Genetic and Immunological Characterization of Brain Metastases from Solid Cancers.

BACKGROUND/AIM: Brain metastasis, a leading cause of cancer death, is a clinical challenge. Recently, genetic characterization of brain metastatic lesions based on next generation sequencing-based advanced technologies, such as single-cell RNA sequencing, has been performed to develop novel efficient therapies. The present study aimed to investigate brain-metastasis-specific biomarkers as well as relevant prognostic factors. PATIENTS AND METHODS: The genetic profiles and expression levels of immune response-associated genes and 820 cancer-associated genes were compared between primary cancer lesions and metastatic cancer lesions obtained from nine cancer patients at the Shizuoka Cancer Center. Cytokine and chemokine marker genes were analyzed via quantitative PCR. T-cell receptor (TCR) repertoire profiling was performed for the same patients. For survival analysis, survival data of 52 cancer patients with brain metastases were utilized. RESULTS: Comparison of driver mutation profiling between primary and metastatic lesions revealed shared core mutations in both lesions and a few new mutations in metastatic lesions. A high tumor mutation burden (TMB) was detected in metastatic lesions. Volcano plot analysis revealed specific features of the metastatic tumor microenvironment, such as cancer signaling promotion and immune suppression due to decreased immune cell infiltration. Survival analysis revealed that three genes, the TREML2 gene, the BTLA gene on activated microglia and the CERS2 gene on metastatic tumor, were potent prognostic factors. CONCLUSION: High TMB in metastatic lesions indicates potential benefit from immune checkpoint inhibitor usage for brain metastasis and TREML2 and BTLA are factors associated with poor prognosis. Activated microglia may be novel targets for the treatment of brain metastasis.

Concordance of ALK fusion gene-rearrangement between immunohistochemistry and next-generation sequencing.

BACKGROUND: Although various companion diagnostic tests of ALK fusion gene-rearrangement are approved, few reports have assessed the concordance of ALK fusion gene-rearrangement in two companion diagnostic tests: next-generation sequencing (NGS) testing and immunohistochemistry (IHC). METHODS: The samples evaluated for gene alterations using NGS testing between May 2019 and November 2021 were included in this study. The inclusion criteria were as follows: samples were diagnosed with non-small cell lung cancer; the results of the NGS analysis were informative; and samples had residual specimens for IHC. We performed IHC on the residual specimens and retrospectively collected sample characteristics from medical records. RESULTS: A total of 185 samples were analyzed using NGS. Twenty-six samples were excluded because of failure to analyze gene alterations using NGS, no residual samples, and inadequate IHC. We analyzed 159 samples. The major histological type was adenocarcinoma (115 samples). The number of surgical and transbronchial lung biopsy specimens was 59 and 56, respectively. ALK fusion gene-rearrangement was detected in four samples using NGS, and five were detected using IHC. The sensitivity and specificity of IHC referred to by NGS were 75.0% and 98.7%, respectively. The concordance rate between IHC and NGS was 98.1%. ALK rearrangement was detected in two patients using IHC but not using NGS. In addition, ALK rearrangement was detected in one patient using NGS but not using IHC. CONCLUSION: Our results suggest that IHC and NGS might be complementary tests. In patients suspected of harboring ALK fusion gene-rearrangement, it should be analyzed using another diagnostic method.

Impact of Mutations in Subunit Genes of the Mammalian SWI/SNF Complex on Immunological Tumor Microenvironment.

