Persistence and enrichment of dominant T cell clonotypes in expanded tumor-infiltrating lymphocytes of breast cancer.

BACKGROUND: Adoptive cell therapy using tumor-infiltrating lymphocytes (TILs) has shown promising results in cancer treatment, including breast cancer. However, clonal dynamics and clinical significance of TIL expansion ex vivo remain poorly understood. METHODS: We investigated T cell receptor (TCR) repertoire changes in expanded TILs from 19 patients with breast cancer. We compared TCR repertoire of TILs at different stages of expansion, including initial (2W TILs) and rapid expansion (REP TILs), and their overlap with formalin fixed paraffin embedded (FFPE) and peripheral blood. Additionally, we examined differences in TCR repertoire between CD4+ and CD8+ REP TILs. RESULTS: In descending order of proportion, average of 60% of the top 10% clonotypes of FFPE was retained in 2W TIL (60% in TRB, 64.7% in TRA). Among the overlapped clonotypes between 2W TILs and REP TILs, 69.9% was placed in top 30% of 2W TIL. The proportion of clonotypes in 2W TIL and REP TIL showed a significant positive correlation. CD4+ and CD8+ T cells show similar results in diversity and CDR3 length. CONCLUSIONS: Our study traces the changes in TILs repertoire from FFPE to 2W TIL and REP TIL and confirmed that clonotypes with high frequencies in TILs have a high likelihood of maintaining their priority throughout culture process.

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.

Tumor-Infiltrating Lymphocytes in Triple-Negative Breast Cancer.

Importance: The association of tumor-infiltrating lymphocyte (TIL) abundance in breast cancer tissue with cancer recurrence and death in patients with early-stage triple-negative breast cancer (TNBC) who are not treated with adjuvant or neoadjuvant chemotherapy is unclear. Objective: To study the association of TIL abundance in breast cancer tissue with survival among patients with early-stage TNBC who were treated with locoregional therapy but no chemotherapy. Design, Setting, and Participants: Retrospective pooled analysis of individual patient-level data from 13 participating centers in North America (Rochester, Minnesota; Vancouver, British Columbia, Canada), Europe (Paris, Lyon, and Villejuif, France; Amsterdam and Rotterdam, the Netherlands; Milan, Padova, and Genova, Italy; Gothenburg, Sweden), and Asia (Tokyo, Japan; Seoul, Korea), including 1966 participants diagnosed with TNBC between 1979 and 2017 (with follow-up until September 27, 2021) who received treatment with surgery with or without radiotherapy but no adjuvant or neoadjuvant chemotherapy. Exposure: TIL abundance in breast tissue from resected primary tumors. Main Outcomes and Measures: The primary outcome was invasive disease-free survival [iDFS]. Secondary outcomes were recurrence-free survival [RFS], survival free of distant recurrence [distant RFS, DRFS], and overall survival. Associations were assessed using a multivariable Cox model stratified by participating center. Results: This study included 1966 patients with TNBC (median age, 56 years [IQR, 39-71]; 55% had stage I TNBC). The median TIL level was 15% (IQR, 5%-40%). Four-hundred seventeen (21%) had a TIL level of 50% or more (median age, 41 years [IQR, 36-63]), and 1300 (66%) had a TIL level of less than 30% (median age, 59 years [IQR, 41-72]). Five-year DRFS for stage I TNBC was 94% (95% CI, 91%-96%) for patients with a TIL level of 50% or more, compared with 78% (95% CI, 75%-80%) for those with a TIL level of less than 30%; 5-year overall survival was 95% (95% CI, 92%-97%) for patients with a TIL level of 50% or more, compared with 82% (95% CI, 79%-84%) for those with a TIL level of less than 30%. At a median follow-up of 18 years, and after adjusting for age, tumor size, nodal status, histological grade, and receipt of radiotherapy, each 10% higher TIL increment was associated independently with improved iDFS (hazard ratio [HR], 0.92 [0.89-0.94]), RFS (HR, 0.90 [0.87-0.92]), DRFS (HR, 0.87 [0.84-0.90]), and overall survival (0.88 [0.85-0.91]) (likelihood ratio test, P < 10e-6). Conclusions and Relevance: In patients with early-stage TNBC who did not undergo adjuvant or neoadjuvant chemotherapy, breast cancer tissue with a higher abundance of TIL levels was associated with significantly better survival. These results suggest that breast tissue TIL abundance is a prognostic factor for patients with early-stage TNBC.

