Evolutionary histories of breast cancer and related clones.

Recent studies have documented frequent evolution of clones carrying common cancer mutations in apparently normal tissues, which are implicated in cancer development1-3. However, our knowledge is still missing with regard to what additional driver events take place in what order, before one or more of these clones in normal tissues ultimately evolve to cancer. Here, using phylogenetic analyses of multiple microdissected samples from both cancer and non-cancer lesions, we show unique evolutionary histories of breast cancers harbouring der(1;16), a common driver alteration found in roughly 20% of breast cancers. The approximate timing of early evolutionary events was estimated from the mutation rate measured in normal epithelial cells. In der(1;16)(+) cancers, the derivative chromosome was acquired from early puberty to late adolescence, followed by the emergence of a common ancestor by the patient's early 30s, from which both cancer and non-cancer clones evolved. Replacing the pre-existing mammary epithelium in the following years, these clones occupied a large area within the premenopausal breast tissues by the time of cancer diagnosis. Evolution of multiple independent cancer founders from the non-cancer ancestors was common, contributing to intratumour heterogeneity. The number of driver events did not correlate with histology, suggesting the role of local microenvironments and/or epigenetic driver events. A similar evolutionary pattern was also observed in another case evolving from an AKT1-mutated founder. Taken together, our findings provide new insight into how breast cancer evolves.

Frequent mutations that converge on the NFKBIZ pathway in ulcerative colitis.

Chronic inflammation is accompanied by recurring cycles of tissue destruction and repair and is associated with an increased risk of cancer1-3. However, how such cycles affect the clonal composition of tissues, particularly in terms of cancer development, remains unknown. Here we show that in patients with ulcerative colitis, the inflamed intestine undergoes widespread remodelling by pervasive clones, many of which are positively selected by acquiring mutations that commonly involve the NFKBIZ, TRAF3IP2, ZC3H12A, PIGR and HNRNPF genes and are implicated in the downregulation of IL-17 and other pro-inflammatory signals. Mutational profiles vary substantially between colitis-associated cancer and non-dysplastic tissues in ulcerative colitis, which indicates that there are distinct mechanisms of positive selection in both tissues. In particular, mutations in NFKBIZ are highly prevalent in the epithelium of patients with ulcerative colitis but rarely found in both sporadic and colitis-associated cancer, indicating that NFKBIZ-mutant cells are selected against during colorectal carcinogenesis. In further support of this negative selection, we found that tumour formation was significantly attenuated in Nfkbiz-mutant mice and cell competition was compromised by disruption of NFKBIZ in human colorectal cancer cells. Our results highlight common and discrete mechanisms of clonal selection in inflammatory tissues, which reveal unexpected cancer vulnerabilities that could potentially be exploited for therapeutics in colorectal cancer.

Age-related remodelling of oesophageal epithelia by mutated cancer drivers.

Clonal expansion in aged normal tissues has been implicated in the development of cancer. However, the chronology and risk dependence of the expansion are poorly understood. Here we intensively sequence 682 micro-scale oesophageal samples and show, in physiologically normal oesophageal epithelia, the progressive age-related expansion of clones that carry mutations in driver genes (predominantly NOTCH1), which is substantially accelerated by alcohol consumption and by smoking. Driver-mutated clones emerge multifocally from early childhood and increase their number and size with ageing, and ultimately replace almost the entire oesophageal epithelium in the extremely elderly. Compared with mutations in oesophageal cancer, there is a marked overrepresentation of NOTCH1 and PPM1D mutations in physiologically normal oesophageal epithelia; these mutations can be acquired before late adolescence (as early as early infancy) and significantly increase in number with heavy smoking and drinking. The remodelling of the oesophageal epithelium by driver-mutated clones is an inevitable consequence of normal ageing, which-depending on lifestyle risks-may affect cancer development.

Time-series blood cytokine profiles correlate with treatment responses in triple-negative breast cancer patients.