BACKGROUND/AIM: Recently, inactivating somatic mutations of SWI/SNF chromatin-remodeling genes in cancers have been reported. However, few studies have been performed regarding the immunological analysis of the tumor microenvironment (TME) in chromatin remodeling complex gene-mutated tumors. In the present study, we identified cancer patients harboring various mammalian SWI/SNF complex mutations and investigated the immunological features in those mutated cancers. PATIENTS AND METHODS: Cancer patients harboring any type of chromatin remodeling complex gene mutation were selected and clinicopathological features were compared between chromatin remodeling complex gene expression-low and expression-high groups. Specifically, expression levels of immune response-associated genes and cancer-associated genes were compared between the SMARCA4 expression-low and expression-high groups using volcano plot analysis. RESULTS: Among cancers harboring PBRM1, SAMRACA4 and ARID2 gene mutations, T-cell marker and mature B-cell marker genes were up-regulated in the tumor. Specifically, T-cell effector genes (CD8B, CD40LG), central memory marker genes (CD27, CCR7) and mature B-cell marker genes (CD20, CD38, CD79 and IRF4) were up-regulated, and cancer-associated genes including MYB, MYC and AURKB genes were down-regulated in the SMARCA4 expression-low group. Remarkably, heatmap of gene expression and immunohistochemistry (IHC) data demonstrated that the tertiary lymphoid structure (TLS) gene signature of mature B cells was up-regulated in SMACA4 gene-mutated stomach cancers. CONCLUSION: These results suggest that immune tumor microenvironment status, such as mature B cell recruitment featuring the TLS gene signature and immune activation mediated by cancer signal down-regulation, might contribute to the classification of SMARCA4 gene-mutated tumors as immune checkpoint blockade therapy-sensitive target tumors.

Prognostic significance of tumor microenvironment assessed by machine learning algorithm in surgically resected non-small cell lung cancer.

BACKGROUND: A methodology to assess the immune microenvironment (IME) of non-small cell lung cancer (NSCLC) has not been established, and the prognostic impact of IME factors is not yet clear. AIMS: This study aimed to assess the IME factors and evaluate their prognostic values. METHODS AND RESULTS: We assessed CD8+ tumor-infiltrating lymphocyte (TIL) density, forkhead box protein P3+ (Foxp3+ ) TIL density, and programmed death receptor ligand-1 (PD-L1) tumor proportion score (TPS) using a machine-learning algorithm in whole-slide imaging (WSI). We dichotomized patients according to TIL density or TPS and compared their clinical outcomes. Between September 2014 and September 2015, 165 patients with NSCLC were enrolled in the study. We assessed IME factors in the epithelium, stroma, and their combination. An improvement in disease-free survival (DFS) was observed in the high CD8+ TIL density group in the epithelium, stroma, and the combination of both. Moreover, the group with high PD-L1 TPS in the epithelium showed better DFS than that with low PD-L1 TPS. In the multivariate analysis, the CD8+ TIL density in the combination of epithelium and stroma and PD-L1 TPS in the epithelium were independent prognostic factors (hazard ratio [HR] = 0.43; 95% confidence interval [CI] = 0.26-0.72; p = .001, HR = 0.49; 95% CI = 0.30-0.81; p = .005, respectively). CONCLUSION: Our approach demonstrated that the IME factors are related to survival in patients with NSCLC. The quantitative assessment of IME factors enables to discriminate patients with high risk of recurrence, who can be the candidates for adjuvant therapy. Assessing the CD8+ TIL density in the combination of epithelium and stroma might be more useful than their individual assessment because it is a simple and time-saving analysis of TILs in WSI.

Immunohistochemical analysis of B7-H3 expression in patients with lung cancer following various anti-cancer treatments.

B7 homolog 3 protein (B7-H3), an immune checkpoint molecule belonging to the B7 family, has been studied as a target for the development of anti-cancer treatment; however, changes in B7-H3 expression during the clinical course remain unknown. This retrospective study aimed to investigate changes in B7-H3 expression of lung cancer specimens in patients with advanced lung cancer following various anti-cancer treatments. The immunohistochemistry (IHC) score was evaluated on a 0-3 scale, and B7-H3 expression was considered positive for grade ≥ 2. The difference in IHC scores before and after anti-cancer treatment was defined as the change in B7-H3 expression. Among 160 patients with lung cancer who received anti-cancer treatment, 88 (55%) and 101 (63%) had B7-H3 expression before and after anti-cancer treatment, respectively. Before treatment, B7-H3 expression was significantly more common in squamous cell carcinoma specimens than in adenocarcinoma specimens (95% vs. 49%, P < 0.001). Of the 19 patients with squamous cell carcinoma, 18 (95%) continued to have high (IHC score: 3) B7-H3 expression following treatment. In contrast, of the 130 patients with adenocarcinoma, 46 (35%) and 17 (13%) showed an increased and a decreased expression, respectively. Patients who received targeted therapy had a significant increase in B7-H3 expression compared with those who received chemotherapy alone (P = 0.015). Overall, squamous cell carcinoma specimens maintained high B7-H3 expression during the clinical course, whereas adenocarcinoma specimens showed changes in expression following anti-cancer treatments. Our results provide the basis for further studies on the development of anti-cancer treatments targeting B7-H3.

