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The standard treatment for hormone receptor-positive breast cancer in good general condition is curative surgery followed by endocrine therapy. However, for older patients, endocrine therapy alone is sometimes chosen instead of curative surgery due to health conditions or personal preference, though this is not yet a standard approach. It is crucial to develop elderly-specific treatment strategies, potentially establishing endocrine therapy alone as a standard option. While endocrine therapy is generally effective, some patients show disease progression from the beginning due to de novo resistance. Hence, identifying such tumors is essential to determine who may benefit from endocrine therapy alone. Fifty-one patients aged over 70 years with estrogen receptor-positive and human epidermal growth factor receptor 2-negative invasive breast cancer who were treated with endocrine therapy instead of curative surgery were retrospectively investigated. Genes possibly related to de novo resistance to endocrine therapy were analyzed using a gene expression panel. Of the 51 patients, three patients showed progressive disease (PD) within 6 months of starting endocrine therapy. Gene expression analysis revealed that some genes, including those related to the cell cycle, such as CDKN3, were expressed at higher levels in the PD group compared with the non-PD group. Among these, CDKN3 retained significantly high expression in the PD group, even after analyzing more samples (log2 fold change, 1.99; P = 0.005). Public mRNA microarray data analysis revealed that patients with high CDKN3 tumors had worse outcomes. We identified several genes possibly involved in the de novo resistance to endocrine therapy. Our data indicate CDKN3 to be a predictive marker for de novo endocrine therapy resistance in older patients with breast cancer. We hope that our data will contribute to further research to establish tailored treatments for elderly breast cancer patients., Competing Interests: Declarations. Competing interests: The authors declare no competing interests. Ethical approval and consent to participate: This study was approved by the institutional review board of our hospital (no: H19-0289). The institutional review board waived the need for written informed consent due to the retrospective nature of the study. Patients could see the research plan on the hospital website and were offered the choice to opt out of the study at any time. This study was performed in accordance with the Declaration of Helsinki., (© 2024. The Author(s).)

Aims: In non-small cell lung cancer, alterations in mesenchymal-epithelial transition (MET) have been recognized as novel therapeutic targets. In particular, the MET exon 14 skipping mutation (METex14s) is a rare oncogenic driver. Targeted therapy with MET tyrosine kinase inhibitors has recently been approved for this mutation. However, c-MET expression and METex14s frequency and their clinicopathological effects in Japanese patients with breast cancer (BC) remain unknown., Methods: Tissue microarray-based immunohistochemistry (IHC) was performed to measure c-MET expression in 930 patients with BC (808 with invasive and 122 with noninvasive BC). Reverse transcription polymerase chain reaction was performed to analyse METex14s in patients exhibiting c-MET expression. Clinicopathological characteristics, including patient prognosis based on c-MET expression and METex14s, were elucidated., Results: IHC staining revealed c-MET expression in 91/930 (9.7%) patients. Notably, IHC expression was frequently observed in apocrine carcinomas (11/26 cases). Among the c-MET IHC-positive cases, METex14s frequency was 25.9% (14/54 cases) in invasive BC and 54.1% (20/37 cases) in noninvasive BC. Furthermore, 4/11 informative noninvasive and invasive BC cases with apocrine differentiation carried METex14s. The nuclear grade was significantly higher in the METex14s-positive group among invasive BC with c-met IHC expression. Furthermore, patients' age and negative rate for PgR IHC was significantly lower in the METex14s-positive group among noninvasive BC. Regarding the factors associated with patient outcomes, both c-MET IHC staining and METex14s expression did not affect survival, regardless of the hormone receptor status., Conclusion: High c-MET expression and METex14s are common in apocrine carcinoma., (© 2024 John Wiley & Sons Ltd.)

Background: Granulomatous mastitis is a relatively rare benign inflammatory disease of the breast, but it is sometimes difficult to distinguish from breast cancer by imaging. We experienced a case that was definitively diagnosed as granulomatous mastitis from the surgical specimen. The mass appeared as a large cystic lesion on imaging, which is unusual for granulomatous mastitis, and was initially suspected to be an encapsulated papillary carcinoma., Case Presentation: A 43-year-old woman presented with a painful mass in her right breast. Ultrasonography revealed a cystic mass lesion with internal solid components, with partially indistinct cyst walls and abundant blood flow. Additionally, lymphadenopathy of one axillary lymph node was observed. Magnetic resonance imaging findings showed irregularly spreading enhanced nodules within the cystic lesion, raising the suspicion of encapsulated papillary carcinoma. Although the histological findings from a needle biopsy were consistent with granulomatous mastitis, the possibility of malignancy could not be ruled out based on imaging, prompting a diagnostic probe lumpectomy. However, the surgical specimens did not reveal any tumorous lesions, and we reached a final diagnosis of granulomatous mastitis. Postoperatively, the patient was followed-up without steroid therapy and has been free from recurrence of mastitis for 22 months after surgery., Conclusions: We report a case of granulomatous mastitis that was detected as a large cystic lesion with a well-defined border on imaging and a definitive diagnosis was made from a surgical specimen., (© 2024. The Author(s).)