BACKGROUND: Triple-negative breast cancer (TNBC) is the most heterogeneous breast cancer subtype. Partly due to its heterogeneity, it is currently challenging to stratify TNBC patients and predict treatment outcomes. METHODS: In this study, we examined blood cytokine profiles of TNBC patients throughout treatments (pre-treatment, during chemotherapy, pre-surgery, and 1 year after the surgery in a total of 294 samples). We analyzed the obtained cytokine datasets using weighted correlation network analyses, protein-protein interaction analyses, and logistic regression analyses. RESULTS: We identified five cytokines that correlate with good clinical outcomes: interleukin (IL)-1α, TNF-related apoptosis-inducing ligand (TRAIL), Stem Cell Factor (SCF), Chemokine ligand 5 (CCL5 also known as RANTES), and IL-16. The expression of these cytokines was decreased during chemotherapy and then restored after the treatment. Importantly, patients with good clinical outcomes had constitutively high expression of these cytokines during treatments. Protein-protein interaction analyses implicated that these five cytokines promote an immune response. Logistic regression analyses revealed that IL-1α and TRAIL expression levels at pre-treatment could predict treatment outcomes in our cohort. CONCLUSION: We concluded that time-series cytokine profiles in breast cancer patients may be useful for understanding immune cell activity during treatment and for predicting treatment outcomes, supporting precision medicine. TRIAL REGISTRATION: The study has been registered with the University Hospital Medical Information Network Clinical Trials Registry ( http://www.umin.ac.jp/ctr/index-j.htm ) with the unique trial number UMIN000023162. The association Japan Breast Cancer Research Group trial number is JBCRG-22. The clinical outcome of the JBCRG-22 study was published in Breast Cancer Research and Treatment on 25 March 2021. https://doi.org/10.1007/s10549-021-06184-w .

Banff 2022 Liver Group Meeting report: Monitoring long-term allograft health.

The Banff Working Group on Liver Allograft Pathology met in September 2022. Participants included hepatologists, surgeons, pathologists, immunologists, and histocompatibility specialists. Presentations and discussions focused on the evaluation of long-term allograft health, including noninvasive and tissue monitoring, immunosuppression optimization, and long-term structural changes. Potential revision of the rejection classification scheme to better accommodate and communicate late T cell-mediated rejection patterns and related structural changes, such as nodular regenerative hyperplasia, were discussed. Improved stratification of long-term maintenance immunosuppression to match the heterogeneity of patient settings will be central to improving long-term patient survival. Such personalized therapeutics are in turn contingent on a better understanding and monitoring of allograft status within a rational decision-making approach, likely to be facilitated in implementation with emerging decision-support tools. Proposed revisions to rejection classification emerging from the meeting include the incorporation of interface hepatitis and fibrosis staging. These will be opened to online testing, modified accordingly, and subject to consensus discussion leading up to the next Banff conference.

An Analysis of 10,000 Cases of Living Donor Liver Transplantation in Japan: Special Reference to the Graft-Versus-Recipient Weight Ratio and Donor Age.

OBJECTIVE: To analyze 10,000 cases of living donor liver transplantation (LDLT) recipient data to elucidate outcomes with special reference to the graft-versus-recipient weight ratio (GRWR), based on the Japanese Liver Transplantation Society (JLTS) registry. BACKGROUND: The JLTS registry has been accurate and complete in characterizing and following trends in patient characteristics and survival of all patients with LDLT. METHODS: Between November 1989 and August 2021, 10,000 patients underwent LDLT in Japan. The procedures performed during the study period included pediatric liver transplantation (age <18 years, n = 3572) and adult liver transplantation (age ≥18 years, n=6428). Factors related to patient survival (PS) and graft survival (GS) were also analyzed. RESULTS: The GRWR was <0.7, 0.7 to <0.8, 0.8 to <3, 3 to <5, and ≥5 in 0.2%, 2.0%, 61.8%, 31.8%, and 2.6% of pediatric patients and <0.6, 0.6 to <0.7, 0.7 to <0.8, and ≥0.8 in 8.0%, 12.7%, 17.7%, and 61.5% of adult patients, respectively. Among pediatric recipients, the PS rate up to 5 years was significantly better in cases with a GRWR ≤5 than in those with a GRWR >5. When the GRWR and donor age were combined, among adult recipients 50 to 60 years old, the early PS and GS up to 5 years were significantly better in cases with a GRWR ≥0.7, than in those with a GRWR <0.7. (P = 0.02). In adults, a multivariate analysis showed that GRWR <0.6, transplant era (<2011), donor age (>60 years), recipient age (>60 years), model for end-stage liver disease score (≥20), and center volume (<10) were significant prognostic factors for long-term PS. CONCLUSION: Although a satisfactory long-term PS and GS, especially in the recent era (2011-2021), was achieved in the JLTS series, a GRWR ≥5 in pediatric cases and relatively old donors with a GRWR <0.7 in adult cases should be managed with caution.

