Single-cell transcriptome analysis reveals subtype-specific clonal evolution and microenvironmental changes in liver metastasis of pancreatic adenocarcinoma and their clinical implications.

BACKGROUND: Intratumoral heterogeneity (ITH) and tumor microenvironment (TME) of pancreatic ductal adenocarcinoma (PDAC) play important roles in tumor evolution and patient outcomes. However, the precise characterization of diverse cell populations and their crosstalk associated with PDAC progression and metastasis is still challenging. METHODS: We performed single-cell RNA sequencing (scRNA-seq) of treatment-naïve primary PDAC samples with and without paired liver metastasis samples to understand the interplay between ITH and TME in the PDAC evolution and its clinical associations. RESULTS: scRNA-seq analysis revealed that even a small proportion (22%) of basal-like malignant ductal cells could lead to poor chemotherapy response and patient survival and that epithelial-mesenchymal transition programs were largely subtype-specific. The clonal homogeneity significantly increased with more prevalent and pronounced copy number gains of oncogenes, such as KRAS and ETV1, and losses of tumor suppressor genes, such as SMAD2 and MAP2K4, along PDAC progression and metastasis. Moreover, diverse immune cell populations, including naïve SELLhi regulatory T cells (Tregs) and activated TIGIThi Tregs, contributed to shaping immunosuppressive TMEs of PDAC through cellular interactions with malignant ductal cells in PDAC evolution. Importantly, the proportion of basal-like ductal cells negatively correlated with that of immunoreactive cell populations, such as cytotoxic T cells, but positively correlated with that of immunosuppressive cell populations, such as Tregs. CONCLUSION: We uncover that the proportion of basal-like subtype is a key determinant for chemotherapy response and patient outcome, and that PDAC clonally evolves with subtype-specific dosage changes of cancer-associated genes by forming immunosuppressive microenvironments in its progression and metastasis.

Identification of maximal tumor size associated with negligible lymph node metastasis for endoscopic submucosal dissection of undifferentiated-type early gastric cancer.

BACKGROUND AND AIMS: When treating undifferentiated-type early gastric cancer (UD-EGC) that is limited to the mucosa (clinically T1a), endoscopic submucosal dissection (ESD) can be considered if the tumor is 2 cm or less and is not ulcerated. However, there is insufficient evidence to determine the relationships between tumor size and oncological safety of ESD in UD-EGC. METHODS: The pathology reports of Korean patients who were diagnosed with UD-EGC (n = 5286) were retrospectively reviewed. The cumulative incidence of lymph node metastasis (LNM) according to tumor size was evaluated in subgroups. The tumor-size cut-off was identified as the upper limit of the 95% confidence interval (CI) of cumulative LNM incidence that did not exceed 1.0%. RESULTS: We identified 1516 patients with non-ulcerated T1a tumors ≤2 cm in size. Among patients without lymphatic invasion, 1.5% (95% CI 0.91-2.16%) had LNM. In patients with poorly differentiated tubular adenocarcinoma (PD), LNM increased from 0 to 0.74% based on a tumor size of 1.0 cm. Regardless of tumor size, smaller percentages of undifferentiated-type (UD) and poorly cohesive carcinoma (PCC) patients experienced LNM than did those with PD. In non-ulcerated mucosal cancer without lymphatic invasion and tumor size ≤0.9 cm, no LNM was observed in patients with UD (95% CI 0-0.53%), PCC (95% CI 0-0.59%), or PD (95% CI 0-0.86%) histologic type. CONCLUSION: In patients diagnosed with non-ulcerated T1a UD-EGC, ESD can be performed if the tumor size is 0.9 cm or less, regardless of histologic type.

Prognostic Effect of Tertiary Lymphoid Structures in Epstein-Barr Virus-associated Gastric Carcinomas measured by Digital Image Analysis.

