in tumor cells was associated with better disease-free survival in patients who had lymph node metastasis (p = 0.009). In conclusion, MHC-II expression in tumor cells was closely associated with an increase in TIL number and interferon signaling in TNBC. Further studies are warranted to improve our understanding regarding TIL influx, as well as patients' responses to immunotherapy.

Glutaminase expression is a poor prognostic factor in node-positive triple-negative breast cancer patients with a high level of tumor-infiltrating lymphocytes.

Glutamine metabolism is emerging as one aspect of dysregulated metabolism of tumors. Triple-negative breast cancer (TNBC) cells are glutamine dependent, whereas luminal-type cells tend to be glutamine independent. Therefore, TNBC patients might benefit from therapies targeting glutamine metabolism. To investigate the clinical significance of glutamine metabolism, we examined expression and prognostic significance of glutaminase in tumor cells and tumor-infiltrating lymphocytes (TILs) in TNBC. We retrieved 658 surgically resected TNBCs and analyzed glutaminase expression in tumor cells and TILs by immunohistochemical staining. Glutaminase expression was observed in 237 cases (36.0%) in tumor cells and 104 cases (15.5%) in TILs. Although glutaminase expression in tumor cells was significantly associated with a low level of TILs (p = 0.018), glutaminase expression in TILs was significantly higher in cases with a high level of TILs (p = 0.031). Glutaminase expression in tumor cells was significantly associated with poor disease-free survival in patients with lymph node metastasis and high levels of TILs (p = 0.020). In addition, it was an independent poor prognostic factor (hazard ratio = 10.643, 95% confidence interval = 1.999-56.668; p = 0.006). Glutaminase expression in tumor cells was observed in a subset of TNBC patients. It was significantly associated with a low level of TILs and poor disease-free survival in TNBCs presenting with lymph node metastasis and high levels of TILs.

Predictive Value of Tertiary Lymphoid Structures Assessed by High Endothelial Venule Counts in the Neoadjuvant Setting of Triple-Negative Breast Cancer.

PURPOSE: The tertiary lymphoid structure (TLS) is an important source of tumor-infiltrating lymphocytes (TILs), which have a strong prognostic and predictive value in triple-negative breast cancer (TNBC). A previous study reported that the levels of CXCL13 mRNA expression were associated with TLSs, but measuring the gene expression is challenging in routine practice. Therefore, this study evaluated the MECA79-positive high endothelial venule (HEV) densities and their association with the histopathologically assessed TLSs in biopsy samples. In addition, the relationship of TLSs with the CXCL13 transcript levels and clinical outcomes were examined. MATERIALS AND METHODS: A total of 108 TNBC patients treated with neoadjuvant chemotherapy (NAC) were studied. The amounts of TILs and TLSs were measured histopathologically using hematoxylin and eosin-stained slides. The HEV densities and TIL subpopulations were measured by immunohistochemistry for MECA79, CD3, CD8, and CD20. CXCL13mRNA expression levels using a NanoString assay (NanoString Technologies). RESULTS: The mean number of HEVs in pre-NAC biopsies was 12 (range, 0 to 72). The amounts of TILs and TLSs, HEV density, and CXCL13 expression showed robust correlations with each other. A lower pre-NAC clinical T stage, higher TIL and TLS levels, a higher HEV density, CD20-positive cell density, and CXCL13 expression were significant predictors of a pathologic complete response (pCR). Higher CD8-positive cell density and levels of CXCL13 expression were significantly associated with a better disease-free survival rate. CONCLUSION: MECA79-positive HEV density in pre-NAC biopsies is an objective and quantitative surrogate marker of TLS and might be a valuable tool for predicting pCR of TNBC in routine pathology practice.