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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.

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.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).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-β1-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.A TP53 mutation-mediated immune-suppressive state in the tumor microenvironment and TGF-β1-PAX7-mediated EMT might contribute to the promotion of recurrence in patients with HNSCC after postoperative radiotherapy.

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.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.In the CD8An 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.

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.

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. 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. Among the 104 patients enrolled, PD-L1 statuses were as follows: tumor proportion score

BACKGROUND Histopathologically, scars can mimic superficial fibromatoses. Superficial fibromatoses are known to show nuclear β-catenin immunoexpression, although the tumor types do not harbor CTNNB1 or APC alterations. This study aimed to evaluate nuclear β-catenin immunoexpression in scars compared to that in superficial fibromatoses. METHODS Immunostaining with an anti-β-catenin antibody, clone 14, was performed on 8 superficial fibromatoses and 22 scars. The extent of β-catenin nuclear staining was classified as negative (

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.

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, We were able to quantitatively evaluate the local presence of IME factors that are difficult to evaluate visually, by using a digital pathology platform. The results showed no associations between IME factors and TMB, but significant associations were found with some clinical factors. It is possible that TMB based on whole exome sequencing was not correlated with neoantigen load.

Background: In IMpassion 130 trial, programmed death-ligand 1 (PD-L1) expression was seen in about 40% of participated patients with triple negative advanced breast cancer (TN-ABC) [Schmid, 2018, N Engl J Med], however, actual situation of PD-L1 expression in patients HER2-negative ABC patients including with TN-ABC has not been well studied. Furthermore, no biomarker related to chemotherapy for HER2-negative ABC patients except for PD-L1 in TN-ABC has been identified. Recently, several reports regarding the relationship between peripheral immune-related markers; such as absolute lymphocyte count (ALC) or neutrophil-to-lymphocyte ratio (NLR), and efficacy of eribulin therapy [Miyoshi, 2020, Breast Cancer; Watanabe, 2020, Breast Cancer Res Treat] or paclitaxel plus bevacizumab (PB) therapy [Nakamoto, 2021, Sci Rep], however, the relationship between the efficacy of PB therapy and peripheral immune related markers including dynamic change during the therapy or local immune-related markers is unclear. Therefore, we conducted multi-institutional, retrospective study 1) to evaluate the actual situation of PD-L1 expression and other immune-related markers of the primary site by central review, and 2) to explore biomarkers for first-line PB therapy using peripheral and local immune-related markers. Patients and methods: We retrospectively reviewed medical records of HER2-negative ABC patients who received PB therapy as first-line (1L) or second-line chemotherapy for ABC. Clinical data including ALC, NLR and serum albumin (Alb) were extracted from medical records, and the pathology of archived tissues of primary and metastatic site (if available) were centrally reviewed including PD-L1 (VentanaR SP142). Statistical analyses were performed using Kaplan-Meier method, log-rank test, Wilcoxon’s test, and Cox hazard model. Mixed-effects model for repeated measures (MMRM) to evaluate the relationships between dynamic change in immune-related markers and time-to treatment termination (TTT) of PB therapy. Results: We identified 156 HER2-negaive ABC patients who underwent PB therapy, and 114 out of 156 patients were eligible for analyses. Of 114 patients, 63 patients (55.3%) had recurrent disease, and 65 (57.0%) patients had visceral disease. Eighty-seven out of 114 (76.3%) patients received PB therapy as 1L chemotherapy. Eighty-four specimens (73.7%) were diagnosed as estrogen-receptor (ER) positive, and PD-L1 positivity rate were 3.6% (1/84) in ER+ subgroup and 30.0% (6/20) in ER- subgroup, respectively. Paired biopsy specimens were eligible in 14 patients, and significant elevation of Ki67 labeling index was noted. In patients who received 1L PB (n = 87), there was no positive relationship between maintained ALC (>1,500 or >1,000/μL at baseline) or low NLR (< 2.5 or < 3) at the initiation of 1L PB therapy and TTTs. However, low NLR (cut-off at 2.5 and 3) at the initiation of second cycle of PB therapy reduced the risk for treatment-termination as; hazard ratio [HR], 0.427; 95% CI 0.218-0.843; P = 0.0147 and HR, 0.344; 95% CI 0.170-0.731; P = 0.0066, respectively. PD-L1 positive patients (n = 5) showed numerically increased risk of treatment-termination (HR, 2.68; 95% CI, 0.922-6.196; P = 0.0674) for 1L PB therapy, however, there was no significant difference in mortality risk regarding PD-L1 statuses. Multivariate analysis using MMRM disclosed that increase of Alb level was predictive factor for 1L PB therapy (HR, 0.41; 95%CI, 0.18-0.93; P = 0.0338). Conclusion: According to our real-world study, 1) PD-L1 positive rate was lower than that of previous reports, 2) low NLR at the initiation of second cycle of PB therapy and dynamic change in albumin level were identified as predictive factors and 3) PD-L1 overexpression was not a prognostic or a predictive factor for PB therapy in patients with HER2-negative ABC. Funding: Chugai Pharmaceutical CO., LTD. Citation Format: Junichiro Watanabe, Takashi Sugino, Koji Muramatsu, Satoshi Morita, Yu Hidaka, Shogo Nakamoto, Mitsuya Ito, Shoichiro Ohtani, Masahiko Ikeda. Identifying immune-related predictive factors for paclitaxel + bevacizumab therapy in patients with HER2-negative advanced breast cancer- A multicenter retrospective study [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-02-48.