A Nationwide Study on HER2-low Breast Cancer in South Korea: Its Incidence of 2022 Real World Data and the Importance of Immunohistochemical Staining Protocols.

Purpose: Notable effectiveness of trastuzumab deruxtecan (T-DXd) in patients with HER2-low advanced breast cancer (BC) has focused pathologists' attention. We studied the incidence and clinicopathologic characteristics of HER2-low BC, and the effects of immunohistochemistry (IHC) associated factors on HER2 IHC results. Materials and Methods: The Breast Pathology Study Group of the Korean Society of Pathologists conducted a nationwide study using real-world data on HER2 status generated between January 2022 and December 2022. Information on HER2 IHC protocols at each participating institution was also collected. Results: Total 11,416 patients from twenty-five institutions included in this study. Of these patients, 40.7% (range: 6.0%-76.3%) were classified as HER2-zero, 41.7% (range: 10.5%-69.1%) as HER2-low, and 17.5% (range: 6.7%-34.0%) as HER2-positive. HER2-low tumors were associated with positive ER and PR statuses (p<0.001 and p<0.001, respectively). Antigen retrieval times (≥ 36 min vs. < 36 min) and antibody incubation times (≥ 12 min vs. < 12 min) affected on the frequency of HER2 IHC 1+ BC at institutions using the PATHWAY HER2 (4B5) IHC assay and BenchMark XT or Ultra staining instruments. Furthermore, discordant results between core needle biopsy (CNB) and subsequent resection specimen HER2 statuses were observed in 24.1% (787/3259) of the patients. Conclusion: The overall incidence of HER2-low BC in South Korea concurs with those reported in previously published studies. Significant inter-institutional differences in HER2 IHC protocols were observed, and it may have impact on HER2-low status. Thus, we recommend standardizing HER2 IHC conditions to ensure precise patient selection for targeted therapy.

Analysis and Development of Antigen-specific T Cells Derived from Peripheral Blood Mononuclear Cells of Healthy Donors.

BACKGROUND/AIM: Adoptive cell therapy using antigen-specific T cells is a promising treatment modality for cancer patients. Various methods to isolate specific T cells and identify corresponding T cell receptor (TCR) sequences are known. This study aimed to identify antigen-specific TCR from T cells isolated using carboxyfluorescein succinimidyl ester (CFSE), which marks proliferating activated T cells. MATERIALS AND METHODS: CFSE stained healthy donor peripheral blood mononuclear cells (PBMCs) were treated with cytomegalovirus (CMV) or Epstein-Barr virus (EBV) peptides for seven days. Then, proliferating T cells with decreased CFSE staining were isolated and single cell VDJ sequencing was performed on isolated T cells to identify antigen-specific TCRs. RESULTS: As antigen-specific TCR candidates, ten TCR clones were selected for the CMV antigen and five for the EBV antigen. The reactivity of ten CMV TCR-transduced T cells and one EBV TCR-transduced T cell toward T2 cells pulsed with CMV or EBV peptide was confirmed via NFAT-luciferase, IFN-γ ELISA, and cytotoxicity assays. CONCLUSION: Identification of antigen-specific TCRs with CFSE staining is a valid method for the development of effective immunotherapy. The identified CMV- or EBV-specific TCRs can be used for adoptive cell therapy to treat cancer.

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.

Proteomic analysis of breast cancer based on immune subtypes.