The efficacy of a machine learning algorithm for assessing tumour components as a prognostic marker of surgically resected stage IA lung adenocarcinoma.

BACKGROUND: The importance of the stromal components in tumour progression has been discussed widely, but their prognostic role in small size tumours with lepidic components is not fully understood. Applying digital tissue image analysis to whole-slide imaging may enhance the accuracy and reproducibility of pathological assessment. This study aimed to evaluate the prognostic value of tumour components of lung adenocarcinoma by measuring the dimensions of the tumour consisting elements separately, using a machine learning algorithm. METHODS: Between September 2002 and December 2016, 317 patients with surgically resected, pathological stage IA adenocarcinoma with lepidic components were analysed. We assessed the whole tumour area, including the lepidic components, and measured the epithelium, collagen, elastin areas and alveolar air space. We analysed the prognostic impact of each tumour component. RESULTS: The dimensions of the epithelium and collagen areas were independent significant risk factors for recurrence-free survival (hazard ratio, 8.38; 95% confidence interval, 1.14-61.88; P = 0.037, and hazard ratio, 2.58; 95% confidence interval, 1.14-5.83; P = 0.022, respectively). According to the subgroup analysis when combining the epithelium and collagen areas as risk factors, patients with tumours consisting of both large epithelium and collagen areas showed significantly poor prognoses (P = 0.002). CONCLUSIONS: We assessed tumour components using a machine learning algorithm to stratify the post-operative prognosis of surgically resected stage IA adenocarcinomas. This method might guide the selection of patients with a high risk of recurrence.

Artificial Intelligence-Powered Prediction of ALK Gene Rearrangement in Patients With Non-Small-Cell Lung Cancer.

PURPOSE: Several studies reported the possibility of predicting genetic abnormalities in non-small-cell lung cancer by deep learning (DL). However, there are no data of predicting ALK gene rearrangement (ALKr) using DL. We evaluated the ALKr predictability using the DL platform. MATERIALS AND METHODS: We selected 66 ALKr-positive cases and 142 ALKr-negative cases, which were diagnosed by ALKr immunohistochemical staining in our institution from January 2009 to March 2019. We generated virtual slide of 300 slides (150 ALKr-positive slides and 150 ALKr-negative slides) using NanoZoomer. HALO-AI was used to analyze the whole-slide imaging data, and the DenseNet network was used to build the learning model. Of the 300 slides, we randomly assigned 172 slides to the training cohort and 128 slides to the test cohort to ensure no duplication of cases. In four resolutions (16.0/4.0/1.0/0.25 µm/pix), ALKr prediction models were built in the training cohort and ALKr prediction performance was evaluated in the test cohort. We evaluated the diagnostic probability of ALKr by receiver operating characteristic analysis in each ALKr probability threshold (50%, 60%, 70%, 80%, 90%, and 95%). We expected the area under the curve to be 0.64-0.85 in the model of a previous study. Furthermore, in the test cohort data, an expert pathologist also evaluated the presence of ALKr by hematoxylin and eosin staining on whole-slide imaging. RESULTS: The maximum area under the curve was 0.73 (50% threshold: 95% CI, 0.65 to 0.82) in the resolution of 1.0 µm/pix. In this resolution, with an ALKr probability of 50% threshold, the sensitivity and specificity were 73% and 73%, respectively. The expert pathologist's sensitivity and specificity in the same test cohort were 13% and 94%. CONCLUSION: The ALKr prediction by DL was feasible. Further study should be addressed to improve accuracy of ALKr prediction.

Immunological and Genetic Characterization of Patients With Head and Neck Cancer who Developed Recurrence.