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 β-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.

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.

Our previous analysis using a mouse metastasis model identified EMI Domain Containing 1 (EMID1) as a potential candidate metastasis-promoting gene. Although EMID1 was isolated as a member of the Emu gene family, which is expressed during mouse embryogenesis, little is known about the molecule. We sought to clarify the molecular function of EMID1 and the protein expression. Overexpression and knockdown studies using mouse 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 β 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.

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.

Pro-gastrin-releasing peptide (ProGRP) is an established tumor marker of small cell lung cancer. The purpose of this study was to determine if ProGRP could serve as a tumor marker for the Ewing sarcoma family of tumors (ESFTs). Sixteen patients with ESFTs (mean age 32 years) were included in this study. As a control group, 42 patients with other tumor types that clinically or pathologically mimic ESFTs were also analyzed. Pre-treatment serum ProGRP and neuron-specific enolase (NSE) levels, the relationships between these levels, and tumor volume were investigated. In addition, serial changes in the serum or plasma ProGRP (6 patients) and NSE levels (5 patients) were measured over the course of treatment. Pre-treatment serum ProGRP levels were higher than the normal range in 8 of 16 patients; for these eight patients, ProGRP levels positively correlated with tumor volume (R = 0.99). In the control group, ProGRP levels were within the normal range, except for the two patients. Changes in ProGRP levels during treatment were consistent with tumor volume. Serum NSE levels were elevated in 14 of 16 patients with ESFTs and 8 of 42 patients with other tumor types. The range of NSE elevation was much smaller compared to that of ProGRP. Our data indicate that ProGRP is superior to NSE in terms of specificity. Serum ProGRP levels were elevated in half of the patients with ESFTs and reflected therapeutic response. ProGRP is a reliable tumor marker for the diagnosis of ESFTs and evaluation of treatment response.

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.

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 is insufficient, and its changes have not been fully evaluated. Methods: We assessed the prevalence and changes in TROP2 expression in lung cancer patients receiving anti-cancer treatments using immunohistochemical (IHC) analysis with an anti-TROP2 (clone: SP295). IHC scores were graded from 0–3; 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 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 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: TROP2 was expressed in most lung cancer specimens before and after anti-cancer treatments. Additionally, some anti-cancer treatments might alter the TROP2 expression. These results may provide a strong rationale for TROP2-directed therapy against advanced lung cancer.

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. 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. 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