Background/aim: Oncotype DX Breast Recurrence Score ® test (ODx) is a gene profiling assay predicting the benefit of adjuvant chemotherapy for early-stage hormone receptor (HR)-positive and human epidermal growth factor receptor 2 (HER2)-negative breast cancer. Meanwhile, to avoid unnecessary financial burden on the patient, many studies have attempted to establish alternatives to ODx using conventional clinicopathological factors, but these have not yet been successful. Thus, we retrospectively investigated clinicopathological factors to establish alternatives to ODx., Patients and Methods: Data from 114 Japanese women who underwent ODx were retrospectively examined to investigate the relationship between ODx recurrence score (RS) and clinicopathological features, including MUC1 staining patterns on immunohistochemical assessment. An RS of 0-25 was defined as low, and 26-100 as high., Results: Ninety patients (79%) had low RS and 24 patients (21%) had high RS. Univariate analysis revealed that low tumor grade, high progesterone receptor (PgR) expression, and low Ki67 labeling index (LI) were significantly associated with low RS (p=0.025, p<0.001, and p<0.001, respectively). Tumors with an apical pattern of MUC1 staining also frequently had a low RS (p=0.024). In multivariate analysis, PgR expression and Ki67 LI were independent factors associated with RS (p<0.001, for both). When the ODx results were categorized with a combination of these two factors, only 2% of the PgR-high and Ki67-low group (one in 51 cases) had a high RS., Conclusion: PgR expression and Ki67 LI were independent factors correlated with RS. MUC1 staining pattern also has the potential to be a useful marker. We believe that it is crucial to continue attempts to identify patients who are unlikely to benefit from ODx., Competing Interests: The Authors have no potential conflicts of interest to disclose., (Copyright 2024, International Institute of Anticancer Research.)

Background: Additional surgical resection is recommended after breast-conserving surgery if the surgical margin is pathologically positive. However, in clinical practice, radiation therapy is sometimes used instead for several reasons. Irradiation may be appropriate for some patients, but real-world data is still insufficient to establish it as standard treatment. We retrospectively investigated the status of local control in patients who received irradiation for positive margins., Methods: We investigated 85 patients with positive margins after curative partial mastectomy who were treated with irradiation instead of additional excision during the period 2006-2013. The patients received whole-breast irradiation (43.2-50 Gy) using photon beams and additional tumour-bed boost (8.1-16 Gy) using electron beams. Intrabreast tumour recurrence was defined as secondary cancer within the ipsilateral conserved breast. Surgical margin was defined as positive if tumour cell exposure was pathologically confirmed on the margin., Results: Seven patients (8.2%) developed intrabreast tumour recurrence during a mean observation period of 119 months. As to components of positive margin, 76 cases were positive for an intraductal component, of which seven (9.2%) developed intrabreast tumour recurrence. Meanwhile, all nine cases positive for an invasive component were free from intrabreast tumour recurrence. Two of the intrabreast tumour recurrence cases seemed to develop new lesions rather than recurrence, considering tumour location. The cumulative incidence of intrabreast tumour recurrence over 10 years was 6.1%. Limited to true recurrence, intrabreast tumour recurrence incidence was 4.9%., Conclusion: Our real-world data supports irradiation as an alternative to additional surgical intervention for positive margins after breast-conserving surgery and offers a basis for further research., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background. Fibroadenoma (FA) and benign phyllodes tumor (PT) of the breast often have similar appearances on imaging. While an exact diagnosis of biopsy specimens is required to choose adequate treatment, including surgical procedures, it is sometimes difficult to pathologically differentiate these 2 tumors due to histological resemblances. To elucidate markers for distinguishing FA from benign PT, we analyzed clinical samples immunohistochemically. Methods. We retrospectively investigated 80 breast fibroepithelial lesions. As a discovery set, 60 surgical excision samples (30 FA and 30 benign PT) were examined. Twenty biopsy samples (10 FA and 10 benign PT) were examined as a validation set. To determine targets for immunohistochemistry, we first tested some proteins based on previous reports. As a result, Ki67 was chosen for differentiating FA and PT; thus further examinations were conducted with this protein. Results. Among the proteins examined, stromal Ki67 was significantly higher in PT than in FA. Benign PT had significantly higher stromal Ki67 expression both at random and at hotspots ( p  < .001 and <.001, respectively). The receiver operating characteristic curve analysis identified 3.5% and 8.5% (at random spots and hotspots, respectively) as the optimal cutoff values of stromal Ki67 for distinguishing between these 2 tumors. In the validation cohort employing needle biopsy specimens, we confirmed that these 2 cutoff values properly classified these 2 tumors ( p  = .043 and .029, respectively). Conclusion. We revealed that stromal Ki67 might be a potential marker for distinguishing FA from benign PT., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Purpose: Breast cancer often metastasizes to the central nervous system. Although the prognosis of brain metastases from breast cancer has been considered poor, and systemic therapy has not contributed to an improved prognosis, newer agents are expected to be more effective. BRCAness is defined as the status of homologous recombination deficiency (HRD) in tumor tissue, regardless of the presence of pathogenic germline BRCA1/2 variants. A study employing next-generation sequencing analysis showed that HRD was found relatively frequently in brain metastases of breast cancer patients. However, there have been no studies evaluating BRCAness in brain metastases of breast cancer with more efficient, rapid, and cost-effective methods., Methods: We retrospectively investigated 17 brain metastases of breast cancer that were surgically resected at our hospital from January 2007 to December 2022. Of these, samples from 15 patients were evaluable for BRCAness by employing multiplex ligation-dependent probe amplification (MLPA) assay., Results: Of the 15 patients, five patients (33%) had tumors with BRCAness. Clinicopathological factors of patients with brain metastases with BRCAness were not statistically different from those of patients who possessed tumors without BRCAness. Patients with brain metastases with BRCAness had shorter overall survival compared to those without BRCAness (BRCAness, median 15 months (95% CI 2-30) vs. non-BRCAness, median 28.5 months (95% CI 10-60); P = 0.013)., Conclusion: In this study, we evaluated BRCAness in brain metastases of breast cancer with the MLPA method, and found that about one-third of patients had BRCAness-positive tumors. The analysis of BRCAness using MLPA has the potential for practical clinical use., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: IgG4-related diseases are characterized by marked infiltration and IgG4-positive plasma cells and fibrosis, and involve multiple organs. However, IgG4-related mastitis is rare. We report a case of mastitis associated with IgG4-related disease., Case Presentation: A 78-year-old woman visited our hospital with a complaint of multiple subcutaneous nodules. A biopsy of a dorsal subcutaneous mass was performed but did not yield a definitive diagnosis. However, blood tests showed a high level of IgG4, thus she was referred to the department of collagen disease for further examination. Computed tomography (CT) showed a thickening of the skin of the right breast, and the patient was referred to our department. On physical examination, a large area of thickened skin was observed in the right breast without inflammatory breast cancer-like redness, and no mass was palpable. A needle biopsy was performed on an indistinct hypoechoic area in the breast, and she was diagnosed with mastitis associated with IgG4-related disease. Systemic steroid therapy was then administered and the symptoms of multiple skin nodules and mastitis improved., Conclusions: We reached the diagnosis based on a biopsy of the mammary gland enabling the patient to begin treatment for IgG4-related disease. This case was characterized by breast skin thickening, which is different from inflammatory breast cancer., (© 2023. The Author(s).)