BACKGROUND/AIM: The recurrence rate of head and neck squamous cell carcinoma (HNSCC) remains high; thus the control of recurrence is a clinical problem to be challenged. To clarify the precise mechanism, specific immunological biomarkers responsible for recurrence were investigated. PATIENTS AND METHODS: The expression levels of immune response-associated and Shizuoka Cancer Center 820 cancer-associated genes, and genetic mutations from whole-exome sequencing were compared between HNSCC patients who developed recurrence (n=8) and HNSCC patients who did not develop recurrence (n=19) using a volcano plot analysis. Cytokine and epithelial-mesenchymal transition marker genes were analyzed using quantitative PCR. Tumor-infiltrating lymphocytes, immune checkpoint molecules, and human papilloma virus status were investigated using immunohistochemistry (IHC). RESULTS: Twenty-seven evaluable patients with HNSCCs received radiation therapy after surgery. Recurrence was identified in 8 patients. TP53 mutations tended to be higher in patients who developed recurrence than in those who did not develop recurrence (75% vs. 31.6%). Gene expression profiling showed the down-regulation of T cell activation genes (ICOS, CD69 and CD83) and the upregulation of the ERBB4, EGFR, VEGF, HIF1A, TGFB1, TWIST1, IL-8, and PAX7 genes, which suggested the activation of the TP53 mutation-TGF-ß1-PAX7 pathway and epithelial-mesenchymal transition. Additionally, IHC indicated a tendency toward a reduction in T cell accumulation and an increase in M2-type macrophage infiltration in tumors that recurred. CONCLUSION: A TP53 mutation-mediated immune-suppressive state in the tumor microenvironment and TGF-ß1-PAX7-mediated EMT might contribute to the promotion of recurrence in patients with HNSCC after postoperative radiotherapy.

Tumor cell enrichment by tissue suspension enables detection of mutations with low variant allele frequency and estimation of germline mutations.

Targeted sequencing offers an opportunity to select specific drugs for cancer patients based on alterations in their genome. However, accurate sequencing cannot be performed in cancers harboring diffuse tumor cells because of low tumor content. We performed tumor cell enrichment using tissue suspension of formalin-fixed, paraffin-embedded (FFPE) tissue sections with low tumor cell content. The enriched fractions were used to efficiently identify mutations by sequencing a target panel of cancer-related genes. Tumor-enriched and residual fractions were isolated from FFPE tissue sections of intestinal and diffuse gastric cancers harboring diffuse tumor cells and DNA of suitable quality was isolated for next-generation sequencing. Sequencing of a target panel of cancer-related genes using the tumor-enriched fraction increased the number of detectable mutations and variant allele frequency. Furthermore, mutation analysis of DNA isolated from tumor-enriched and residual fractions allowed us to estimate germline mutations without a blood reference. This approach of tumor cell enrichment will not only enhance the success rate of target panel sequencing, but can also improve the accuracy of detection of somatic mutations in archived specimens.

Expression of N-Terminal-Deficient E-Cadherin Protein in Invasive Lobular Carcinoma of the Breast.

Invasive lobular carcinoma (ILC) of the breast is characterized by the discohesive growth of tumor cells, which is mainly associated with the complete loss of E-cadherin (E-cad) expression. However, some aberrant expression patterns of E-cad protein that are inconsistent with their morphologies have been reported in ILC. We report herein ILC cases expressing a new type of abnormal E-cad protein that lacks the N-terminal domain, but conserves the C-terminal domain on the cell membrane. Immunohistochemical staining of 299 ILC cases using specific antibodies against the N-terminal or C-terminal region of E-cad revealed that 227 (76%) cases showed loss of the membranous expression of both terminuses (N-/C-) and 72 (24%) cases showed expression of only the C-terminus (N-/C+). In all cases, the expression of p120-catenin and ß-catenin coincided with the expression of the C-terminus of E-cad. Clinicopathologic analysis revealed that N-/C+ expression in ILC cells was significantly associated with the histologic subtype (especially mixed-type ILC with another histologic type) and immunohistochemical molecular subtype (especially the triple-negative subtype), but not with prognostic factors (pT or pN). In addition, 12 of 15 cases (80%) with aberrant cytoplasmic localization of the N-terminal of E-cad showed diffuse membranous expression of the C-terminal domain. Additional immunohistochemistry using an antibody recognizing the extracellular juxtamembrane region showed that 28 (39%) of the N-/C+ cases had lost membranous expression, suggesting diversity in the deletion pattern of the N-terminal region. Our findings provide a novel mechanism for the loss of E-cad function because of N-terminal-deficient E-cad protein in ILC.

Trophoblast cell-surface antigen 2 expression in lung cancer patients and the effects of anti-cancer treatments.