Cancer metastasis shows great diversity in target organs, routes and molecular mechanisms depending on the type of cancer and even on the individual patients. To identify key molecules involved in metastasis, we constructed a murine model system including multiple sublines with different organotropism and pathways of metastasis. We selected metastatic sublines from a murine mammary tumor cell line MCH66. Using this model, we extracted metastasis-related molecules by gene expression screening methods and verified their metastasis-promoting effects by gene knockdown or overexpression experiments. For the candidates promoting metastasis, we analyzed molecular functions involved in metastasis: cell growth, motility and invasive activity. We established a metastasis model including low metastatic sublines (66C8, 66LM, 66-4) and highly metastatic counterparts with various organotropism, such as to the lung (66Lu10), liver (HM-KAN5) and general organs (66HM and its clones: HM1-6 and HM1-7). The sublines basically exhibited the invasion-independent metastasis pathway characterized by endothelial cell-covered tumor emboli, whereas 66HM and HM-KAN5 showed an alternative metastasis pathway based on invasion in part and in whole, respectively. Comprehensive gene analysis extracted several molecular candidates responsible for metastasis. S100A14 was identified as one of the promissing candidates promoting lung-metastasis, which was verified by gene knockdown experiments in vivo. In addition, in vivo and in vitro functional analyses demonstrated that S100A14 enhanced scattering, motility and invasiveness of mouse tumor cells. Our model system may be adaptable to the diversity of metastasis in human cancers and useful for exploring the molecular mechanism responsible for metastasis.

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.

Pleomorphic lobular carcinoma (PLC) of the breast is a variant of lobular carcinoma, characterized by loss of E-cadherin expression and high-grade morphologies. Whether the pathogenesis of PLC is in the ductal or the lobular lineage has been discussed. In this report, a case of PLC combined with apocrine carcinoma is presented. Histologically, the tumor showed two distinct carcinoma components: one was a typical apocrine carcinoma, and the other was a pleomorphic invasive lobular carcinoma. The former showed complete membranous expression of E-cadherin, whereas the latter aberrantly expressed it not on the cell membrane, but in the cytoplasm. Both components were triple-negative and strongly positive for GCDFP-15, suggesting apocrine differentiation. The intraductal component showed only a feature of apocrine ductal carcinoma in situ. This case suggests that apocrine carcinoma could be an origin of PLC.

We analyzed serum ProGRP levels in patients with Ewing sarcoma, and found that 5 out of 9 patients had elevated levels; the values range equally with those of patients with limited disease of small-cell lung carcinoma. Serum ProGRP levels in patients with bone and soft tissue malignancies other than Ewing sarcoma are not elevated. Immunohistochemical studies demonstrated that ProGRP-like immunoreactivities were detected in Ewing sarcoma tissues obtained from 2 patients with elevated serum ProGRP levels, suggesting that ProGRP is a product of tumor cells of Ewing sarcoma. These results indicate that serum ProGRP could serve as a specific tumor marker for Ewing sarcoma. Since ProGRP is a major hormonal product of tumor cells of small-cell lung carcinoma, a typical neuroendocrine carcinoma, it is reasonable to postulate that the present study provides an evidence for Ewing sarcoma to possess neuroendocrine differentiation.

Adenosquamous carcinoma (ASC) is a rare malignant neoplasm of the head and neck regions. We elucidated the relationship between ASC and MUC molecule expression. We selected 14 cases of ASC in the head and neck, and examined them immunohistohcmically. Seven cases of tongue, 3 cases of larynx and 4 cases of hypopharynx carcinoma were selected. Nine (64.3%) of 14 cases showed lymph node metastasis at the diagnosis. Laryngeal and hypopharyngeal cases showed a higher stage. Six cases (66.7%), all of which showed lymph node metastases, died of disease. Immunohistochemical examinations showed that ASC was positive for both markers of squamous cell carcinoma and adenocarcinoma. ASC showed positivity for MUC1 (13 cases: 92.3%), MUC1core (12 cases: 85.2%) and MUC4 (12 cases: 85.7%). In rare ASC of the head and neck region, it was revealed that MUC1 and MUC4 expression might be associated with its nodal status.