A Deep Learning-Based Assay for Programmed Death Ligand 1 Immunohistochemistry Scoring in Non-Small Cell Lung Carcinoma: Does it Help Pathologists Score?

Several studies have developed various artificial intelligence (AI) models for immunohistochemical analysis of programmed death ligand 1 (PD-L1) in patients with non-small cell lung carcinoma; however, none have focused on specific ways by which AI-assisted systems could help pathologists determine the tumor proportion score (TPS). In this study, we developed an AI model to calculate the TPS of the PD-L1 22C3 assay and evaluated whether and how this AI-assisted system could help pathologists determine the TPS and analyze how AI-assisted systems could affect pathologists' assessment accuracy. We assessed the 4 methods of the AI-assisted system: (1 and 2) pathologists first assessed and then referred to automated AI scoring results (1, positive tumor cell percentage; 2, positive tumor cell percentage and visualized overlay image) for final confirmation, and (3 and 4) pathologists referred to the automated AI scoring results (3, positive tumor cell percentage; 4, positive tumor cell percentage and visualized overlay image) while determining TPS. Mixed-model analysis was used to calculate the odds ratios (ORs) with 95% CI for AI-assisted TPS methods 1 to 4 compared with pathologists' scoring. For all 584 samples of the tissue microarray, the OR for AI-assisted TPS methods 1 to 4 was 0.94 to 1.07 and not statistically significant. Of them, we found 332 discordant cases, on which the pathologists' judgments were inconsistent; the ORs for AI-assisted TPS methods 1, 2, 3, and 4 were 1.28 (1.06-1.54; P = .012), 1.29 (1.06-1.55; P = .010), 1.28 (1.06-1.54; P = .012), and 1.29 (1.06-1.55; P = .010), respectively, which were statistically significant. For discordant cases, the OR for each AI-assisted TPS method compared with the others was 0.99 to 1.01 and not statistically significant. This study emphasized the usefulness of the AI-assisted system for cases in which pathologists had difficulty determining the PD-L1 TPS.

Adverse effects of graft congestion and ameliorative effects of hepatocyte growth factor after liver transplantation in rats.

Living donor liver transplantation (LT) and deceased donor split-LT often result in congestion within liver grafts. The regenerative process and function of congested areas, especially graft congestion associated with LT, are not well understood. Therefore, we created new rat models with congested areas in partially resected livers and orthotopically transplanted these livers into syngeneic rats to observe liver regeneration and function in congested areas. This study aimed to compare liver regeneration and the function of congested areas after liver resection and LT, and to explore a new approach to ameliorate the adverse effects of graft congestion. Although the congested areas after liver resection regenerated normally on postoperative day 7, the congested areas after LT had poor regeneration with abscess development on postoperative day 7. Necrotic areas in congested areas were larger after LT than after liver resection on postoperative days 1, 3, and 7 ( p < 0.05, p < 0.05, and p < 0.01, respectively). Although congested areas after liver resection did not affect survival, in the LT model, the survival of rats with congested areas was significantly poorer even with larger grafts than that of rats with smaller noncongested grafts ( p = 0.04). Hepatocyte growth factor administration improved the survival rate of rats with congested grafts from 41.7% to 100%, improved the regeneration of congested areas, and significantly reduced the size of necrotic areas ( p < 0.05). Thus, congested areas in liver grafts may negatively impact recipients. Short-term administration of hepatocyte growth factor may improve postoperative outcomes of recipients with graft congestion and contribute to more effective use of liver grafts (approval number: MedKyo-23137, Institutional Ethics Committee/Kyoto University).

Comprehensive analyses of the clinicopathological features and genomic mutations of combined hepatocellular-cholangiocarcinoma.