BACKGROUND: Trophoblast cell-surface antigen 2 (TROP2) is expressed on the surface of trophoblast cells and many malignant tumor cells. However, data on TROP2 expression in advanced lung cancer are insufficient, and its changes have not been fully evaluated. METHODS: We assessed the prevalence and changes in TROP2 expression in patients with lung cancer who received anti-cancer treatments using immunohistochemical (IHC) analysis with an anti-TROP2 antibody (clone: SP295). IHC scores were graded from 0 to 3; grade ≥ 2 was considered positive for TROP2 expression. We defined a difference in IHC score, before and after anti-cancer treatments, as the change in TROP2 expression. RESULTS: Before anti-cancer treatment, TROP2 expression was observed in 89% (143/160) of the patients and was significantly more common in adenocarcinoma and squamous cell carcinoma than in neuroendocrine carcinoma (P < 0.001). After anti-cancer treatment, TROP2 expression was observed in 87% (139/160) of the patients. The distribution of TROP2 expression in post-treatment samples was analogous to that in pre-treatment samples when compared using the Wilcoxon signed-rank test (P = 0.509). However, an increase in TROP2 expression was seen in 19 (12%), and a decrease in 20 (13%) patients. Patients treated with targeted therapy showed significantly higher changes in TROP2 expression (P = 0.019) and thoracic radiotherapy was more likely to increase TROP2 expression than chemotherapy alone. CONCLUSION: Although some anti-cancer treatments might alter the TROP2 expression, TROP2 was expressed in most lung cancer specimens before and after anti-cancer treatments. These results support the development of TROP2-directed therapy against advanced lung cancer in various treatment lines.

Development of an Automatic Measurement Method for CD8 and PD-1 Positive T Cells Using Image Analysis Software.

BACKGROUND/AIM: With the progress in cancer immunotherapy using immune checkpoint blockade (ICB) therapy, histological observations of tumor-infiltrating lymphocyte (TIL) status are needed to evaluate the antitumor effect of ICB using imaging analysis software. MATERIALS AND METHODS: Formalin-fixed paraffin-embedded sections obtained from colorectal cancer and gastric cancer patients with more than 500 single nucleotide variants were stained with anti-CD8 and anti-PD-1 antibodies. Based on our own algorithm and imaging analysis software, an automatic TIL measurement method was established and compared to the manual counting methods. RESULTS: In the CD8+ T cell number measurement, there was a good correlation (r=0.738 by Pearson test) between the manual and automated counting methods. However, in the PD-1+ T cell measurement, there was a large difference in TIL numbers in both groups. After adjustment of the parameter settings, the correlation between the manual and automated methods in the PD-1+ T cell measurements improved (r=0.668 by Pearson test). CONCLUSION: An imaging software-based automatic measurement could be a simple and useful tool for evaluating the therapeutic effect of cancer immunotherapies in terms of TIL status.

EMID1, a multifunctional molecule identified in a murine model for the invasion independent metastasis pathway.

EMI Domain Containing 1 (EMID1) was identified as a potential candidate metastasis-promoting gene. We sought to clarify the molecular function of EMID1 and the protein expression. Overexpression and knockdown studies using mouse tumor cell lines identified two novel functions of EMID1: intracellular signaling involving enhancement of cell growth via cell cycle promotion and suppression of cell motility, and inhibition of cell-matrix adhesion by extracellularly secreted EMID1. EMID1 deposited on the culture dish induced self-detachment of cells that overexpressed the protein and inhibited adhesion of additionally seeded cells. This multifunctional property involving both intracellular signaling and the extracellular matrix suggests that EMID1 may be a matricellular proteins. Expression analysis using immunohistochemical staining revealed expression of EMID1 that was limited to chief cells of the gastric fundic gland and ß cells of the pancreatic islets in normal adult human tissues, implying cell-specific functions of this molecule. In addition, increased expression of EMID1 protein detected in some cases of human cancers implies that EMID1 might be a new therapeutic target for cancer treatment.

Comparison of Immunohistochemical Expression of Cytokeratin 19, c-KIT, BerEP4, GATA3, and NUTM1 Between Porocarcinoma and Squamous Cell Carcinoma.