BACKGROUND: Immunotherapy is applied to breast cancer to resolve the limitations of survival gain in existing treatment modalities. With immunotherapy, a tumor can be classified into immune-inflamed, excluded and desert based on the distribution of immune cells. We assessed the clinicopathological features, each subtype's prognostic value and differentially expressed proteins between immune subtypes. METHODS: Immune subtyping and proteomic analysis were performed on 56 breast cancer cases with neoadjuvant chemotherapy. The immune subtyping was based on the level of tumor-infiltrating lymphocytes (TILs) and Klintrup criteria. If the level of TILs was ≥ 10%, it was classified as immune-inflamed type without consideration of the Klintrup criteria. In cases of 1-9% TIL, Klintrup criteria 1-3 were classified as the immune-excluded subtype and Klintrup criteria not available (NA) was classified as NA. Cases of 1% TILs and Klintrup 0 were classified as the immune-desert subtype. Mass spectrometry was used to identify differentially expressed proteins in formalin-fixed paraffin-embedded biopsy tissues. RESULTS: Of the 56 cases, 31 (55%) were immune-inflamed, 21 (38%) were immune-excluded, 2 (4%) were immune-desert and 2 (4%) were NA. Welch's t-test revealed two differentially expressed proteins between immune-inflamed and immune-excluded/desert subtypes. Coronin-1A was upregulated in immune-inflamed tumors (adjusted p = 0.008) and α-1-antitrypsin was upregulated in immune-excluded/desert tumors (adjusted p = 0.008). Titin was upregulated in pathologic complete response (pCR) than non-pCR among immune-inflamed tumors (adjusted p = 0.036). CONCLUSIONS: Coronin-1A and α-1-antitrypsin were upregulated in immune-inflamed and immune-excluded/desert subtypes, respectively. Titin's elevated expression in pCR within the immune-inflamed subtype may indicate a favorable prognosis. Further studies involving large representative cohorts are necessary to validate these findings.

CCR10-mediated Enhancement of T Cell Trafficking for Improved Tumor Immunotherapy.

BACKGROUND/AIM: The effectiveness of adoptive T cell therapy for solid tumors remains suboptimal, partly attributed to insufficient T cell infiltration into the tumor site. A promising strategy involves directing T cells towards the tumor utilizing tumor-specific chemokine receptors. MATERIALS AND METHODS: We analyzed chemokine receptor expression in activated T cells and chemokine expression in breast and lung cancer using The Cancer Genome Atlas (TCGA) data. Subsequently, we generated 1G4 T cell receptor-engineered T (TCR-T) cells with CCR10 and performed in vitro and in vivo efficacy tests. RESULTS: CCR10 exhibited insufficient expression in various human T cells. Analysis of TCGA RNA sequencing data revealed elevated expression of the chemokine CCL28, the corresponding chemokine for CCR10, in breast and lung cancer. Consequently, we generated CCR10-1G4 TCR-T cells. CCR10-1G4 dual expressing TCR-T cells exhibited comparable cellular cytotoxicity but increased mobility compared to 1G4 TCR-T cells in vitro. Furthermore, injecting CCR10-1G4 dual expressing TCR-T cells into a xenograft tumor model demonstrated enhanced in vivo trafficking and a greater reduction of tumor burden. CONCLUSION: This study highlights the potential of CCR10 for developing efficient adoptive T-cell treatments targeting solid tumors.

Prognostic Significance of Tumor-Infiltrating Lymphocytes and High-Risk Human Papillomavirus in Ocular Sebaceous Carcinoma: A Comprehensive Analysis.