e14732 Background: Human trophoblast cell surface glycoprotein (TROP2) is expressed on the cell surface of trophoblasts and various solid cancer cells. Recently, an antibody-drug conjugate (ADC) targeting TROP2 has been developed. The objective of this retrospective study is to evaluate the prevalence of TROP2 expression in the specimen of lung cancer and its changes during anticancer treatments. Methods: TROP2 expression was evaluated prior to and following anticancer treatment by immunohistochemical (IHC) analysis using an anti-TROP2 antibody (SP295). IHC scores were graded from 0 to 3 and grade ≥2 was considered to be positive. Pre- and post-treatment IHC scores of TROP2 expression were compared by Wilcoxon signed rank test. Results: Among 160 patients with evaluable tissue samples, median age was 65 (range, 39-89) years with 69% in stage III-IV. Histological diagnosis included 81% adenocarcinoma (Ad), 12% squamous cell carcinoma (Sq), 5% neuroendocrine carcinoma (NEC), and others. Among 130 Ad patients, 59% had EGFR mutations. Anticancer treatments included cytotoxic chemotherapy (77%), targeted therapy (42%), and radiotherapy (19%). TROP2 was expressed in 143 (89%) and 139 (87%) in pre- and post-treatment samples, respectively. Pre-treatment TROP2 positive expression were more frequently observed in Ad (92%) or Sq (100%) than NEC histology (25%, p < 0.001). There was no significant change in TROP2 expression after treatment (p = 0.509). Conclusions: TROP2 was commonly observed in the specimen of lung cancer especially in non-NEC histology regardless of anticancer treatments. TROP2-targeted ADC might have a therapeutic potential to be tested in TROP2-expressing lung cancer. [Table: see text]

Background Salivary duct carcinoma with rhabdoid features is extremely rare. Methods We report 2 cases of salivary duct carcinoma with rhabdoid features treated at our institution. Results Case 1 was a 44-year-old Japanese man who had swelling in the left parotid region. This tumor consisted of residual pleomorphic adenoma and widely invasive carcinoma, which showed a diffuse growth pattern by atypical rhabdoid cells. Case 2 was a 66-year-old Japanese man who had swelling of the right cervical region. This submandibular tumor was also composed of both residual pleomorphic adenoma region and invasive adenocarcinoma components, whereas some metastatic lesions were purely composed of rhabdoid cells. Such cells were strongly and diffusely positive for cytokeratins (CKs), gross cystic disease fluid protein-15 (GCDFP), and androgen receptor (AR). Case 1 was also positive for Her-2 and p53. Conclusion Both patients were diagnosed with carcinoma ex pleomorphic adenoma and their carcinomatous components were composed of salivary duct carcinoma with rhabdoid features, which is a highly aggressive tumor, similar to salivary duct carcinoma. © 2013 The Authors. Head & Neck published by Wiley Periodicals, Inc. Head Neck 36: E28–E35, 2014

We report a case of diffuse idiopathic pulmonary neuroendocrine cell hyperplasia (DIPNECH). We performed immunohistochemical analysis of 17 neuropeptides and human gonadotropin-alpha (hCGα), a trophoblastic peptide that promotes the proliferation of neuroendocrine cells. A 51-year-old woman with no history of smoking was found to have a nodule in the right middle lobe. Upon examination, the nodule was found to comprise diffuse linear and nodular neuroendocrine cell hyperplasia (NECH), numerous pulmonary tumorlets merging with one peripheral carcinoid, and an additional central carcinoid. Immunohistochemical analysis revealed diffuse but intense expression of the general neuroendocrine markers CD56, synaptophysin, and chromogranin A, together with gastrin-releasing peptide (GRP), calcitonin, and hCGα throughout the carcinoids, tumorlets, and NECH. Positive staining was also noted for adrenocorticotropic hormone, corticotropin-releasing hormone, met-enkephalin, vasoactive intestinal polypeptide, neurotensin, and growth hormone-releasing hormone in a few isolated cells of the carcinoids and the tumorlets, but staining for these proteins was entirely negative in the NECH lesions. The presence of these neuropeptides in neuroendocrine tumors might explain the presence of neuropeptide-producing tumors of the lungs, cases of which have been reported over the last 30 years. The preoperative serum proGRP level was high but returned to normal after surgical intervention, indicating that GRP was produced and secreted by carcinoids, tumorlets, and/or NECH lesions. It is also probable that neuroendocrine cells secreted GRP into the interstitium in a paracrine manner, leading to the development of dense fibrosis around the tumorlets. During the preoperative and postoperative periods, no evidence of bronchiolitis obliterans was noted, in contrast to some previously reported cases of DIPNECH.