Epstein-Barr virus-associated gastric carcinoma (EBVaGC) is characterized by prominent tumor-infiltrating lymphocytes (TIL) and has a favorable prognosis. Tertiary lymphoid structures (TLS), characterized by ectopic aggregated lymphocytes with high endothelial venules (HEV), are associated with favorable outcomes in various solid tumors. We hypothesized that EBVaGC, characterized by intense TIL, may be closely associated with TLS or HEV. To test this hypothesis, we digitally analyzed the TLS, HEV, and TIL in 73 surgically resected advanced EBVaGCs. For HEV, dual MECA-79 and CD31 dual immunohistochemistry were performed, and the ectopic expression of MECA-79 in tumor cells was measured. In 73 patients with EBVaGC, a high TLS ratio was found in 29 (39.7%) cases, high tumor-associated HEV density in 44 (60.3%) cases, and high CD8+ TIL density in 38 (52.1%) cases. Ectopic tumor expression of MECA-79 was observed in 36 patients (49.3%) cases. A low TLS ratio and tumor-associated HEV density were significantly associated with lymph node metastasis (p=0.005 and 0.042, respectively). Ectopic MECA-79 expression was significantly associated with lymph node metastasis (p=0.003). Patients with a low TLS ratio (p=0.038), low HEV density (p=0.042), and ectopic tumor MECA-79 expression (p=0.032) had significantly worse prognoses. In conclusion, the TLS ratio and HEV density affect the survival of patients with EBVaGC and may be related to the immune response that interrupts lymph node metastasis.

Interpretation of PD-L1 expression in gastric cancer: summary of a consensus meeting of Korean gastrointestinal pathologists.

Nivolumab plus chemotherapy in the first-line setting has demonstrated clinical efficacy in patients with human epidermal growth factor receptor 2-negative advanced or metastatic gastric cancer, and is currently indicated as a standard treatment. Programmed death-ligand 1 (PD-L1) expression is an important biomarker for predicting response to anti-programmed death 1/PD-L1 agents in several solid tumors, including gastric cancer. In the CheckMate-649 trial, significant clinical improvements were observed in patients with PD-L1 combined positive score (CPS) ≥ 5, determined using the 28-8 pharmDx assay. Accordingly, an accurate interpretation of PD-L1 CPS, especially at a cutoff of 5, is important. The CPS method evaluates both immune and tumor cells and provides a comprehensive assessment of PD-L1 expression in the tumor microenvironment of gastric cancer. However, CPS evaluation has several limitations, one of which is poor interobserver concordance among pathologists. Despite these limitations, clinical indications relying on PD-L1 CPS are increasing. In response, Korean gastrointestinal pathologists held a consensus meeting for the interpretation of PD-L1 CPS in gastric cancer. Eleven pathologists reviewed 20 PD-L1 slides with a CPS cutoff close to 5, stained with the 28-8 pharmDx assay, and determined the consensus scores. The issues observed in discrepant cases were discussed. In this review, we present cases of gastric cancer with consensus PD-L1 CPS. In addition, we briefly touch upon current practices and clinical issues associated with assays used for the assessment of PD-L1 expression in gastric cancer.

Prognostic and predictive impact of sex in locally advanced microsatellite instability high gastric or gastroesophageal junction cancer: An individual patient data pooled analysis of randomized clinical trials.

BACKGROUND: Surgery plus peri-operative/adjuvant chemotherapy is the standard of care for locally advanced GC/GEJC, though with unsatisfactory results. dMMR/MSI-high tumors have better prognosis and scant benefit from chemotherapy as compared to pMMR/MSS ones. The differential outcome of therapies in terms of safety and efficacy according to sex is still debated in GC/GEJC patients. METHODS: We previously performed an individual patient data pooled analysis of MAGIC, CLASSIC, ITACA-S, and ARTIST trials including GC/GEJC patients treated with surgery alone or surgery plus peri-operative/adjuvant chemotherapy to assess the value of MSI status. We performed a secondary analysis investigating the prognostic and predictive role of sex (female versus male) in the pooled analysis dataset in the overall population and patients stratified for MSI status (MSI-high versus MSS/MSI-low). Disease-free (DFS) and overall survival (OS) were calculated. RESULTS: Patients with MSI-high tumors had improved survival as compared to MSS/MSI-low ones irrespective of sex, whereas in those with MSS/MSI-low tumors, females had numerically longer OS and DFS (5-year OS was 63.2% versus 57.6%, HR 0.842; p = 0.058, and 5-year DFS was 55.8% versus 50.8%, HR 0.850; p = 0.0504 in female versus male patients). The numerical difference for the detrimental effect of chemotherapy in MSI-high GC was higher in females than males, while the significant benefit of chemotherapy over surgery alone was confirmed in MSS/MSI-low GC irrespective of sex. CONCLUSIONS: This pooled analysis including four randomized trials highlights a relevant impact of sex in the prognosis and treatment efficacy of MSI-high and MSS/MSI-low non-metastatic GC/GEJC.

Establishment of a patient-specific avatar organoid model derived from endoscopic ultrasonography-guided fine needle biopsy for timely clinical application in pancreatic ductal adenocarcinoma.