AIM: Combined hepatocellular-cholangiocarcinoma (cHCC-CCA) is a rare primary liver cancer that has two different tumor phenotypes in a single tumor nodule. The relationship between genetic mutations and clinicopathological features of cHCC-CCA remains to be elucidated. METHODS: Whole-exome sequencing analyses were carried out in 13 primary and 2 recurrent cHCC-CCAs. The whole-exome analyses and clinicopathological information were integrated. RESULTS: TP53 was the most frequently mutated gene in this cohort, followed by BAP1, IDH1/2, and NFE2L2 mutations in multiple cases. All tumors with diameters <3 cm had TP53 mutations. In contrast, six of seven tumors with diameters ≥3 cm did not have TP53 mutations, but all seven tumors had mutations in genes associated with various pathways, including Wnt, RAS/PI3K, and epigenetic modulators. In the signature analysis, the pattern of mutations shown in the TP53 mutation group tended to be more similar to HCC than the TP53 nonmutation group. Mutations in recurrent cHCC-CCA tumors were frequently identical to those in the primary tumor, suggesting that those tumors originated from identical clones of the primary cHCC-CCA tumors. Recurrent and co-occurrent HCC tumors in the same patients with cHCC-CCA had either common or different mutation patterns from the primary cHCC-CCA tumors in each case. CONCLUSIONS: The study suggested that there were two subtypes of cHCC-CCA, one involving TP53 mutations in the early stage of the carcinogenic process and the other not involving such mutations. The comparison of the variants between primary and recurrent tumors suggested that cHCC-CCA was derived from an identical clone.

Efficacy of steroid therapy for improving native liver survival after pediatric acute liver failure with immune activation.

AIM: Recent evidence suggests that acute liver failure (ALF) in some patients may reflect a dysregulated immune response, and that corticosteroids improve survival of the native liver in ALF patients with high serum alanine aminotransferase levels, which are an indication of liver inflammation. However, it is unclear whether steroids are effective for pediatric acute liver failure (PALF). The aim of this retrospective case-control study is to examine whether steroid therapy for PALF accompanied by immune activation improves the survival of native liver and to identify factors that predict responses to steroid treatment. METHODS: Of 38 patients with PALF treated at Kyoto University Hospital from February 2006 to August 2022, 19 receiving steroids who met the specific criteria for identifying the pathophysiology of immune activity in the liver (the "Steroid group"), and seven steroid-free patients who also met the criteria ("Nonsteroid group") were enrolled. Patients in the "Steroid group" were categorized as "responders" or "nonresponders" according to treatment outcome. Clinical and histological data were analyzed. RESULTS: Survival of the native liver in the Steroid group was significantly higher than that in the Nonsteroid group (68% vs. 0%, respectively; p = 0.0052). Nonresponders were significantly younger, with higher Model for End-stage Liver Disease and pediatric end-stage liver disease scores, higher prothrombin time - international normalized ratio, and higher serum ferritin levels than responders. Massive hepatic necrosis was more common in nonresponders. CONCLUSION: Steroid therapy is effective for PALF patients with liver inflammation; however, liver transplantation should be prioritized for young children with ALF accompanied by severe coagulopathy or massive hepatic necrosis.

Polymorphic lymphoproliferative disorder arising in a rheumatoid arthritis patient, presenting as fibrin-associated large B-cell lymphoma-like lesions in aortic and mitral valves.

We herein report a case of methotrexate-associated lymphoproliferative disorder (MTX-LPD) showing fibrin-associated large B-cell lymphoma-like heart valve lesions, and Epstein-Barr virus (EBV)-positive mucocutaneous ulcer-like cutaneous and oral mucosal lesions. MTX-LPD is a critical complication that can occur in RA patients who are treated with MTX. EBV also plays a defining or important role in LPDs. Among the sites of MTX-LPD, 40-50% occur in extranodal sites, including the gastrointestinal tract, skin, liver, lung, and kidney. There are few reports of MTX-LPDs involving the heart valves, and to the best of our knowledge, this is the first case to be reported in the English literature. The possibility of EBV-positive LPD should be considered in RA patients, even in patients with an atypical site, as in this case.

Lynch syndrome screening in patients with young-onset extra-colorectal Lynch syndrome-associated cancers.