ABSTRACT: Distinguishing porocarcinoma from squamous cell carcinoma (SCC) is clinically significant; however, differential diagnosis can often be challenging. This study sought to confirm the diagnostic utility of cytokeratin 19, c-KIT, BerEP4, GATA3, and NUTM1 immunohistochemistry in distinguishing porocarcinoma from SCC. Immunohistochemical analysis of cytokeratin 19, c-KIT, BerEP4, GATA3, and NUTM1 in 14 porocarcinomas and 22 SCCs was performed; the extents and intensities of expression of these markers were recorded. The statistical associations of the immunoexpression between porocarcinoma and SCC were analyzed using the Pearson χ2 test. Cytokeratin 19 was positive in 13 (92.9%) of 14 porocarcinomas, and for all the positive cases, staining was strong and evident in >20% of the tumor cells. By contrast, 9 (40.9%) of 22 SCCs expressed cytokeratin 19 (P = 0.0018), of which 6 showed extremely focal (≤10% of the tumor cells) expression. Of the 14 porocarcinomas, 11 (78.6%) cases showed c-KIT positivity, whereas only 3 of 22 SCCs (13.6%) expressed c-KIT focally (P = 0.0001). In addition, BerEP4 immunostaining differed between porocarcinomas and SCCs (57.1% vs. 9.1%, respectively; P = 0.0017). However, no significant difference between the groups was reported in terms of GATA3 expression (57.1% vs. 72.7%, respectively; P = 0.3336). NUTM1 was expressed in 4/14 (28.6%) porocarcinomas but not in the SCCs. Immunohistochemistry for cytokeratin 19, c-KIT, and BerEP4 could be helpful in distinguishing porocarcinomas from SCCs. In addition, NUTM1 immunoexpression is highly specific, although not sensitive, to porocarcinomas. GATA3 immunohistochemistry has no meaningful implications in the differential diagnosis of porocarcinoma and SCC.

Detection of programmed cell death-ligand 1 using 22C3 antibody in patients with unresectable stage III non-small cell lung cancer receiving chemoradiotherapy.

BACKGROUND: The expression of programmed cell death-ligand 1 (PD-L1) is a biomarker for administering immune check point inhibitors in patients with advanced stage non-small cell lung cancer. Although the consolidation therapy of durvalumab after definitive chemoradiotherapy has become the new standard of care for patients with unresectable stage III non-small cell lung cancer, the prevalence and prognostic role of PD-L1 expression in this population remain unclear. METHODS: We retrospectively reviewed data from patients with unresectable stage III non-small cell lung cancer who received definitive chemoradiotherapy at our institution between 2012 and 2017. Levels of PD-L1 were assessed using 22C3 antibody, and associations of progression-free and overall survival rates with PD-L1 statuses at a tumor proportion score cutoff of 1% were analyzed. RESULTS: Among the 104 patients enrolled, PD-L1 statuses were as follows: tumor proportion score < 1%, 73 (70.2%); 1-49%, 21 (20.2%); and ≥ 50%, 10 (9.6%). The number of patients with stage III non-small cell lung cancer with pretreatment PD-L1 tumor proportion score ≥ 1% was less than the number with advanced stage disease. There was no association between patient characteristics and PD-L1 status, and no significant differences were observed in progression-free and overall survival rates relative to PD-L1 status. CONCLUSION: Expression of PD-L1 in patients with stage III non-small cell cancer before chemoradiotherapy should be assessed because of the low prevalence of tumors with tumor proportion scores ≥ 1%. Further studies are needed to clarify whether durvalumab improves survival after definitive chemoradiotherapy, irrespective of tumor PD-L1 expression.

Nuclear ß-catenin immunoexpression in scars.