High-risk human papillomavirus (hrHPV) and tumor-infiltrating lymphocytes (TILs) are known to have prognostic significance in oropharyngeal squamous cell carcinoma. However, their significance in ocular sebaceous carcinoma (OSC) remains unverified because of the rarity of the condition. This study aimed to investigate the association between clinicopathologic features, biomarkers, and hrHPV infection and their potential to predict prognosis in OSC patients. We analyzed the clinicopathologic features of 81 OSC patients from Asan Medical Center between 2000 and 2022. Seventeen biomarkers and hrHPV were examined using immunohistochemistry and DNA in situ hybridization on tissue microarray cores. hrHPV was identified in 31 cases (38.3%). Univariate analysis revealed that hrHPV infection was associated with comedonecrosis (P = .032), high Ki-67 labeling index (≥30%, P = .042), lower expression of E-cadherin (P = .033), and loss of expression of zinc finger protein 750 (P = .023). Multivariate analysis revealed that loss of expression of zinc finger protein 750 (P = .026) remained an independently associated factor for hrHPV. Progression-free survival analysis was performed on 28 patients who were continuously observed for more than 5 years. During a median follow-up duration of 86 months, recurrence or metastasis developed in 14 patients (50%) within the survival cohort, occurring at a median time of 48 months after excision. Univariate analysis indicated that recurrence or metastasis was associated with tumor size (P = .010), high TILs (≥10%; P = .025), lymphovascular invasion (P = 0.043), site of origin (P = .025), and high expression of bcl-2-associated athanogene 3 (P = .039). Multivariate analysis demonstrated that high TILs (P = .017) and site of origin (P = .025) were independent prognostic factors. The prognosis of OSC was hrHPV-independent, and a better prognosis was associated with the site of origin in the order of the gland of Zeis, meibomian gland, and multicentric site, as well as with high TILs.

21-gene expression assay and clinical outcomes of premenopausal patients with hormone receptor-positive breast cancer.

The prognostic role of the recurrence score (RS) based on the 21-gene expression assay in premenopausal women is not well delineated, and we investigated the association of outcomes and the RS in premenopausal patients who had 21-gene expression assay at Asan Medical Center, Seoul, Korea, between June 2005 and July 2018. Invasive breast cancer-free survival (IBCFS) by STEEP version 2.0 was compared according to the RS and clinical risk factors. A total of 554 patients were included in our study and 116 patients (20.9%) had age <40 years, 238 patients (43.0%) had luminal B subtype (Ki67 ≥ 20%), and 83 patients (15.0%) had RS >25. All patients received adjuvant tamoxifen ± chemotherapy. Overall, patients with RS >25 showed trend toward worse IBCFS from multivariable analysis (adjusted HR 1.89 [95% CI: 0.95-3.73], P = .069). When comparing outcomes according to age and luminal subtypes, patients with luminal B subtype and age <40 years (n = 60) showed significantly worse outcomes compared to the others (luminal A or luminal B + age ≥40 years, n = 494; adjusted HR 2.95 [95% CI: 1.49-5.82], log-rank P < .001). Among patients with luminal B subtype and age <40 years, there was no significant association observed between IBCFS and the RS (log-rank P = .51). In conclusion, while RS >25 showed association with poor outcomes in premenopausal women, it may have less prognostic significance among those with luminal B subtype and age <40 years.

Association of cyclooxygenase-2 expression with endoplasmic reticulum stress and autophagy in triple-negative breast cancer.

Cyclooxygenase-2 plays a role in oncogenesis and its overexpression is associated with triple-negative breast cancer. However, the mechanisms whereby cyclooxygenase-2 contribute to breast cancer are complex and not well understood. Cyclooxygenase-2 overexpression causes hypoxia, oxidative stress, and endoplasmic reticulum stress. The aim of this study is to investigate the correlations among cyclooxygenase-2 expression, endoplasmic reticulum stress-associated molecules, and autophagy-associated molecules in triple-negative breast cancer. Surgical specimens from two cohorts of triple-negative breast cancer patients without neoadjuvant systemic therapy were analyzed: cohorts 1 and 2 consisted of 218 cases from 2004 to 2006 and 221 cases from 2007 to 2009, respectively. Specimens were evaluated by immunohistochemical examination of cyclooxygenase-2, endoplasmic reticulum stress markers, and autophagy markers expression using tissue microarrays. Cyclooxygenase-2 was overexpressed in 29.8% and 23.9% of cases in cohorts 1 and 2, respectively; and it was positively correlated with two out of three endoplasmic reticulum stress-associated molecules (XBP1, p = 0.025 and p = 0.003 in cohort 1 and cohort 2, respectively; PERK, p < 0.001 in both cohorts). Cyclooxygenase-2 was also positively correlated with two out of three autophagy markers (p62, p = 0.002 and p = 0.003 in cohort 1 and cohort 2, respectively; beclin1, p < 0.001 in both cohorts). Although cyclooxygenase-2 was not an independent prognostic factor for distant metastasis free survival and overall survival, its expression was associated with the expression of endoplasmic reticulum stress and autophagy molecules in triple-negative breast cancer.