BACKGROUND AND AIMS: Pancreatic ductal adenocarcinoma (PDAC) has the worst survival rate among tumors. At the time of diagnosis, more than 80% of PDACs are considered to be surgically unresectable, and there is an unmet need for treatment options in these inoperable PDACs. This study aimed to establish a patient-derived organoid (PDO) platform from endoscopic ultrasound-guided fine needle biopsy (EUS-FNB) collected at diagnosis and to determine its clinical applicability for the timely treatment of unresectable PDAC. METHODS: Patients with suspected PDAC were prospectively enrolled at the Samsung Medical Center from 2015 to 2019. PDAC tissues were acquired by means of EUS-FNB to establish PDAC PDOs, which were comprehensively analyzed for histology, genomic sequencing, and high-throughput screening (HTS) drug sensitivity test. RESULTS: PDAC PDOs were established with a success rate of 83.2% (94/113). It took approximately 3 weeks from acquiring minimal EUS-FNB specimens to generating sufficient PDAC PDOs for the simultaneous HTS drug sensitivity test and genomic sequencing. The high concordance between PDAC tissues and matched PDOs was confirmed, and whole-exome sequencing revealed the increased detection of genetic alterations in PDOs compared with EUS-FNB tissues. The HTS drug sensitivity test showed clinical correlation between the ex vivo PDO response and the actual chemotherapeutic response of the study patients in the real world (13 out of 15 cases). In addition, whole-transcriptome sequencing identified candidate genes associated with nab-paclitaxel resistance, such as ITGB7, ANPEP, and ST3GAL1. CONCLUSIONS: This PDAC PDO platform allows several therapeutic drugs to be tested within a short time window and opens the possibility for timely personalized medicine as a "patient avatar model" in clinical practice.

Comparative analysis of ARID1A mutations with mRNA levels and protein expression in gastric carcinoma.

The ARID1A gene is pivotal in chromatin remodeling and genomic integrity and is frequently mutated in various cancer types. ARID1A mutation is the second most frequently mutated tumor suppressor gene and has been suggested as a predictor of immunotherapeutic responsiveness in gastric carcinoma (GC). Despite its significance, the relationship among ARID1A somatic mutations, RNA expression levels, and protein expression remains unclear, particularly in GC. For this purpose, we performed comparative study in two cohorts. Cohort 1 used next-generation sequencing (NGS) to identify 112 GC cases with ARID1A mutations. These cases were compared with ARID1A immunohistochemistry (IHC) results. Cohort 2 employed microarray gene expression data to assess ARID1A RNA levels and compare them with ARID1A IHC results. In Cohort 1, 38.4% of ARID1A-mutated GC exhibited a complete loss of ARID1A protein when assessed by IHC, whereas the remaining 61.6% displayed intact ARID1A. Discordance between NGS and IHC results was not associated with specific mutation sites, variant classifications, or variant allele frequencies. In Cohort 2, 24.1% of the patients demonstrated a loss of ARID1A protein, and there was no significant difference in mRNA levels between the ARID1A protein-intact and -loss groups. Our study revealed a substantial discrepancy between ARID1A mutations detected using NGS and protein expression assessed using IHC in GC. Moreover, ARID1A mRNA expression levels did not correlate well with protein expression. These findings highlighted the complexity of ARID1A expression in GC.

Patient-derived tumor spheroid-induced angiogenesis preclinical platform for exploring therapeutic vulnerabilities in cancer.

This study addresses the demand for research models that can support patient-treatment decisions and clarify the complexities of a tumor microenvironment by developing an advanced non-animal preclinical cancer model. Based on patient-derived tumor spheroids (PDTS), the proposed model reconstructs the tumor microenvironment with emphasis on tumor spheroid-driven angiogenesis. The resulting microfluidic chip system mirrors angiogenic responses elicited by PDTS, recapitulating patient-specific tumor conditions and providing robust, easily quantifiable outcomes. Vascularized PDTS exhibited marked angiogenesis and tumor proliferation on the microfluidic chip. Furthermore, a drug that targets the vascular endothelial growth factor receptor 2 (VEGFR2, ramucirumab) was deployed, which effectively inhibited angiogenesis and impeded tumor invasion. This innovative preclinical model was used for investigating distinct responses for various drug combinations, encompassing HER2 inhibitors and angiogenesis inhibitors, within the context of PDTS. This integrated platform could potentially advance precision medicine by harmonizing diverse data points within the tumor microenvironment with a focus on the interplay between cancer and the vascular system.