BACKGROUND: Lynch syndrome (LS) is a hereditary cancer syndrome caused by pathogenic germline variants in mismatch repair (MMR) genes, which predisposes to various types of cancers showing deficient MMR (dMMR). Identification of LS probands is crucial to reduce cancer-related deaths in affected families. Although universal screening is recommended for colorectal and endometrial cancers, and age-restricted screening is proposed as an alternative, LS screening covering a broader spectrum of cancer types is needed. In the current study, we elucidated the rate of dMMR tumors and evaluated the outcome of LS screening in young-onset extra-colorectal LS-associated cancers. METHODS: Immunohistochemistry for MMR proteins were retrospectively performed in a total of 309 tissue samples of endometrial, non-mucinous ovarian, gastric, urothelial, pancreatic, biliary tract, and adrenal cancers in patients < 50 years of age. Clinicopathological information and the results of genetic testing were obtained from medical charts. RESULTS: There were 24 dMMR tumors (7.8%) including 18 endometrial, three ovarian, two urothelial, and one gastric cancer. Co-occurrence of colorectal cancer and family history of LS-associated cancers was significantly enriched in patients with dMMR tumors. Among the 16 patients with dMMR tumors who were informed of the immunohistochemistry results, five with endometrial and one with urothelial cancer were diagnosed as LS with positive pathogenic variants in MMR genes. CONCLUSIONS: We report the outcome of immunohistochemistry for MMR proteins performed in multiple types of young-onset extra-colorectal LS-associated cancers. Our study demonstrates the feasibility of a comprehensive LS screening program incorporating young-onset patients with various types of extra-colorectal LS-associated cancers.

Tertiary Lymphoid Tissues Are Microenvironments with Intensive Interactions between Immune Cells and Proinflammatory Parenchymal Cells in Aged Kidneys.

SIGNIFICANCE STATEMENT: Ectopic lymphoid structures called tertiary lymphoid tissues (TLTs) develop in several kidney diseases and are associated with poor renal prognosis. However, the mechanisms underlying TLT expansion and their effect on renal regeneration remain unclear. The authors report that single-nucleus RNA sequencing and validation experiments demonstrate that TLTs potentially amplify inflammation in aged injured kidneys. Lymphocytes within TLTs promote proinflammatory phenotypes of the surrounding proximal tubules and fibroblasts within the TLTs via proinflammatory cytokine production. These proinflammatory parenchymal cells then interact with immune cells by chemokine or cytokine production. Such cell-cell interactions potentially increase inflammation, expand TLTs, and exacerbate kidney injury. These findings help illuminate renal TLT pathology and suggest potential therapeutic targets. BACKGROUND: Ectopic lymphoid structures called tertiary lymphoid tissues (TLTs) develop in several kidney diseases and are associated with poor renal prognosis. However, the mechanisms that expand TLTs and underlie exacerbation of kidney injury remain unclear. METHODS: We performed single-nucleus RNA sequencing (snRNA-seq) on aged mouse kidneys with TLTs after ischemia-reperfusion injury. The results were validated using immunostaining, in situ hybridization of murine and human kidneys, and in vitro experiments. RESULTS: Using snRNA-seq, we identified proinflammatory and profibrotic Vcam1+ injured proximal tubules (PTs) with NF κ B and IFN-inducible transcription factor activation. VCAM1 + PTs were preferentially localized around TLTs and drove inflammation and fibrosis via the production of multiple chemokines or cytokines. Lymphocytes within TLTs expressed Tnf and Ifng at high levels, which synergistically upregulated VCAM1 and chemokine expression in cultured PT cells. In addition, snRNA-seq also identified proinflammatory and profibrotic fibroblasts, which resided within and outside TLTs, respectively. Proinflammatory fibroblasts exhibited STAT1 activation and various chemokine or cytokine production, including CXCL9/CXCL10 and B cell-activating factor, contributing to lymphocyte recruitment and survival. IFN γ upregulated the expression of these molecules in cultured fibroblasts in a STAT1-dependent manner, indicating potential bidirectional interactions between IFN γ -producing CXCR3 + T cells and proinflammatory fibroblasts within TLTs. The cellular and molecular components described in this study were confirmed in human kidneys with TLTs. CONCLUSIONS: These findings suggest that TLTs potentially amplify inflammation by providing a microenvironment that allows intense interactions between renal parenchymal and immune cells. These interactions may serve as novel therapeutic targets in kidney diseases involving TLT formation.