An extremely rare case of mixed acinar-endocrine carcinoma (MAEC) arising from the stomach in a 56-year-old Japanese woman is herein presented. An endoscopic examination and computed tomography showed a protruding gastric tumor and a large extragastric mass, respectively. Macroscopic observation on the surgical specimen revealed the extragastric cystic mass was continued to the intragastric tumor. Histologically, the intragastric tumor consisted of large or small solid nests with acinar appearance. The cancer cells had an ovoid nuclei and polygonal cytoplasm, which was frequently amphophilic. Immunohistochemical examination showed that the cancer cells were positive for chromogranin-A, synaptophysin, alpha-amylase, lipase, and alpha-fetoprotein (AFP) but were negative for CD56, insulin, and other hormones. Ultrastructurally, the cancer cells contained 500-nm electron-lucent zymogen granules and 230-nm electron-dense neuroendocrine granules. This tumor was finally diagnosed to be MAEC with AFP production of the stomach. Although no ectopic pancreas was found in the stomach, this tumor may originate from ectopic pancreas. As another theory, it is possible for this tumor to originate from the pluripotent stem cells in the stomach. A gastric MAEC with AFP production has not been reported previously.

A pleomorphic adenoma (PA) is the most common epithelial tumor in the salivary glands, but it frequently shows a mesenchyme-like histology, including the presence of myxoid and chondroid areas. Cartilage-specific matrix proteins are deposited in PA. Aggrecan is a major component of cartilage-specific proteoglycans. The present study examined the ultrastructure of the stromal areas in ten salivary PA specimens and investigated the distribution of aggrecan by immunoelectron microscopy. Aggrecan was deposited in the myxoid and chondroid stroma of PA. Ultrastructural observations revealed many proteoglycan cores and fibrils in the myxoid stroma and some spindle-shaped neoplastic myoepithelial cells with vacuoles and actin filaments in the myxoid areas. By immunoelectron microscopy, positivity for aggrecan was observed in the vacuoles of neoplastic myoepithelial cells, which coexisted with the viscous materials, and it was also frequently seen in electron-dense crystals in the myxoid stroma. These findings suggest that neoplastic myoepithelial cells produce aggrecan and release it from vacuoles, and aggrecan is then deposited in the myxoid stroma. Aggrecan deposition is therefore considered to play an important role in the formation of the mesenchyme-like stroma, especially the myxoid stroma.

Matrix-producing carcinoma of the breast is a well-established entity in the group of metaplastic carcinoma, which is histologically characterized by myxochondroid matrix formation and is extremely rare. We describe here four additional cases of matrix-producing carcinoma of the breast. All cases of matrix-producing carcinoma show nest-like, sheet-like, and cord-like growth of tumor cells with cellular atypia, in addition to scattered cancer cells within myxoid or myxohyalinous stroma. Three of four cases showed an acellular or oligocellular matrix-rich zone in the center of the tumor. Immunohistochemically, cancer cells of all cases were positive for cytokeratins and epithelial membrane antigens and partially positive for sox9 and p63. Aggrecan and type II collagen, which are cartilage-specific matrix molecules, were deposited in the stroma of all cases. Type I and type IV collagens were also deposited on the stroma of all cases. These findings suggest that, although cancer cells of matrix-producing carcinoma of the breast are epithelial, they transdifferentiate to chondrocyte-like cells and produce cartilage-specific matrix molecules, which are useful markers for diagnosing matrix-producing carcinoma.

Alveolar soft part sarcoma (ASPS) is a rare mesenchymal neoplasm of uncertain origin. In this article, we report a case of ASPS occurring in the larynx, an extremely rare location for this rather unusual tumor.The patient was a 34-year-old Japanese woman who requested an examination for hoarseness. The tumor showed a proliferation of large polygonal cells with periodic-acid-Schiff-positive diastase-resistant intracytoplasmic granules, arranged in an alveolar growth pattern. The cytoplasm of the tumor cells was eosinophilic. Tumor cells were positive for vimentin and titin. Nuclear immunoreactivity for TFE3 was observed, and the Ki-67 labeling index was 14.7%. Ultrastructurally, electron-dense rod-shaped crystals were infrequently observed in the cytoplasm. This case was finally diagnosed as ASPS of the larynx.We discuss the histogenesis and differential diagnosis of ASPS with immunohistochemical and ultrastructural findings. TFE3 immunohistochemistry was found to be a very useful marker for the diagnosis of ASPS.