Early Immune Remodeling Steers Clinical Response to First-Line Chemoimmunotherapy in Advanced Gastric Cancer.

Adding anti-programmed cell death protein 1 (anti-PD-1) to 5-fluorouracil (5-FU)/platinum improves survival in some advanced gastroesophageal adenocarcinomas (GEA). To understand the effects of chemotherapy and immunotherapy, we conducted a phase II first-line trial (n = 47) sequentially adding pembrolizumab to 5-FU/platinum in advanced GEA. Using serial biopsy of the primary tumor at baseline, after one cycle of 5-FU/platinum, and after the addition of pembrolizumab, we transcriptionally profiled 358,067 single cells to identify evolving multicellular tumor microenvironment (TME) networks. Chemotherapy induced early on-treatment multicellular hubs with tumor-reactive T-cell and M1-like macrophage interactions in slow progressors. Faster progression featured increased MUC5A and MSLN containing treatment resistance programs in tumor cells and M2-like macrophages with immunosuppressive stromal interactions. After pembrolizumab, we observed increased CD8 T-cell infiltration and development of an immunity hub involving tumor-reactive CXCL13 T-cell program and epithelial interferon-stimulated gene programs. Strategies to drive increases in antitumor immune hub formation could expand the portion of patients benefiting from anti-PD-1 approaches. SIGNIFICANCE: The benefit of 5-FU/platinum with anti-PD-1 in first-line advanced gastric cancer is limited to patient subgroups. Using a trial with sequential anti-PD-1, we show coordinated induction of multicellular TME hubs informs the ability of anti-PD-1 to potentiate T cell-driven responses. Differential TME hub development highlights features that underlie clinical outcomes. This article is featured in Selected Articles from This Issue, p. 695.

Clinical Implication of Concurrent Amplification of MET and FGFR2 in Metastatic Gastric Cancer.

BACKGROUND: c-mesenchymal epithelial transition factor receptor (c-MET) and fibroblast growth factor receptor 2 (FGFR2) amplification have been identified as factors associated with advanced stage and poor prognosis in gastric cancer (GC). While they are typically considered mutually exclusive, concurrent amplifications have been reported in a small subset of GC patients. METHODS: in this retrospective study, we analyzed the clinical outcomes of GC patients with MET and FGFR2 amplification using the next-generation sequencing (NGS) database cohort at Samsung Medical Center, which included a total of 2119 patients between October 2019 and April 2021. RESULTS: Of 2119 cancer patients surveyed, the number of GC patients was 614 (29.0%). Out of 614 GC patients, 39 (6.4%) had FGFR2 amplification alone, 22 (3.6%) had MET amplification, and 2 GC patients (0.3%) had concurrent FGFR2 and MET amplification. Two patients with concurrent FGFR2 and MET amplification did not respond to first-line chemotherapy. These two patients had significantly shorter overall survival (3.6 months) compared to patients with FGFR2 or MET amplification alone (13.6 months and 8.4 months, respectively) (p = 0.004). Lastly, we tested the existence of FGFR2 and MET in tumor specimens from different organ sites. Initially, the NGS was tested in a primary tumor specimen from stomach cancer, where the MET copy number was 14.1 and the FGFR2 copy number was 5.3. We confirmed that both MET and FGFR2 were highly amplified in the primary tumor using FISH (MET-CEP7 ratio = 5 and FGFR2-CEP7 ratio = 3). However, although the MET copy number was normal in peritoneal seeding using FISH, FGFR2 remained amplified using FISH (FGFR2-CEP7 ratio = 7) with high FGFR2 protein overexpression. Hence, there was intra-patient molecular heterogeneity. CONCLUSIONS: our findings suggest that concurrent amplification of FGFR2 and MET in GC patients is associated with clinical aggressiveness and may contribute to non-responsiveness to chemotherapy or targeted therapy.

Prevalence of RAF1 Aberrations in Metastatic Cancer Patients: Real-World Data.