Aims: Tyrosine kinase (TK) alterations, such as anaplastic lymphoma kinase (ALK) fusion, neurotrophic tyrosine receptor kinase (NTRK) fusion, c-ros oncogene 1 (ROS1) fusion and mesenchymal-epithelial transition factor (MET) exon 14 skipping, have been reported in colorectal cancers (CRC). We have previously reported CRCs with NTRK fusion among our cohort. However, their clinicopathological features have not been fully elucidated., Methods and Results: Tissue microarray (TMA)-based immunohistochemistry (IHC) was performed on 951 CRC lesions from 944 patients. IHC was evaluated as positive or negative for ALK and ROS1 and 0 to 3+ for c-MET. For ALK and ROS1 IHC-positive cases, RNA-based imbalanced gene expression assays, Archer FusionPlex assays and reverse transcription-polymerase chain reaction (RT-PCR) followed by Sanger sequencing were performed. For c-MET IHC 3+ cases, RT-PCR followed by Sanger sequencing were performed. ALK IHC was positive in three cases (0.2%) and all showed imbalanced ALK gene expression. The following ALK fusions were confirmed: EML4 (exon 21)::ALK (exon 20), EML4 (exon 6)::ALK (exon 19) and HMBOX1 (exon 6)::ALK (exon 20). Two showed microsatellite instability-high/mismatch repair (MMR)-deficient, and all were located in the right colon. ROS1 IHC was positive in one case; however, imbalanced expression and ROS1 fusion was negative. Forty-two cases (4.4%) showed c-MET IHC3+. MET exon 14 skipping was confirmed in nine cases. All cases were microsatellite stable/MMR-proficient, and eight were located in the left colon and rectum., Conclusions: CRCs with these TK alterations had distinct clinicopathological features. Together with our previous study, 15 cases (1.6%) harboured targetable TK alterations (three NTRK fusion, three ALK fusion, nine MET exon 14 skipping)., (© 2023 John Wiley & Sons Ltd.)