Predictive modelling for the diagnosis of oral and laryngeal premalignant and malignant lesions using p53 and Ki-67 expression.

Oral and laryngeal epithelial lesions are currently diagnosed using histological criteria based on the World Health Organization (WHO) classification, which can cause interobserver variability. An integrated diagnostic approach based on immunohistochemistry (IHC) would aid in the interpretation of ambiguous histological findings of epithelial lesions. In the present study, IHC was used to evaluate the expression of p53 and Ki-67 in 114 cases of oral and laryngeal epithelial lesions in 104 patients. Logistic regression analysis and decision tree algorithm were employed to develop a scoring system and predictive model for differentiating the epithelial lesions. Cohen's kappa coefficient was used to evaluate interobserver variability, and next-generation sequencing (NGS) and IHC were used to compare TP53 mutation and p53 expression patterns. Two expression patterns for p53, namely, diffuse expression type (pattern HI) and null type (pattern LS), and the pattern HI for Ki-67 were significantly associated with high-grade dysplasia (HGD) or squamous cell carcinoma (SqCC). With an accuracy and area under the receiver operating characteristic curve (AUC) of 84.6% and 0.85, respectively, the scoring system based on p53 and Ki-67 expression patterns classified epithelial lesions into two types: non-dysplasia (ND) or low-grade dysplasia (LGD) and SqCC or HGD. The decision tree model constructed using the p53 and Ki-67 expression patterns classified epithelial lesions into ND, LGD, and group 2, including HGD or SqCC, with an accuracy and AUC of 75% and 0.87, respectively. The integrated diagnosis had a better correlation with near perfect agreement (weighted kappa 0.92, unweighted kappa 0.88). The patterns HI and LS for p53 were confirmed to be correlated with missense mutations and nonsense/frameshift mutations, respectively. A predictive model for diagnosis was developed based on the correlation between TP53 mutation and p53 expression patterns. These results indicate that the scoring system based on p53 and Ki-67 expression patterns can differentiate epithelial lesions, especially in cases when the morphological features are ambiguous.

Ki-67, 21-Gene Recurrence Score, Endocrine Resistance, and Survival in Patients With Breast Cancer.