PURPOSE: Therapeutic targeting of RAF1 is a promising cancer treatment, but the relationship between clinical features and RAF1 aberrations in terms of the MAPK signaling pathway is poorly understood in various solid tumors. METHODS: Between October 2019 and June 2023 at Samsung Medical Center, 3895 patients with metastatic solid cancers underwent next-generation sequencing (NGS) using TruSight Oncology 500 (TSO500) assays as routine clinical practice. We surveyed the incidence of RAF1 aberrations including mutations (single-nucleotide variants [SNVs]), amplifications (copy number variation), and fusions. RESULTS: Among the 3895 metastatic cancer patients, 77 (2.0%) exhibited RAF1 aberrations. Of these 77 patients, 44 (1.1%) had RAF1 mutations (SNV), 25 (0.6%) had RAF1 amplifications, and 10 (0.3%) had RAF1 fusions. Among the 10 patients with RAF1 fusions, concurrent RAF1 amplifications and RAF1 mutations were detected in one patient each. The most common tumor types were bladder cancer (11.5%), followed by ampulla of Vater (AoV) cancer (5.3%), melanoma (3.0%), gallbladder (GB) cancer (2.6%), and gastric (2.3%) cancer. Microsatellite instability high (MSI-H) tumors were observed in five of 76 patients (6.6%) with RAF1 aberrations, while MSI-H tumors were found in only 2.1% of patients with wild-type RAF1 cancers (p < 0.0001). CONCLUSION: We demonstrated that approximately 2.0% of patients with metastatic solid cancers have RAF1 aberrations according to NGS of tumor specimens.

Comprehensive molecular analysis to predict the efficacy of chemotherapy containing bevacizumab in patients with metastatic colorectal cancer.

Background: Although bevacizumab is an important treatment for metastatic colorectal cancer (CRC), not all patients with CRC benefit from it; in unselected patient populations, only modest survival benefits have been reported. Methods: We evaluated clinical outcomes in 110 patients using comprehensive molecular characterization to identify biomarkers for a response to bevacizumab-containing treatment. The molecular analysis comprised whole-exome sequencing, ribonucleic acid sequencing, and a methylation array on patient tissues. Results: Genomic and molecular characterization was successfully conducted in 103 patients. Six of 103 CRC samples were hypermutated, and none of the non-hypermutant tumors were microsatellite unstable. Among those 103 patients, 89 had adenocarcinoma (ADC), 15 were diagnosed with mucinous ADC, and six had signet-ring cell carcinoma (SRCC). Consensus molecular subtype (CMS) 2 was unique to ADC. Of the four SRCCs, two were CMS1, one was CMS4, and the other was CMS3. APC mutation status was a significantly enriched factor in responders to bevacizumab treatment. Fibroblast growth factor receptor (FGFR) 1/2 signaling was upregulated in non-responders, whereas cell cycle, transfer ribonucleic acid processing, nucleotide excision repair, and oxidative phosphorylation pathways were enriched in responders. In addition, IGF1 was differentially expressed in non-responders (log2 fold change = -1.43, p = 4.11 × 10-5, false discovery rate = 0.098), and FLT1 was highly methylated in non-responders (p = 7.55 × 10-3). When the molecular pathways were reanalyzed separately according to the backbone chemotherapy (FOLFOX vs. FOLFIRI), the significance of the molecular pathways varied according to the backbone chemotherapy. Conclusions: This study sought a subset of CRC patients with a distinct clinical response to chemotherapy containing bevacizumab. Our results need to be validated in a large group of homogenous patient cohort and examined according to the different chemotherapy backbones to create personalized therapeutic opportunities in CRC.

Impact of programmed death-ligand 1 (PD-L1) positivity on clinical and molecular features of patients with metastatic gastric cancer.

BACKGROUND: Programmed death-ligand 1 (PD-L1) is an important screening biomarker to select patients with gastric cancer (GC) for optimized treatment, including immune checkpoint inhibitors (ICI). METHODS: In this single-institution retrospective cohort study, patients with metastatic GC with available PD-L1 results between October 2019 and September 2021 were identified by reviewing their electronic medical records. Genomic data were obtained from the Samsung Medical Center Clinical Sequencing Platform. RESULTS: Among the 399 patients, 276 (69%) had a PD-L1 combined positive score (CPS) ≥1, 155 (39%) had a CPS between 1 and 5, and 121 (30%) had a CPS ≥5. Of the 121 patients with CPS ≥5, 28 (23%) had a known etiology for "inflamed tumor," with Epstein-Barr virus (EBV) positivity (N = 11) or high tumor mutational burden (TMB) (N = 17), which included microsatellite instability (MSI) (N = 9). PD-L1 CPS ≥5 was observed in 11/11 (100%) patients with EBV positivity, 9/12 (75%) patients with MSI, and 17/33 (52%) patients with high TMB. For the 108 patients who received ICI therapy, CPS ≥5 was the only predictor significantly associated with survival in multivariable analyses, including TMB, MSI, or EBV. Objective response rate (ORR) was 49% in patients with CPS ≥5, 30% in patients with 1 ≤ CPS <5, and 19% in patients with CPS <1. Among the 31 responders to ICI therapy, 27 (87%) had a CPS of ≥1. Mutations in TET2, IRS2, DOT1L, PTPRT, and LRP1B were associated with a higher ORR (63%-100%), whereas MDC1 mutations were associated with a low ORR (22%). CONCLUSIONS: PD-L1 expression is an independent and sensitive biomarker for ICI therapy. Considering its significant association with several gene alterations, including PIK3CA mutations and MET amplification, combining ICI therapy with other targeted agents may be a promising therapeutic strategy for GC.