Heparanase (HPSE) is the only mammalian endo -β-glucuronidase known to catalyze the degradation of heparan sulfate. Dysfunction of HPSE activity has been linked to several disease states, resulting in HPSE becoming the target of numerous therapeutic programs, yet no drug has passed clinical trials to date. Pentosan polysulfate sodium (PPS) is a heterogeneous, FDA-approved drug for the treatment of interstitial cystitis and a known HPSE inhibitor. However, due to its heterogeneity, characterization of its mechanism of HPSE inhibition is challenging. Here, we show that inhibition of HPSE by PPS is complex, involving multiple overlapping binding events, each influenced by factors such as oligosaccharide length and inhibitor-induced changes in the protein secondary structure. The present work advances our molecular understanding of the inhibition of HPSE and will aid in the development of therapeutics for the treatment of a broad range of pathologies associated with enzyme dysfunction, including cancer, inflammatory disease, and viral infections.

Subtypes of small cell lung carcinoma (SCLC) are defined by the expression of ASCL1, NEUROD1, and POU2F3 markers. The aim of our study was to explore the extent to which the intratumoral heterogeneity of ASCL1, NEUROD1, and POU2F3 may lead to discrepancies in expression of these markers in surgical samples and their matched tissue microarray (TMA) and lymph node (LN) metastatic sites. METHODS AND RESULTS: The cohort included 77 patients with SCLC. Immunohistochemical examinations were performed on whole slides of the primary tumour, paired TMAs, and metastatic LN sites. Samples with H-scores >50 were considered positive. Based on the ASCL1, NEUROD1, and POU2F3 staining pattern, we grouped the tumours as follows: ASCL1-dominant (SCLC-A), NEUROD1-dominant (SCLC-N), ASCL1/NEUROD1 double-negative with POU2F3 expression (SCLC-P), and negative for all three markers (SCLC-I). In whole slides, 40 SCLC-A (52%), 20 SCLC-N (26%), 15 SCLC-P (20%), and two SCLC-I (3%) tumours were identified. Comparisons of TMAs or LN metastatic sites and corresponding surgical specimens showed that positivity for ASCL1, NEUROD1, and POU2F3 in TMAs (all P < 0.0001) or LN metastatic sites (ASCL1, P = 0.0047; NEUROD1, P = 0.0069; POU2F3, P < 0.0001) correlated significantly with that of corresponding surgical specimens. CONCLUSION: The positivity for these markers in TMAs and LN metastatic sites was significantly correlated with that of corresponding surgical specimens, indicating that biopsy specimens could be used to identify molecular subtypes of SCLC in patients., (© 2023 John Wiley & Sons Ltd.)

Adjuvant chemotherapy has played a major role in the treatment of hormone receptor-positive breast cancer for many years. To better determine which patient subsets need adjuvant chemotherapy, various gene expression analyses have been developed, but cost-effective tools to identify such patients remain elusive. In the present report, we retrospectively investigated immunohistochemical expression and subcellular localization of MUC1 in primary tumors and examined their relationship to tumor malignancy, chemotherapy effect and patient outcomes. We retrospectively examined three patient cohorts with hormone receptor-positive/human epidermal growth factor receptor 2-negative invasive breast cancer: 51 patients who underwent 21-gene expression analysis (multi-gene assay-cohort), 96 patients who received neoadjuvant chemotherapy (neoadjuvant chemotherapy-cohort), and 609 patients whose tumor tissue was used in tissue-microarrays (tissue-microarray-cohort). The immunohistochemical staining pattern of the anti-MUC1 monoclonal antibody, Ma695, was examined in cancer tissues, and subcellular localization was determined as apical, cytoplasmic or negative. In the multi-gene assay-cohort, tumors with apical patterns had the lowest recurrence scores, reflecting lower tumor malignancy, and were significantly lower than MUC1-negative tumors (P = 0.038). In the neoadjuvant chemotherapy-cohort, there was no correlation between MUC1 staining patterns and effects of chemotherapy. Finally, in the tissue-microarray-cohort, we found that patients with apical MUC1 staining patterns had significantly longer disease-free-survival and overall survival than other patterns (P = 0.020 and 0.039, respectively). Our data suggest that an apical MUC1 staining pattern indicates luminal A-likeness. Assessment of the subcellular localization of MUC1 glycoprotein may be useful for identifying patients who can avoid adjuvant chemotherapy., (© 2023. The Author(s).)

Background: With the improvement of optimal perioperative drug therapy for breast cancer patients, physicians now have to treat the adverse effects and comorbidities associated with long-term treatments. We report a case who suffered cardiac arrest due to acute myocarditis developed after initiation of adjuvant treatment., Case Presentation: After completing preoperative chemotherapy and undergoing curative surgery for right breast cancer, a 46-year-old female patient started adjuvant tamoxifen and resumed trastuzumab. Two months later, she complained fever and dyspnea. Blood tests showed a marked increase in hepatic enzymes, and the patient was rushed to our emergency room on suspicion of drug-induced liver injury. In the emergency room, the patient went into cardiac arrest shortly after tachycardia with ST-segment elevation appeared on the monitored electrocardiogram. Resuscitation was started immediately and tracheal intubation, intra-aortic balloon pumping, and extracorporeal membrane oxygenation were started. Coronary angiography results were negative for ischemic heart disease. A diagnosis of fulminant myocarditis was made and steroid pulse therapy and immunoglobulin therapy were started. After the start of treatment, the symptoms of heart failure improved steadily and the patient was discharged on the 28th day. Histological findings of the myocardial biopsy revealed degeneration and necrosis of myocardial cells with marked lymphocytic infiltration, consistent with the histology of lymphocytic myocarditis. Serum cytomegalovirus, coxsackie B virus and adenovirus antibodies were all elevated and these findings were consistent with acute viral myocarditis., Conclusions: We report a case with strong indications for therapy-induced liver damage, who was ultimately diagnosed with acute viral myocarditis and successfully treated with multidisciplinary therapy. We believe that our findings would be useful for other clinicians in managing similar patients., (© 2023. The Author(s).)