Prevention of quality of life (QOL) deterioration is associated with the inhibition of geriatric diseases and the regulation of brain function. However, no substance is known that prevents the aging of both body and brain. It is known that polyamine concentrations in somatic tissues (including the brain) decrease with increasing age, and polyamine-rich foods enhance longevity in yeast, worms, flies, and mice, and protect flies from age-induced memory impairment. A main source of exogenous polyamines is the intestinal lumen, where they are produced by intestinal bacteria. We found that arginine intake increased the concentration of putrescine in the colon and increased levels of spermidine and spermine in the blood. Mice orally administered with arginine in combination with the probiotic bifidobacteria LKM512 long-term showed suppressed inflammation, improved longevity, and protection from age-induced memory impairment. This study shows that intake of arginine and LKM512 may prevent aging-dependent declines in QOL via the upregulation of polyamines.

Background Evidence suggests that intestinal microbiota play an important role in the pathogenesis of atopic dermatitis (AD) through induction of immunosuppression and immune tolerance; however, the exact underlying mechanism is unclear. Few studies to date have examined the effects of probiotics on adult-type AD. Objective To examine the effects of the probiotic Bifidobacterium animalis subsp lactis LKM512 on adult-type AD and the expression of metabolites that are known to be influenced by gut microbiota in fecal samples. Methods Forty-four patients were randomly assigned to receive LKM512 or a placebo and underwent medical examinations. Fecal microbiota were analyzed with real-time polymerase chain reaction. Metabolomic analysis was conducted to search for antipruritic metabolites produced by intestinal bacteria using feces derived from 3 patients whose itch scores had improved using capillary electrophoresis with time-of-flight mass spectrometry. Antipruritic effects of kynurenic acid were observed using AD-induced NC/Nga mice. Results LKM512 administration alleviated itch in AD patients compared with controls and improved the dermatology-specific quality-of-life scores when compared with the controls. Administration of LKM512 also increased the expression of the antipruritic and antinociceptive metabolite kynurenic acid (KYNA) in patients whose itch score had improved after LKM512 treatment. In mouse experiments, scratching behavior counts tended to be decreased by KYNA injection when compared with no treatment. Conclusion LKM512 administration may exert antipruritic effects by increasing KYNA production. LKM512 could therefore be a potentially effective therapeutic candidate for the reduction of pruritus. Trial Registration umin.ac.jp/ctr Identifier: UMIN000005695.

Adenosquamous carcinoma (ASC) is a rare malignant neoplasm of the head and neck regions. We elucidated the relationship between ASC and MUC molecule expression. We selected 14 cases of ASC in the head and neck, and examined them immunohistohcmically. Seven cases of tongue, 3 cases of larynx and 4 cases of hypopharynx carcinoma were selected. Nine (64.3%) of 14 cases showed lymph node metastasis at the diagnosis. Laryngeal and hypopharyngeal cases showed a higher stage. Six cases (66.7%), all of which showed lymph node metastases, died of disease. Immunohistochemical examinations showed that ASC was positive for both markers of squamous cell carcinoma and adenocarcinoma. ASC showed positivity for MUC1 (13 cases: 92.3%), MUC1core (12 cases: 85.2%) and MUC4 (12 cases: 85.7%). In rare ASC of the head and neck region, it was revealed that MUC1 and MUC4 expression might be associated with its nodal status.

Proneurotensin/neuromedin N (proNT/NMN), the precursor of neurotensin (NT) and neuromedin N (NMN), is produced by cancer tissues derived from the pancreas and colon. NT stimulates tumor growth and proliferation through its receptors; however, little is known about the precursor molecule in cancer tissues. We previously demonstrated that proNT/NMN is secreted from small cell lung carcinoma (SCLC) cell lines in serum-free conditioned medium, but not from non-small cell lung carcinoma (NSCLC) cell lines. It was suggested that this precursor may serve as a tumor marker for SCLC. In this study, we established in vivo xenograft models to evaluate the possibility of proNT/NMN as a specific tumor marker. SBC3 cells, derived from human SCLC, were inoculated into mice, and the proNT/NMN levels in plasma and tumor tissues were detected using specific antibodies. In contrast to control mouse plasma, the proNT/NMN levels in tumor-bearing mice increased as the tumors grew, and the elevated plasma proNT/NMN levels were decreased by tumor resection. Moreover, proNT/NMN was expressed in SBC3 tumors, suggesting that proNT/NMN was secreted into blood from the tumor, and this secretion may be specific to SCLC.