Importance: Both high 21-gene recurrence score (RS) and high Ki-67 level are poor prognostic factors in patients with estrogen receptor (ER)-positive ERBB2-negative (ER+/ERBB-) breast cancer; however, a discrepancy between the 2 has been noted. Survival differences according to these 2 biomarkers are not well known. Objective: To assess the associations between RS and Ki-67 expression and between Ki-67 expression and recurrence-free survival in patients with ER+/ERBB- breast cancer with low RS. Design, Setting, and Participants: This cohort study included women treated for ER+/ERBB2- breast cancer who underwent the 21-gene RS test from March 2010 to December 2020 in 2 hospitals in Korea. Exposures: Recurrence score and Ki-67 level. Main Outcomes and Measures: A Cox proportional hazards regression model was used to examine the association of Ki-67 with recurrence-free survival (RFS), while a binary logistic regression model was used to examine the association between Ki-67 and secondary endocrine resistance. High Ki-67 expression was defined as 20% or greater, and low genomic risk as an RS of 25 or less. Secondary endocrine resistance was defined as breast cancer recurrence that occurred after at least 2 years of endocrine therapy and during or within the first year after completing 5 years of adjuvant endocrine therapy. Results: A total of 2295 female patients were included (mean [SD] age, 49.8 [9.3] years), of whom 1948 (84.9%) were in the low genomic risk group and 1425 (62.1%) had low Ki-67 level. The median follow-up period was 40 months (range, 0-140 months). The RS and Ki-67 level had a moderate correlation (R = 0.455; P < .001). Of the patients with low Ki-67 level, 1341 (94.1%) had low RS, whereas 607 of 870 patients with high Ki-67 level (69.8%) had low RS. In patients with low RS, the RFS differed significantly according to Ki-67 level (low Ki-67, 98.5% vs high Ki-67, 96.5%; P = .002). Among the 1807 patients with low genomic risk who did not receive chemotherapy, high Ki-67 level was independently associated with recurrence (hazard ratio, 2.51; 95% CI, 1.27-4.96; P = .008). Recurrence after 3 years differed significantly according to Ki-67 level (low Ki-67, 98.7% vs high Ki-67, 95.7%; P = .003), whereas recurrence within 3 years did not differ (low Ki-67, 99.3% vs high Ki-67, 99.3%; P = .90). In addition, Ki-67 was associated with secondary endocrine resistance in patients with low RS who did not receive chemotherapy (odds ratio, 2.49; 95% CI, 1.13-5.50; P = .02). Conclusions and Relevance: In this cohort study of patients with ER+/ERBB2- breast cancer, a moderate correlation was observed between Ki-67 and RS, and high Ki-67 level in patients with low genomic risk was associated with increased risk of secondary endocrine resistance.

Prognostic implications of HER2 changes after neoadjuvant chemotherapy in patients with HER2-zero and HER2-low breast cancer.

BACKGROUND: The transition of human epidermal growth factor receptor 2 (HER2) status after neoadjuvant chemotherapy (NAC) in HER2-low breast cancer has not been thoroughly evaluated. Here, we evaluated the HER2 transition among HER2-zero and HER2-low breast cancer cases post-NAC and its impact on clinical outcomes. METHODS: We included 1288 patients with HER2-low or zero breast cancer who underwent NAC and surgery between 2014 and 2018 and had paired pre- and post-therapeutic HER2 status results. RESULTS: Among patients who were HER2-zero pre-NAC (n = 650), 68% and 29% were HER2-zero and HER2-low, respectively, post-NAC. Among patients who were HER2-low pre-NAC (n = 638), 32% of patients showed HER2 changes (low to zero), and 59% of patients had a constant HER2-low status post-NAC. Patients with constant HER2-low or transitions from HER2-low to zero had a higher proportion of hormone receptor positivity (84% and 79%) than those with changes from HER2-zero to low (77%) or with constant HER2-zero (56%), respectively. Multivariable logistic regression analysis revealed that patients with oestrogen receptor positivity had a higher probability of gaining HER2-low expression than those with oestrogen receptor negativity (odds ratio 2.48). No significant differences were observed in terms of overall survival or disease-free survival between patients with and without HER2-changes according to their hormone receptor status, except in the post-therapeutic HER2-low, hormone receptor-negativity subset. CONCLUSION: Temporal heterogeneity of HER2-low expression is observed in substantial numbers of post-NAC breast cancer patients. Clinical outcomes show no significant associations, except in the post-therapeutic HER2-low, hormone receptor negativity subset. The prognostic implications of HER2 transition in HER2-low breast cancer require further investigation.

Randomized phase III trial of a neoadjuvant regimen of four cycles of adriamycin plus cyclophosphamide followed by four cycles of docetaxel (AC4-D4) versus a shorter treatment of three cycles of FEC followed by three cycles of docetaxel (FEC3-D3) in node-positive breast cancer (Neo-shorter; NCT02001506).