Photolysis under optimised flow conditions of metal carbonyls [{L n }M(CO) x ] [{L n }M(CO) x = Cr(CO) 6 , Mo(CO) 6 , W(CO) 6 , Mn(CO) 3 (η-C 5 H 4 Me), Re(CO) 3 (η-C 5 H 5 )] in tetrahydrofuran (THF) conveniently affords the synthetically versatile and labile solvento complexes [{L n }M(CO) x -1 (THF)], thereby obviating many of the caveats associated with photochemical syntheses using either 'batch' or falling film techniques. Conversions were optimised and yields assayed by a combination of in situ infrared spectroscopy and derivatisation as the corresponding triphenylphosphine complexes [{L n }M(CO) x -1 (PPh 3 )].

Background: Rating lymphocytes (TILs) are a prognostic marker in breast cancer and high TIL infiltration correlates with better patient outcomes. Meanwhile, parameters involving immune cells in peripheral blood have also been established as prognostic markers. High platelet-to-lymphocyte ratios (PLRs) and neutrophil-to-lymphocyte ratios (NLRs) are related to poor outcomes in breast cancer, but their mechanisms remain unknown. To date, TILs and these parameters have been examined separately., Methods: We investigated the relationship between TILs and the peripheral blood markers, PLR and NLR, in the same patients, using surgical specimens from 502 patients with invasive breast carcinoma without preoperative chemotherapy. For analysis of triple-negative breast cancer (TNBC) patient outcomes, 59 patients who received preoperative chemotherapy were also examined. For immune cell profiling, multiplexed fluorescent immunohistochemistry (mfIHC) of CD3, CD4, CD8, FOXP3 and T-bet, was conducted., Results: A positive correlation between PLR and TIL was observed in TNBC (P = 0.013). On mfIHC, tumors in patients with high PLR and NLR contained more CD3 + CD4 + FOXP3 + T-cells (P = 0.049 and 0.019, respectively), while no trend was observed in CD8 + T-cells. TNBC patients had different patterns of outcomes according to TIL and PLR, with the TIL-high/PLR-low group having the lowest rate of disease relapse and death, and the longest distant metastasis-free and overall survivals, while the TIL-low/PLR-high group had the shortest survivals., Conclusions: Our data suggest that the combination of PLR with TIL assessment may enable more accurate prediction of patient outcomes with TNBC., (© 2022. The Author(s).)

Background: We examined the relationship between the tumour microenvironment and the clinical efficacy of neoadjuvant chemotherapy in patients with cT2-4aN0M0 bladder cancer using multiplex fluorescence immunohistochemistry., Methods: The study retrospectively evaluated 51 patients who underwent radical cystectomy following neoadjuvant chemotherapy for cT2-4aN0M0 muscle-invasive bladder cancer. Patients were divided into responders (+ T cell (p = 0.0056) and CD204 + cell (p = 0.0394) densities were significantly higher in the intratumoural area in non-responders than in responders. Patients with higher CD204 + cell densities in cancerous areas had worse prognosis., Conclusions: This comprehensive analysis of the immune microenvironment of a muscle-invasive bladder cancer specimen revealed that preexisting tumour-infiltrating proliferating CD8 + T cells and CD204 + cells are indicators of the response to neoadjuvant chemotherapy and that CD204 + cells can be considered an unfavourable prognostic factor in these patients., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