PIK3CA mutation subtype delineates distinct immune profiles in gastric carcinoma.

PIK3CA mutations in cancer regulate tumour immunogenicity. Given that PIK3CA mutation subtypes influence therapeutic responses to AKT inhibitor and that H1047R mutation confers selective growth advantages after immunotherapy, we hypothesised that immune phenotypes may depend on PIK3CA mutation subtypes. We investigated 133 gastric cancers (GCs) harbouring PIK3CA mutation [21 E542K (15.8%), 36 E545X (27.1%), 26 H1047X (19.5%), and 46 others (34.6%)]. Four patients (3.0%) had a combination of mutations (E542K + E545K in 3 patients and E545K + H1047R in 1 patient). Epstein-Barr virus (EBV) and microsatellite instability (MSI) status, PD-L1 (programmed death-ligand 1) combined positive score (CPS), and stromal tumour-infiltrating lymphocytes (TILs) were assessed. Concurrent genomic alterations, GeoMx digital spatial profiling (DSP), and OPAL multiplex immunohistochemistry (mIHC) were analysed, and correlation between the two assays was investigated. Of the 133 PIK3CA-mutant (PIK3CAm ) GCs, MSI-high GC was significantly frequent in the H1047X mutation subtype (p = 0.005), while EBV positivity did not affect the mutation subtypes. There was no significant survival difference between the E542K, E545X, and H1047X subgroups. However, in the subgroup analysis for EBV-positive GC, H1047Xm GC showed a trend towards shorter survival than E542K and E545Xm GC (p = 0.090 and 0.062). With DSP analysis, H1047Xm GC showed elevated VISTA (p = 0.0003), granzyme B (p < 0.0001), CD4 (p = 0.0001), and CD45 (p < 0.0001) expression compared with the E542Km or E545Xm GC subgroups, and only VISTA expression remained significant (p < 0.0001) using OPAL mIHC. DSP and OPAL analyses showed a moderate correlation of CD4 (ρ = 0.42, p = 0.004) and CD8 (ρ = 0.62, p < 0.001) expression levels in a comparison of six antibodies. Immune-related protein expression levels were evident when classified by the three PIK3CA hotspot mutations, and H1047Xm GC showed the highest immune-related protein expression compared with E542Km or E545Xm GC. Our results demonstrated distinct immune profiles in GC with PIK3CA hotspot mutations using GeoMx DSP and OPAL mIHC, and there was a correlation between the two multiplex platforms. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Lymphocyte-activating gene 3 expression in tumor cells predicts immune checkpoint inhibitor response in triple negative breast cancer.

Introduction: Immune checkpoint inhibitor (ICI) is one of the standard treatment strategies in triple negative breast cancer (TNBC). However, the benefit of ICI with chemotherapy is limited in metastatic TNBC. In this study, we evaluated the effect of PD-L1 and LAG-3 expression on tissue microenvironment of mTNBC treated with ICI. Methods: We reviewed representative formalin-fixed paraffin embedded specimens from metastatic or archival tumor tissues of TNBCs who treated with PD-1/PD-L1 inhibitors in metastatic setting. We used the Opal multiplex Detection kit with six antibodies (anti-PD-L1, anti-LAG-3, anti-CD68, anti-panCK, anti-CD8, anti-CD107a/LAMP antibody). Results: We evaluated the association between LAG-3+cells and survival outcome regarding CK expression. Stromal LAG-3+/CK+ and LAG-3+/CK- cells were not associated with ICI-progression free survival(PFS) (P=0.16). However, LAG-3+ cell distributions in the tumor area impacted on ICI-PFS. A high density of LAG-3+CK+ cells was associated with shorter ICI-PFS compared with low densities of both LAG-3+CK+ and LAG-3+CK- cells (1.9 vs. 3.5 months). In addition, a high density of LAG-3+CK- cells had a relatively longer ICI-PFS compared with other groups (P=0.01). In terms of total area, the pattern of densities of LAG-3+CK+ cells and LAG-3+CK- cells were similar to those in the tumor area In addition, ICI-PFS of LAG-3+CK- and LAG-3+CK+ cell densities in the total area was equal to that in the tumor area. Discussion: In conclusion, our findings revealed tumor-intrinsic LAG-3 expression was the resistance mechanism toward PD-1/PD-L1 inhibitors in mTNBCs. Multivariate analysis also suggested that LAG-3 expression in tumor cells was an independent predictive biomarker.