BACKGROUND: Histopathologically, scars can mimic superficial fibromatoses. Superficial fibromatoses are known to show nuclear ß-catenin immunoexpression, although the tumor types do not harbor CTNNB1 or APC alterations. This study aimed to evaluate nuclear ß-catenin immunoexpression in scars compared to that in superficial fibromatoses. METHODS: Immunostaining with an anti-ß-catenin antibody, clone 14, was performed on 8 superficial fibromatoses and 22 scars. The extent of ß-catenin nuclear staining was classified as negative (<10%), focally positive (10-49%), or diffusely positive (50-100%). ß-catenin staining intensity was semi-quantitatively graded as weak, moderate, or strong. RESULTS: In 21 (95%) scars, nuclear ß-catenin immunoexpression was detected in fibroblasts/myofibroblasts, with mainly diffuse (16/21) and moderate (14/21) to strong (5/21) staining. In contrast, seven (88%) of the eight superficial fibromatoses expressed ß-catenin in the nuclei of the lesional spindle cells, at varying levels of staining intensity. Fibroblasts in normal papillary dermis always showed nuclear ß-catenin expression to varying degrees but those in the reticular dermis did not. CONCLUSIONS: Scars typically exhibit nuclear ß-catenin expression similar to that in superficial fibromatoses. Thus, ß-catenin immunohistochemistry is not suitable for distinguishing superficial fibromatoses from scars.

Effect of preoperative chemoradiotherapy on the immunological status of rectal cancer patients.

The aim of the study was to investigate the effect of chemo-radiation on the genetic and immunological status of rectal cancer patients who were treated with preoperative chemoradiotherapy (CRT). The expression of immune response-associated genes was compared between rectal cancer patients treated (n = 9) and not-treated (n = 10) with preoperative CRT using volcano plot analysis. Apoptosis and epithelial-to-mesenchymal transition (EMT) marker genes were analysed by quantitative PCR (qPCR). Other markers associated with the tumor microenvironment (TME), such as tumor-infiltrating lymphocytes (TIL) and immune checkpoint molecules, were investigated using immunohistochemistry (IHC). The clinical responses of preoperative CRT for 9 rectal cancer patients were all rated as stable disease, while the pathological tumor regression score (TRG) revealed 6 cases of grade2 and 3 cases of grade1. According to the genetic signature of colon cancers, treated tumors belonged to consensus molecular subtype (CMS)4, while not-treated tumors had signatures of CMS2 or 3. CRT-treated tumors showed significant upregulation of EMT-associated genes, such as CDH2, TGF-beta and FGF, and cancer stem cell-associated genes. Additionally, qPCR and IHC demonstrated a suppressive immunological status derived from the upregulation of inflammatory cytokines (IL-6, IL-10 and TGF-beta) and immune checkpoint genes (B7-H3 and B7-H5) and from M2-type macrophage accumulation in the tumor. The induction of EMT and immune-suppressive status in the tumor after strong CRT treatment urges the development of a novel combined therapy that restores immune-suppression and inhibits EMT, ultimately leading to distant metastasis control.

Metabolic Profiling of Human Gastric Cancer Cells Treated With Salazosulfapyridine.

PURPOSE: The adhesion molecule cluster of differentiation 44v9 interacts with and stabilizes the cystine/glutamate exchanger protein, which functions as a transporter of cystine. Stabilized cystine/glutamate exchanger increases extracellular cystine uptake and enhances glutathione production. Augmented levels of reduced glutathione mitigate reactive oxygen species and protect cancer cells from apoptosis. Salazosulfapyridine blocks cystine/glutamate exchanger activity and mitigates the supply of cystine to increase intracellular ROS production, thereby increasing cell susceptibility to apoptosis. This enhances the effect of anticancer drugs such as cisplatin. Currently, salazosulfapyridine is being developed as a promising anticancer agent. In the present study, we elucidated the molecular mechanism associated with salazosulfapyridine by investigating the salazosulfapyridine-induced changes in glutathione metabolism in cultured gastric cancer cell lines. METHODS: The effect of salazosulfapyridine treatment on glutathione metabolism was investigated in 4 gastric cancer (AGS, MKN1, MKN45, and MKN74) and 2 colorectal cancer (HCT15 and HCT116) cell lines using metabolomic analyses. In addition, the effect of inhibition of the reduced form of nicotinamide adenine dinucleotide phosphate by 2-deoxyglucose on glutathione metabolism was evaluated. RESULTS: Under hypoxia, enhanced glycolysis resulted in lactate accumulation with an associated reduction in nicotinamide adenine dinucleotide phosphate. Salazosulfapyridine treatment decreased the cysteine content and inhibited the formation of glutathione. Combined treatment with salazosulfapyridine and 2-deoxyglucose significantly inhibited cell proliferation. 2-Deoxyglucose, an inhibitor of glycolysis, depleted nicotinamide adenine dinucleotide phosphate required for the formation of glutathione. CONCLUSIONS: Our results indicate that in cancer cells having a predominant glycolytic pathway, metabolomic analyses under hypoxic conditions enable the profiling of global metabolism. In addition, inhibiting the supply of nicotinamide adenine dinucleotide phosphate by blocking glycolysis is a potential treatment strategy for cancer, in addition to cystine blockade by salazosulfapyridine.