The utility of ATP-dependent multi-enzymatic reactions is limited by their requirement for stoichiometric amounts of this expensive cofactor or additional purified enzymes for its recycling. Here we describe a simple method for the production of recombinant cell-free extracts (or lysates) of E. coli that support ATP-dependent biotransformations. The inexpensive preparation described is obtained with modest processing from a single recombinant bacterial culture of E. coli. In addition to recombinantly overexpressed enzymes that catalyze the primary ATP-dependent reactions of interest, endogenous kinases that are naturally present in the extract catalyze recycling of the requisite ATP. This means that only catalytic amounts of cofactor are necessary to drive the biotransformation, and without the requirement for additional purified enzymes. This approach has been applied successfully to an array of in vitro enzymatic cascades with multiple ATP-dependent steps., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Tumour-infiltrating lymphocyte (TIL)-high breast tumours have a high rate of pathological complete response (pCR) with neoadjuvant chemotherapy. In our routine pathological diagnoses of biopsy specimens from pCR cases, we have observed a high infiltration of plasma cells (PCs). A positive correlation of PCs with favourable patient outcome has recently been reported, but little is known about how PCs contribute to local tumour immunity., Methods: We retrospectively examined biopsy specimens from 146 patients with invasive breast cancer who received neoadjuvant chemotherapy. CD138 + PC infiltration was assessed by immunohistochemistry. Multiplexed fluorescent immunohistochemistry (mfIHC) with T and B cell markers was also conducted to elucidate the profile of immune cells., Results: Greater PC infiltration was observed in the pCR group (p = 0.028) and this trend was confirmed in another patient cohort. With mfIHC, we observed significantly more CD8 + , T-bet + CD4 + , and CD8 + FOXP3 + T cells, total B cells and PCs in pCR cases. Such cases were also characterised by high expression of both PD-1 and PD-L1 on B cells and PCs. In patients with hormone receptor-negative tumours, high PC infiltration was correlated with significantly longer disease-free survival (p = 0.034)., Conclusions: We found that higher PC infiltration in biopsy specimens before neoadjuvant chemotherapy was associated with pCR. With mfIHC, we also revealed that the local cytotoxic immune response was clearly enhanced in pCR cases, as was the infiltration of B cells including PCs. Moreover, higher PC levels were correlated with favourable outcomes in hormone receptor-negative breast cancer patients., (© 2021. The Author(s).)

Introduction: This study investigates the immune profile of the primary lung tumors and the corresponding brain metastasis from patients with NSCLC using multiplex fluorescence immunohistochemistry., Methods: The study evaluated 34 patients who underwent autopsy or surgical resection for brain metastasis and autopsy, surgical resection, or core biopsy for primary lung cancer. We compared the densities of various immune cells in the primary tumors and the brain metastases by multiplex fluorescence immunohistochemical analysis., Results: The density of CD4-positive (CD4 + ) T-cells, CD8-positive T-cells, and CD4 + Foxp3-positive T-cells were statistically higher in both tumor and stromal areas in primary lung cancer specimens when compared with brain metastases samples ( p < 0.0001). Only CD204-positive cells were statistically higher in the tumor areas of the brain metastases ( p  = 0.0118). Tumor-infiltrating lymphocytes associated with brain metastases positively correlated with overall survival, but primary lung tumor-infiltrating lymphocytes did not. The density of CD4 + and CD4 + Foxp3-positive T-cells in brain metastases with radiation was statistically higher in the carcinoma and stromal areas compared with those without radiation ( p  = 0.0343, p  = 0.0173)., Conclusions: Our findings that CD204-positive cells were higher in brain metastases may have broader implications for treatment as these macrophages may be immunosuppressive and make the immune environment less reactive. Furthermore, the finding that the density of CD4 + T-cells was higher in cancer and stroma areas of brain metastases after radiotherapy supports the addition of immunotherapy to radiation therapy in the treatment of brain metastases in NSCLC., (© 2021 THE AUTHORS.)

Introduction and Importance: Primary tumors of the pleura are rare, with malignant mesothelioma being the most common of these neoplasms. Pathological diagnosis of sarcomatoid mesothelioma can be more challenging than that of epithelioid malignant mesothelioma because of its similarities with true sarcomas and restricted or inconsistent expression of mesothelial markers in immunohistochemistry analysis., Presentation of Case: Here, we present an unusual case of malignant pleural mesothelioma concomitant with lung adenocarcinoma in a 72-year-old Japanese man, a smoker with no family history of cancer and asbestos exposure. Malignant pleural mesothelioma is composed of epithelial and spindle-shaped cells. Spindle-shaped cells with scant eosinophilic cytoplasm and hyperchromatic nuclei proliferated in abundant myxoid stroma containing thin-walled blood vessels, mimicking myxofibrosarcoma. The loss of BAP1 (BRCA1-associated protein 1) expression, as assessed by immunohistochemistry, and homozygous deletions of CDKN2A, detected using fluorescence in situ hybridization (FISH), were observed in both components. Targeted sequencing revealed that lung adenocarcinoma harbored EGFR mutations, whereas no mutations were detected in either component of biphasic mesothelioma., Discussion: Although alcian blue-stained mucins were detected in biphasic mesothelioma subsets, the clinicopathological significance of myxoid stroma in biphasic and sarcomatoid mesothelioma remains largely unknown., Conclusion: Our case presented a unique morphology mimicking myxofibrosarcoma in a sarcomatoid component of biphasic mesothelioma; therefore, it raises a question on the clinicopathological significance of myxoid stroma in sarcomatous areas of biphasic and sarcomatoid mesothelioma., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Although sulfated xylooligosaccharides are promising therapeutic leads for a multitude of afflictions, the structural complexity and heterogeneity of commercially deployed forms (e. g. Pentosan polysulfate 1) complicates their path to further clinical development. We describe herein the synthesis of the largest homogeneous persulfated xylooligomers prepared to date, comprising up to eight xylose residues, as standards for biological studies. Near quantitative sulfation was accomplished using a remarkably mild and operationally simple protocol which avoids the need for high temperatures and a large excess of the sulfating reagent. Moreover, the sulfated xylooligomer standards so obtained enabled definitive identification of a pyridinium contaminant in a sample of a commercially prepared Pentosan drug and provided significant insights into the conformational preferences of the constituent persulfated monosaccharide residues. As the spatial distribution of sulfates is a key determinant of the binding of sulfated oligosaccharides to endogenous targets, these findings have broad implications for the advancement of Pentosan-based treatments., (© 2021 Wiley-VCH GmbH.)