TP53 mutations predict poor response to immunotherapy in patients with metastatic solid tumors.

BACKGROUND: TP53 is the most commonly mutated gene across all cancer types. R175H mutation was considered structural mutation where the mutation causes misfolding of the protein and leads to a significant conformational alterations within p53's DNA binding domain. The aim of this study was to explain the reason why R175H worse the response to immunotherapy by analyzing tumor immune microenvironment through the expression of immune cells and PD-1. MATERIALS AND METHODS: Patients diagnosed with metastatic carcinoma, including colorectal cancer (CRC), breast cancer (BRCA), gastric cancer (GC), non-small cell lung cancer (NSCLC), and 20 other cancer types, treated in a palliative setting at Samsung Medical Center between October 2019 and April 2021, were enrolled. Of these patients, those who underwent TDS analysis (TruSight™ Oncology 500 assay [TSO 500]) were finally analyzed. RESULTS: Of 1770 patients, 1012 (57.2%) harbored genetic alterations in TP53. All mutations were single nucleotide variants (SNVs), and the most frequent SNV was R175H (n = 84, 7.5%) which was known as one of the most common hotspot TP53 mutation. The overall survival of patients with TP53 R175H mutations was significantly worse following chemotherapy (606 vs. 456 days, p < 0.001) or immunotherapy (822 vs. 350 days, p < 0.001) compared to those with TP53 mutation in other loci. RNA sequencing indicated that the immune response-related pathways were downregulated in tumors harboring TP53 R175H mutation. Moreover, the expression of CD8(+) T cells PD-1 were lowered in R175H mutation tumors. In the analysis of TP53 structural domain, compared to those having TP53 mutation in other domain, patients with mutations occurring in the nuclear exporter signal (NES) and E4F1-binding domains had significantly worse overall survival following chemotherapy (NES: 606 vs. 451 days, p = 0.043; E4F1: 606 vs. 469 days, p = 0.046) and immunotherapy (NES: 822 vs. 403 days, p < 0.001; E4F1: 822 vs. 413 days, p < 0.001). In addition, tumors with TP53 mutation and co-existing copy number amplification of CCND1, FGF4, and FGF19 in chromosome 11 conferred worse prognosis than those with only TP53 mutation (p < 0.050). DISCUSSION: Each TP53 mutations indicated differential treatment outcomes following chemotherapy or immunotherapy in patients with metastatic cancer. Functional analysis including RNASeq suggested that TP53 mutation downregulated immune response. CONCLUSION: Overall, we found each TP53 mutation to indicate different prognoses in patients with metastatic tumors undergoing chemotherapy and ICI treatment. Further validations, including a prospective cohort study or a functional study, would be particularly valuable in advancing the knowledge on this aspect and developing improved prognostic parameters.

Clinicopathologic significance of the delta-like ligand 4, vascular endothelial growth factor, and hypoxia-inducible factor-2α in gallbladder cancer.

BACKGROUND: Gallbladder cancer (GBC) is usually detected in advanced stages with a low 5-year survival rate. Delta-like ligand 4 (DLL4), vascular endothelial growth factor (VEGF), and hypoxia-inducible factor-2alpha (HIF2α) have been studied for their role in tumorigenesis and potential for therapeutic target, and multiple clinical trials of the agents targeting them are ongoing. We investigated the expression of these markers in surgically resected GBC and tried to reveal their association with the clinicopathologic features, mutual correlation of their expression, and prognosis of the GBC patients by their expression. METHODS: We constructed the tissue microarray blocks of 99 surgically resected GBC specimens and performed immunohistochemistry of DLL4, VEGF, and HIF2α. We used the quantitative digital image analysis to evaluate DLL4 and VEGF expression, while the expression of HIF2α was scored manually. RESULTS: The expression of VEGF and HIF2α showed a significant trend with tumor differentiation (p= .028 and p= .006, respectively). We found that the high DLL4 and VEGF expression were significantly correlated with lymph node metastasis (p= .047, both). The expression of VEGF and HIF2α were significantly correlated (p < .001). The GBC patients with low HIF2α expression showed shorter recurrence-free survival than those with high HIF2α expression. CONCLUSIONS: This study suggested the possibility of the usage of DLL4 and VEGF to predict the lymph node metastasis and the possibility of VEGF and HIF2α to predict the expression level mutually. Further studies may be needed to validate our study results and eventually accelerate the introduction of the targeted therapy in GBC.