PURPOSE: To determine whether six cycles of FEC3-D3 has a comparable efficacy to eight of AC4-D4. METHODS: The enrolled patients (pts) were clinically diagnosed with stage II or III breast cancer. The primary endpoint was a pathologic complete response (pCR), and the secondary endpoints were 3 year disease-free survival (3Y DFS), toxicities, and health-related quality of life (HRQoL). We calculated that 252 pts were needed in each treatment group to enable the detection of non-inferiority (non-inferiority margin of 10%). RESULTS: In terms of ITT analysis, 248 pts were finally enrolled. The 218 pts who completed the surgery were included in the current analysis. The baseline characteristics of these subjects were well balanced between the two arms. By ITT analysis, pCR was achieved in 15/121 (12.4%) pts in the FEC3-D3 arm and 18/126 (14.3%) in the AC4-D4 arm. With a median follow up of 64.1 months, the 3Y DFS was comparable between the two arms (75.8% in FEC3-D3 vs. 75.6% in AC4-D4). The most common adverse event (AE) was Grade 3/4 neutropenia, which arose in 27/126 (21.4%) AC4-D4 arm pts vs 23/121 (19.0%) FEC3-D3 arm cases. The primary HRQoL domains were similar between the two groups (FACT-B scores at baseline, P = 0.35; at the midpoint of NACT, P = 0.20; at the completion of NACT, P = 0.44). CONCLUSION: Six cycles of FEC3-D3 could be an alternative to eight of AC4-D4. Trial registration ClinicalTrials.gov NCT02001506. Registered December 5,2013. https://clinicaltrials.gov/ct2/show/NCT02001506.

Tumor-infiltrating lymphocyte therapy: Clinical aspects and future developments in this breakthrough cancer treatment.

Tumor-infiltrating lymphocyte (TIL) therapy is a promising approach for treating refractory or advanced solid cancers by using autologous TILs harvested from cancer tissues. Despite the heterogeneity of cancer, TIL therapy can potentially produce a positive therapeutic response, including complete remission. After decades of research on lymphocyte functions, culture/expansion methods, therapeutic protocols, and multiple clinical trials, TIL therapy has finally reached a stage where it can be formally approved for clinical use. TIL therapy is expected to hold a unique position among anti-cancer therapeutic options as a standard intervention. To successfully introduce TIL therapy into clinical settings, there is a need to expand therapeutic indications and set up the best protocols for cancer tissue sampling and manufacturing, and related clinical trials. Moreover, studies on next-generation TIL therapy have already begun, and post-approval real-world data will promote and support further research.

Immune repertoire and responses to neoadjuvant TCHP therapy in HER2-positive breast cancer.

Background: Despite the introduction of trastuzumab, pathologic complete response (pCR) is not attained in approximately 30-40% of Human epithelial growth factor receptor-2-positive breast cancer. Tumor-infiltrating lymphocytes (TIL) have been suggested as a predictive marker of treatment response, albeit not always effective. We investigated the relationship between trastuzumab, docetaxel, carboplatin, and pertuzumab (TCHP) treatment and immune repertoire as a treatment response predictor. Design: In all, 35 cases were divided into two experimental groups: 10 and 25 cases in the preliminary and main experiments, respectively. In the preliminary experiment, the biopsy tissues before TCHP treatment and the surgical tissues after TCHP treatment were compared. In the main experiment, the biopsy tissues before TCHP treatment were compared according to the TCHP treatment response. Methods: The T-cell repertoire for TRA, TRB, TRG, and TRD, and B-cell repertoire for immunoglobulin heavy, immunoglobulin kappa, and immunoglobulin lambda were evaluated. Whole transcriptome sequencing was also performed. Results: In the preliminary experiment, the density and richness of the T-cell receptor (TCR) and B-cell receptor (BCR) repertoires decreased after treatment, regardless of TCHP response. In the main experiment, the Shannon's entropy index, density, and length of CDR3 of the TCR and BCR repertoires did not differ significantly in patients who did and did not achieve pCR. The pCR and non-pCR subgroups according to the level of TILs revealed that the non-pCR/lowTIL group had a higher proportion of low-frequency clones than the pCR/lowTIL group in TRA (non-pCR/lowTIL versus pCR/lowTIL, 0.01-0.1%, 63% versus 45.3%; <0.01%, 32.9% versus 51.8%, p < 0.001) and TRB (non-pCR/lowTIL versus pCR/lowTIL, 0.01-0.1%, 26.5% versus 14.7%; <0.01%, 72.0% versus 84.1%, p < 0.001). Conclusions: The role of the diversity, richness, and density of the TCR and BCR repertoires as predictive markers for TCHP response was not identified. Compositions of low-frequency clones could be candidates for predictive factors of TCHP response; however, validation studies and further research are necessary.