Aims: In the evolving era of precision medicine, increasing emphasis is placed on detecting molecular alterations driving the development of specific cancers and targeting them with matched therapies that can yield the best outcomes for patients. Lung adenocarcinomas with uncommon actionable alterations, including MET exon 14 skipping (METex14), ERBB2 and BRAF mutations, are rare and poorly characterised cancers., Methods and Results: To more clearly understand the histopathological features of lung adenocarcinoma with uncommon actionable alterations, we compared the histological features of 678 cases with mitogenic driver alterations from 996 surgically resected lung adenocarcinomas. Genomic data from our cohort revealed METex14, ERBB2 and BRAF mutations in 13, 16 and 15 cases, respectively. Patients who had lung adenocarcinoma with METex14 were often elderly females. Histological features such as clear cell features (23%), hyaline globules (31%) and nuclear pleomorphism (39%) were the most frequently identified in METex14-positive cases; among those, three cases (23%) had tumour cells with bizarre giant or multilobulated nuclei. Additionally, the micropapillary pattern was the most frequently identified in ERBB2-mutated lung adenocarcinoma (31%). Lung adenocarcinoma with BRAF mutations tended to be less invasive, and the BRAF V600E mutation was identified in only one case with lepidic adenocarcinoma. Immunohistochemically, all METex14, ERBB2 and BRAF-positive tumours, except for invasive mucinous adenocarcinoma, were positive for thyroid transcription factor 1 (TTF-1)., Conclusions: Our data from Japanese patients showed that lung adenocarcinoma with METex14 had unique clinicopathological characteristics: tumour cells with marked nuclear pleomorphism, hyaline globules and expression of TTF-1 in elderly women who never or lightly smoked., (© 2020 John Wiley & Sons Ltd.)

Background: Primary breast lymphoma is rare. Occurrence rates of malignant breast tumors in children are also quite low. We herein report a B-lymphoblastic lymphoma of the breast arisen in an adolescent girl. To the best of our knowledge, this is the youngest case with primary breast non-Hodgkin's lymphoma., Case Presentation: A 14-year-old Japanese girl felt a lump in her right breast and came to our hospital. A circumscribed soft mass, 30 mm in diameter, was palpable. Histological examination revealed atypical lymphoid cells diffusely spreading into the breast tissue. Based on results of immunohistochemistry and flow cytometry, her disease was diagnosed as B-lymphoblastic lymphoma (stage I). She was then referred to the pediatric department and received combination chemotherapy, based on a chemotherapy regimen for children with acute lymphoblastic leukemia. Following remission induction therapy, we confirmed no FDG uptake in the right breast on PET-CT scan., Conclusions: We have described a rare malignant lymphoma arising in the breast of an adolescent female. Histological assessment is necessary for diagnosis of breast lymphoma. However, it can be challenging with several reasons, and clinical information may contribute to the assessment. Moreover, treatments for lymphoma vary according to disease types. Thus, surgeons should collaborate closely with pathologists, pediatricians, and hematologists.

Peptides featuring an N-terminal cysteine residue and the unnatural amino acid 3-(2-cyano-4-pyridyl)alanine (Cpa) cyclize spontaneously in aqueous solution at neutral pH. Cpa is readily available and easily introduced into peptides using standard solid-phase peptide synthesis. The reaction is orthogonal to all proteinogenic amino acids, including cysteine residues that are not at the N-terminus. A substrate peptide of the Zika virus NS2B-NS3 protease cyclized in this way produced an inhibitor of high affinity and proteolytic stability.