Deceased donor non-composite split liver and intestinal transplantation for children.

Children with intestinal failure suffer liver damage associated with parenteral nutrition: a condition known as intestinal failure-associated liver disease (IFALD), which requires transplantation of both liver and intestine. In many countries, simultaneous transplantation of these two organs is performed using grafts from a deceased donor, but there have been no such cases in Japan, and the details of the procedure are not clear. Recently, we performed simultaneous split liver and intestinal transplantation in two premature infants with IFALD, using organs from identical deceased donors and achieved good results. These are the first two cases of this procedure being performed in Japan. We report these cases and discuss the important aspects of the surgical and perioperative management.

M2-like tumor-associated macrophages promote epithelial-mesenchymal transition through the transforming growth factor ß/Smad/zinc finger e-box binding homeobox pathway with increased metastatic potential and tumor cell proliferation in lung squamous cell carcinoma.

Epithelial-mesenchymal transition (EMT) promotes primary tumor progression toward a metastatic state. The role of tumor-associated macrophages (TAMs) in inducing EMT in lung squamous cell carcinoma (LUSC) remains unclear. We aimed to clarify the significance of TAMs in relation to EMT in LUSC. We collected 221 LUSC specimens from patients who had undergone surgery. Immunohistochemistry was performed to evaluate M1-like and M2-like TAM distribution and EMT by E-cadherin and vimentin staining. Human LUSC cell lines (H226 and EBC-1) and a human monocyte cell line (THP-1) were used for in vitro experiments. M2-like polarization of TAMs and EMT marker expression in LUSC cells were evaluated by western blotting. The biological behavior of LUSC cells was evaluated by migration, invasion, and cell proliferation assays. Immunohistochemical analysis showed that 166 (75.1%) tumors were E-cadherin-positive and 44 (19.9%) were vimentin-positive. M2-like TAM density in the tumor stroma was significantly associated with vimentin positivity and worse overall survival. Western blotting demonstrated higher levels of CD163, CD206, vascular endothelial growth factor, and transforming growth factor beta 1 (TGF-ß1) in TAMs versus unstimulated macrophages. Furthermore, increased TGF-ß1 secretion from TAMs was confirmed by ELISA. TAM-co-cultured H226 and EBC-1 cells exhibited EMT (decreased E-cadherin, increased vimentin). Regarding EMT-activating transcriptional factors, phosphorylated Smad3 and ZEB-family proteins were higher in TAM-co-cultured LUSC cells than in parental cells. TAM-co-cultured H226 and EBC-1 cells demonstrated enhanced migration and invasion capabilities and improved proliferation. Overall, the present study suggests that TAMs can induce EMT with increased metastatic potential and tumor cell proliferation in LUSC.

A risk-based subgroup analysis of the effect of adjuvant S-1 in estrogen receptor-positive, HER2-negative early breast cancer.

PURPOSE: The Phase III POTENT trial demonstrated the efficacy of adding S-1 to adjuvant endocrine therapy for estrogen receptor-positive, HER2-negative early breast cancer. We investigated the efficacy of S-1 across different recurrence risk subgroups. METHODS: This was a post-hoc exploratory analysis of the POTENT trial. Patients in the endocrine-therapy-only arm were divided into three groups based on composite risk values calculated from multiple prognostic factors. The effects of S-1 were estimated using the Cox model in each risk group. The treatment effects of S-1 in patients meeting the eligibility criteria of the monarchE trial were also estimated. RESULTS: A total of 1,897 patients were divided into three groups: group 1 (≤ lower quartile of the composite values) (N = 677), group 2 (interquartile range) (N = 767), and group 3 (> upper quartile) (N = 453). The addition of S-1 to endocrine therapy resulted in 49% (HR: 0.51, 95% CI: 0.33-0.78) and 29% (HR: 0.71, 95% CI 0.49-1.02) reductions in invasive disease-free survival (iDFS) events in groups 2 and 3, respectively. We could not identify any benefit from the addition of S-1 in group 1. The addition of S-1 showed an improvement in iDFS in patients with one to three positive nodes meeting the monarchE cohort 1 criteria (N = 290) (HR: 0.47, 95% CI: 0.29-0.74). CONCLUSIONS: The benefit of adding adjuvant S-1 was particularly marked in group 2. Further investigations are warranted to explore the optimal usage of adjuvant S-1.