A Standardized Pathology Report for Gastric Cancer: 2nd Edition.

The first edition of 'A Standardized Pathology Report for Gastric Cancer' was initiated by the Gastrointestinal Pathology Study Group of the Korean Society of Pathologists and published 17 years ago. Since then, significant advances have been made in the pathologic diagnosis, molecular genetics, and management of gastric cancer (GC). To reflect those changes, a committee for publishing a second edition of the report was formed within the Gastrointestinal Pathology Study Group of the Korean Society of Pathologists. This second edition consists of two parts: standard data elements and conditional data elements. The standard data elements contain the basic pathologic findings and items necessary to predict the prognosis of GC patients, and they are adequate for routine surgical pathology service. Other diagnostic and prognostic factors relevant to adjuvant therapy, including molecular biomarkers, are classified as conditional data elements to allow each pathologist to selectively choose items appropriate to the environment in their institution. We trust that the standardized pathology report will be helpful for GC diagnosis and facilitate large-scale multidisciplinary collaborative studies.

Comprehensive immunoprofile analysis of prognostic markers in pancreaticobiliary tract cancers.

Pancreaticobiliary tract cancer has a poor prognosis with unmet needs in a new target treatment. Some studies have reported that an enhancement of T-cell immunity is associated with a good prognosis. The aim of this study is to investigate the immunoprofile as a prognostic marker of pancreaticobiliary tract cancers. Unresectable pancreatic ductal adenocarcinoma (PDAC, n = 80) and biliary tract cancer (BTC, n = 74) diagnosed between January 2012 and December 2018 in Samsung Medical Center were analyzed. Expression levels of CD8, FOXP3, PD-1, PD-L1, and CXCL13 in PDAC and BTC tissue samples were examined with immunohistochemical staining, which was evaluated with various clinical factors. In PDAC, higher degree of PD-L1 expression was significantly associated with shorter overall survival (OS) (p = 0.0095). On the other hand, higher infiltrations of PD-1+ immune cells (p = 0.0002) and CD8+ T cells (p = 0.0067) were associated with longer OS. In BTC, higher FOXP3+ (p = 0.0343) and CD8+ (p = 0.0028) cell infiltrations were associated with better survival. Low infiltration of CD8+ (p = 0.0148), FOXP3+ (p = 0.0208), PD-1+ (p = 0.0318) cells in PDAC, and FOXP3+ cells (p = 0.005) in BTC were considerably related to metastasis. In a combined evaluation of clinical factors and immunoprofiles, univariate analysis revealed that operation after chemotherapy (p < 0.0001), mass size (p = 0.0004), metastasis (p = 0.006), PD-L1 (p < 0.0001), PD-1 (p = 0.003) and CD8 (p = 0.0063) was significantly associated with OS in PDAC, and CD8 (p = 0.007) was statistically related to OS in BTC. In multivariate analysis, prognostic factors were operation after chemotherapy (p = 0.021) in PDAC and CD8 (p = 0.037) in BTC. Therefore, immunoprofile analysis of cells expressing CD8, FOXP3, PD-1, and PD-L1 might have prognostic values in patients with pancreaticobiliary tract cancers.

A standardized pathology report for gastric cancer: 2nd edition.

The first edition of 'A Standardized Pathology Report for Gastric Cancer' was initiated by the Gastrointestinal Pathology Study Group of the Korean Society of Pathologists and published 17 years ago. Since then, significant advances have been made in the pathologic diagnosis, molecular genetics, and management of gastric cancer (GC). To reflect those changes, a committee for publishing a second edition of the report was formed within the Gastrointestinal Pathology Study Group of the Korean Society of Pathologists. This second edition consists of two parts: standard data elements and conditional data elements. The standard data elements contain the basic pathologic findings and items necessary to predict the prognosis of GC patients, and they are adequate for routine surgical pathology service. Other diagnostic and prognostic factors relevant to adjuvant therapy, including molecular biomarkers, are classified as conditional data elements to allow each pathologist to selectively choose items appropriate to the environment in their institution. We trust that the standardized pathology report will be helpful for GC diagnosis and facilitate large-scale multidisciplinary collaborative studies.