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.

Establishment of a platform for measuring mitochondrial oxygen consumption rate for cardiac mitochondrial toxicity.

The heart has an abundance of mitochondria since cardiac muscles require copious amounts of energy for providing continuous blood through the circulatory system, thereby implying that myocardial function is largely reliant on mitochondrial energy. Thus, cardiomyocytes are susceptible to mitochondrial dysfunction and are likely targets of mitochondrial toxic drugs. Various methods have been developed to evaluate mitochondrial toxicity by evaluating toxicological mechanisms, but an optimized and standardized assay for cardiomyocytes remains unmet. We have therefore attempted to standardize the evaluation system for determining cardiac mitochondrial toxicity, using AC16 human and H9C2 rat cardiomyocytes. Three clinically administered drugs (acetaminophen, amiodarone, and valproic acid) and two anticancer drugs (doxorubicin and tamoxifen) which are reported to have mitochondrial effects, were applied in this study. The oxygen consumption rate (OCR), which directly reflects mitochondrial function, and changes in mRNA levels of mitochondrial respiratory complex I to complex V, were analyzed. Our results reveal that exposure to all five drugs results in a concentration-dependent decrease in the basal and maximal levels of OCR in AC16 cells and H9C2 cells. In particular, marked reduction in the OCR was observed after treatment with doxorubicin. The reduction in OCR after exposure to mitochondrial toxic drugs was found to be associated with reduced mRNA expression in the mitochondrial respiratory complexes, suggesting that the cardiac mitochondrial toxicity of drugs is majorly due to dysfunction of mitochondrial respiration. Based on the results of this study, we established and standardized a protocol to measure OCR in cardiomyocytes. We expect that this standardized evaluation system for mitochondrial toxicity can be applied as basic data for establishing a screening platform to evaluate cardiac mitochondrial toxicity of drugs, during the developmental stage of new drug discovery.

Trophoblast cell-surface antigen 2 (TROP2) expression in triple-negative breast cancer.

BACKGROUND: Trophoblast cell-surface antigen 2 (TROP2) is related to tumor proliferation enhancement and poor prognosis. An antibody targeting TROP2 was developed to treat metastatic triple-negative breast cancer (TNBC) which has a limited treatment modality. To characterize the TROP2 expressing tumors in TNBC, we analyzed TROP2 expression in three cohorts; (1) primary tumor without neoadjuvant chemotherapy, (2) primary tumor with neoadjuvant chemotherapy, and (3) metastatic tumor. METHODS: A total of 807 TNBC cases were evaluated for TROP2 immunohistochemical expression. We evaluated the TROP2 H-score distribution in the three cohorts. Tumors were divided into two groups based on TROP2 expression (high vs. low). We analyzed the relationship between clinicopathologic features and markers, including epidermal growth factor receptor, cytokeratin 5/6, p53, and Ki-67, and prognostic significance at high vs. low TROP2 expression. RESULTS: There was no difference in TROP2 H-score distribution between the three cohorts. Moderate-to-strong membranous expression of TROP2 in at least 10% of tumor cells was present in 662 cases (82.0%) in Cohort 1, 59 cases (89.4%) in Cohort 2, and 23 cases (88.5%) in Cohort 3. There was no significant difference in clinicopathologic features between high vs. low TROP2 in all cohorts. TROP2 H-score was an independent poor prognostic factor for overall survival in Cohort 3. CONCLUSIONS: TNBC showed similar TROP2 expression regardless of neoadjuvant treatment or primary tumor/metastasis. Although the prognostic significance of TROP2 expression in metastatic TNBC has been revealed, further evaluation of the predictive value of TROP2 expression for targeted therapy is needed.