Deep Learning for Predicting Effect of Neoadjuvant Therapies in Non-Small Cell Lung Carcinomas With Histologic Images.

Neoadjuvant therapies are used for locally advanced non-small cell lung carcinomas, whereby pathologists histologically evaluate the effect using resected specimens. Major pathological response (MPR) has recently been used for treatment evaluation and as an economical survival surrogate; however, interobserver variability and poor reproducibility are often noted. The aim of this study was to develop a deep learning (DL) model to predict MPR from hematoxylin and eosin-stained tissue images and to validate its utility for clinical use. We collected data on 125 primary non-small cell lung carcinoma cases that were resected after neoadjuvant therapy. The cases were randomly divided into 55 for training/validation and 70 for testing. A total of 261 hematoxylin and eosin-stained slides were obtained from the maximum tumor beds, and whole slide images were prepared. We used a multiscale patch model that can adaptively weight multiple convolutional neural networks trained with different field-of-view images. We performed 3-fold cross-validation to evaluate the model. During testing, we compared the percentages of viable tumor evaluated by annotator pathologists (reviewed data), those evaluated by nonannotator pathologists (primary data), and those predicted by the DL-based model using 2-class confusion matrices and receiver operating characteristic curves and performed a survival analysis between MPR-achieved and non-MPR cases. In cross-validation, accuracy and mean F1 score were 0.859 and 0.805, respectively. During testing, accuracy and mean F1 score with reviewed data and those with primary data were 0.986, 0.985, 0.943, and 0.943, respectively. The areas under the receiver operating characteristic curve with reviewed and primary data were 0.999 and 0.978, respectively. The disease-free survival of MPR-achieved cases with reviewed and primary data was significantly better than that of the non-MPR cases (P<.001 and P=.001), and that predicted by the DL-based model was almost identical (P=.005). The DL model may support pathologist evaluations and can offer accurate determinations of MPR in patients.

Risk factors for antibody-mediated rejection in ABO blood-type incompatible and donor-specific antibody-positive liver transplantation.

Antibody-mediated rejection (AMR) is a refractory rejection after ABO blood-type incompatible (ABOi) or donor-specific antibody (DSA)-positive liver transplantation (LT). Pretransplant rituximab desensitization dramatically reduced posttransplant AMR development; however, risk factors for AMR in the rituximab era remain unclear in both ABOi living-donor LT (ABOi-LDLT) and preformed DSA-positive LT (pDSA-LT). Of our 596 adult LDLTs (≥18 y) after rituximab introduction (2004-2019), 136 were ABOi-LDLT (22.8%). After excluding retransplants (9), acute liver failure (7), and protocol deviations (16), 104 ABOi-LDLTs were finally enrolled. Of these, 19 recipients developed AMR, 18 of which occurred within 2 weeks after transplantation (95%). ABOi-AMR significantly worsened graft and recipient survival than those without ( p =0.02 and 0.04, respectively). Model for End-stage Liver Disease (MELD) ≤13 (OR: 5.15 [1.63-16.3], p =0.005) and pre-rituximab anti-ABO IgM-titer ≥128 (OR: 3.25 [1.05-10.0], p =0.03) were identified as independent risk factors for ABOi-AMR development. Recipients fulfilling both factors showed significantly worse survival rates than those who did not ( p =0.003). Of 352 adult LTs, after introducing the LABScreen Single Ag method (2009-2019), pDSA with mean fluorescence intensity (MFI) ≥500 was detected in 50 cases (14.2%). After excluding 10 ABOi-LDLTs, 40 pDSA-LTs were finally analyzed, of which 5 developed AMR. The combination of high-titer (sum-MFI ≥10,000) and multi-loci pDSAs was a significant risk factor for pDSA-AMR development ( p <0.001); however, it did not affect the 5-year recipient survival compared with those without ( p =0.56). In conclusion, preoperative MELD ≤13 and pre-rituximab anti-ABO IgM-titer ≥128 for ABOi-LDLT, and the combination of sum-MFI ≥10,000 and multi-loci pDSAs for pDSA-LT, are risk factors for AMR in the era of rituximab desensitization. Characteristically, ABOi-AMR significantly deteriorated graft and recipient survival, whereas pDSA-AMR did not.