Assessment of associations between clinical and immune microenvironmental factors and tumor mutation burden in resected nonsmall cell lung cancer by applying machine learning to whole-slide images.

BACKGROUND: It is unclear whether clinical factors and immune microenvironment (IME) factors are associated with tumor mutation burden (TMB) in patients with nonsmall cell lung cancer (NSCLC). MATERIALS AND METHODS: We assessed TMB in surgical tumor specimens by performing whole exome sequencing. IME profiles, including PD-L1 tumor proportion score (TPS), stromal CD8 tumor-infiltrating lymphocyte (TIL) density, and stromal Foxp3 TIL density, were quantified by digital pathology using a machine learning algorithm. To detect factors associated with TMB, clinical data, and IME factors were assessed by means of a multiple regression model. RESULTS: We analyzed tumors from 200 of the 246 surgically resected NSCLC patients between September 2014 and September 2015. Patient background: median age (range) 70 years (39-87); male 37.5%; smoker 27.5%; pathological stage (p-stage) I/II/III, 63.5/22.5/14.0%; histological type Ad/Sq, 77.0/23.0%; primary tumor location upper/lower, 58.5/41.5%; median PET SUV 7.5 (0.86-29.8); median serum CEA (sCEA) level 3.4 ng/mL (0.5-144.3); median serum CYFRA 21-1 (sCYFRA) level 1.2 ng/mL (1.0-38.0); median TMB 2.19/ Mb (0.12-64.38); median PD-L1 TPS 15.1% (0.09-77.4); median stromal CD8 TIL density 582.1/mm2 (120.0-4967.6);, and median stromal Foxp3 TIL density 183.7/mm2 (6.3-544.0). The multiple regression analysis identified three factors associated with higher TMB: smoking status: smoker, increase PET SUV, and sCEA level: >5 ng/mL (P < .001, P < .001, and P = .006, respectively). CONCLUSIONS: The IME factors assessed were not associated with TMB, but our findings showed that, in addition to smoking, PET SUV and sCEA levels may be independent predictors of TMB. TMB and IME factors are independent factors in resected NSCLC.

Pretreatment intratumoral susceptibility signals correlate with response to high-dose methotrexate and progression-free survival in primary central nervous system lymphoma.

We aimed to estimate the frequency of intratumoral susceptibility signals (ITSS) in susceptibility-weighted imaging (SWI) in consecutive patients with primary central nervous system lymphoma (PCNSL), and to determine if pretreatment heterogeneity of PCNSL is predictive of response to chemotherapy by using ITSS on SWI. We retrospectively examined 29 immunocompetent patients with PCNSL who underwent SWI-MRI before treatment. A univariate analysis was conducted with Fisher's exact test. Progression free survival (PFS) was calculated by the Kaplan-Meier method and compared by the log rank test. The patients, including 16 males, were initially treated at a median age of 69 years. All tissue types were diffuse large B-cell lymphoma. Nineteen patients (66%) presented lesions with ITSS. Sixteen patients (55%) received initial treatment with R-MTX (rituximab plus high-dose methotrexate). Seven out of nine patients with ITSS exhibited a poor response, whereas all seven without ITSS exhibited a good response to R-MTX. Regarding the absence of ITSS, the sensitivity, specificity, and diagnostic accuracy for a good response to R-MTX were 0.78, 1.00, and 0.88, respectively. Patients without ITSS showed significantly longer PFS compared to patients with ITSS (median PFS: 28.9 vs 2.1 months, P < 0.01). In conclusion, ITSS in PCNSL patients were more common than previously reported. We have to be careful to use ITSS for differentiating PCNSL and glioblastoma. Presence of ITSS correlated significantly with therapeutic response to R-MTX. ITSS may be a new marker for the response to chemotherapy in patients with PCNSL. A prospective multi-institutional analysis is needed.