Galactosidase-catalyzed fluorescence amplification method (GAFAM): sensitive fluorescent immunohistochemistry using novel fluorogenic ß-galactosidase substrates and its application in multiplex immunostaining.

Multiplex immunohistochemistry/multiplex immunofluorescence (mIHC/mIF) enables the simultaneous detection of multiple markers in a single tissue section by visualizing the markers in different colors. Currently, tyramide signal amplification (TSA) is the most commonly used method because it is heat resistant to multiplexing. SPiDER-ßGal (6'-(diethylamino)-4'-(fluoromethyl)spiro[isobenzofuran-1(3H),9'-[9H]xanthen]-3'-yl ß-D-galactopyranoside), a novel fluorogenic substrate of ß-galactosidase (ß-gal) was reported recently. Its properties are favorable for application in sensitive mIF based on quinone methide chemistry. Combining SPiDER-ßGal with its related substrates, a novel, sensitive fluorescent IHC method for formalin-fixed paraffin-embedded (FFPE) sections was developed, named the galactosidase-catalyzed fluorescence amplification method (GAFAM). Evaluation of GAFAM indicated the following characteristics: (1) the entire GAFAM procedure was complete within a few hours; (2) the optimal working concentration of the substrates was 20 µM; (3) the fluorescent product was heat resistant; (4) the GAFAM exhibited sensitivity comparable with that of TSA, which was higher than that of conventional IF; and (5) the GAFAM was applicable to mIF and multispectral imaging. GAFAM is expected to be applicable to IF (or mIF in combination with TSA), and is a promising tool for facilitating morphological research in various fields of life science.

An exploratory study for tuft cells in the breast and their relevance in triple-negative breast cancer: the possible relationship of SOX9.

BACKGROUND: Breast cancer is highly heterogeneous, suggesting that small but relevant subsets have been under-recognized. Rare and mainly triple-negative breast cancers (TNBCs) were recently found to exhibit tuft cell-like expression profiles, including POU2F3, the tuft cell master regulator. In addition, immunohistochemistry (IHC) has identified POU2F3-positive cells in the normal human breast, suggesting the presence of tuft cells in this organ. METHODS: Here, we (i) reviewed previously identified POU2F3-positive invasive breast cancers (n = 4) for POU2F3 expression in intraductal cancer components, (ii) investigated a new cohort of invasive breast cancers (n = 1853) by POU2F3-IHC, (iii) explored POU2F3-expressing cells in non-neoplastic breast tissues obtained from women with or without BRCA1 mutations (n = 15), and (iv) reanalyzed publicly available single-cell RNA sequencing (scRNA-seq) data from normal breast cells. RESULTS: Two TNBCs of the four previously reported invasive POU2F3-positive breast cancers contained POU2F3-positive ductal carcinoma in situ (DCIS). In the new cohort of invasive breast cancers, IHC revealed four POU2F3-positive cases, two of which were triple-negative, one luminal-type, and one triple-positive. In addition, another new POU2F3-positive tumor with a triple-negative phenotype was found in daily practice. All non-neoplastic breast tissues contained POU2F3-positive cells, irrespective of BRCA1 status. The scRNA-seq reanalysis confirmed POU2F3-expressing epithelial cells (3.3% of all epithelial cells) and the 17% that co-expressed the other two tuft cell-related markers (SOX9/AVIL or SOX9/GFI1B), which suggested they were bona fide tuft cells. Of note, SOX9 is also known as the "master regulator" of TNBCs. CONCLUSIONS: POU2F3 expression defines small subsets in various breast cancer subtypes, which can be accompanied by DCIS. The mechanistic relationship between POU2F3 and SOX9 in the breast warrants further analysis to enhance our understanding of normal breast physiology and to clarify the significance of the tuft cell-like phenotype for TNBCs.