Cyanuric acid is a metabolic intermediate of s -triazines, such as atrazine (a common herbicide) and melamine (used in resins and plastics). Cyanuric acid is mineralized to ammonia and carbon dioxide by the soil bacterium Pseudomonas sp. strain ADP via three hydrolytic enzymes (AtzD, AtzE, and AtzF). Here, we report the purification and biochemical and structural characterization of AtzE. Contrary to previous reports, we found that AtzE is not a biuret amidohydrolase, but instead it catalyzes the hydrolytic deamination of 1-carboxybiuret. X-ray crystal structures of apo AtzE and AtzE bound with the suicide inhibitor phenyl phosphorodiamidate revealed that the AtzE enzyme complex consists of two independent molecules in the asymmetric unit. We also show that AtzE forms an α2β2 heterotetramer with a previously unidentified 68-amino acid-long protein (AtzG) encoded in the cyanuric acid mineralization operon from Pseudomonas sp. strain ADP. Moreover, we observed that AtzG is essential for the production of soluble, active AtzE and that this obligate interaction is a vestige of their shared evolutionary origin. We propose that AtzEG was likely recruited into the cyanuric acid-mineralizing pathway from an ancestral glutamine transamidosome that required protein-protein interactions to enforce the exclusion of solvent from the transamidation reaction., Competing Interests: The authors declare that they have no conflicts of interest with the contents of this article., (© 2018 Esquirol et al.)

Carbamate kinases catalyze the conversion of carbamate to carbamoyl phosphate, which is readily transformed into other compounds. Carbamate forms spontaneously from ammonia and carbon dioxide in aqueous solutions, so the kinases have potential for sequestrative utilization of the latter compounds. Here, we compare seven carbamate kinases from mesophilic, thermophilic, and hyperthermophilic sources. In addition to the known enzymes from Enterococcus faecalis and Pyrococcus furiosus , the previously unreported enzymes from the hyperthermophiles Thermococcus sibiricus and Thermococcus barophilus , the thermophiles Fervidobacterium nodosum and Thermosipho melanesiensis , and the mesophile Clostridium tetani were all expressed recombinantly, each in high yield. Only the clostridial enzyme did not show catalysis. In direct assays of carbamate kinase activity, the three hyperthermophilic enzymes display higher specific activities at elevated temperatures, greater stability, and remarkable substrate turnover at alkaline pH (9.9 to 11.4). Thermococcus barophilus and Thermococcus sibiricus carbamate kinases were found to be the most active when the enzymes were tested at 80°C, and maintained activity over broad temperature and pH ranges. These robust thermococcal enzymes therefore represent ideal candidates for biotechnological applications involving aqueous ammonia solutions, since nonbuffered 0.0001 to 1.0 M solutions have pH values of approximately 9.8 to 11.8. As proof of concept, here we also show that carbamoyl phosphate produced by the Thermococcus barophilus kinase is efficiently converted in situ to carbamoyl aspartate by aspartate transcarbamoylase from the same source organism. Using acetyl phosphate to simultaneously recycle the kinase cofactor ATP, at pH 9.9 carbamoyl aspartate is produced in high yield and directly from solutions of ammonia, carbon dioxide, and aspartate. IMPORTANCE Much of the nitrogen in animal wastes and used in fertilizers is commonly lost as ammonia in water runoff, from which it must be removed to prevent downstream pollution and evolution of nitrogenous greenhouse gases. Since carbamate kinases transform ammonia and carbon dioxide to carbamoyl phosphate via carbamate, and carbamoyl phosphate may be converted into other valuable compounds, the kinases provide a route for useful sequestration of ammonia, as well as of carbon dioxide, another greenhouse gas. At the same time, recycling the ammonia in chemical synthesis reduces the need for its energy-intensive production. However, robust catalysts are required for such biotransformations. Here we show that carbamate kinases from hyperthermophilic archaea display remarkable stability and high catalytic activity across broad ranges of pH and temperature, making them promising candidates for biotechnological applications. We also show that carbamoyl phosphate produced by the kinases may be efficiently used to produce carbamoyl aspartate., (Copyright © 2018 American Society for Microbiology.)

The peptide hormone calcitonin is intimately connected with human cancer development and proliferation. Its biosynthesis is reasoned to proceed via glycine-, α-hydroxyglycine-, glycyllysine-, and glycyllysyllysine-extended precursors; however, as a result of the limitations of current analytical methods, until now, there has been no procedure capable of detecting these individual species in cell or tissue samples. Therefore, their presence and dynamics in cancer had not been established. Here, we report the first methodology for the separation, detection, and quantification of calcitonin and each of its precursors in human cancer cells. We also report the discovery and characterization of O-glycosylated calcitonin and its analogous biosynthetic precursors. Through direct and simultaneous analysis of the glycosylated and nonglycosylated species, we interrogate the hormone biosynthesis. This shows that the cellular calcitonin level is maintained to mitigate effects of biosynthetic enzyme inhibitors that substantially change the proportions of calcitonin-related species released into the culture medium.

The photoinduced interconversion between cinnamido-substituted cyclodextrins constitutes a gating switch through which the substituent moves to open or block access to the cyclodextrin cavity. Most unusually for a cyclodextrin-based device, the operation of this gate is solvent-independent and unaffected by potentially competitive guests. It occurs in MeOH and DMSO, as well as in water. This contrasts with other cyclodextrin inclusion phenomena that are usually driven by hydrophobic effects and limited to aqueous media., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The interactions of nineteen peptide substrates and fifteen analogous peptidomimetic glycolate inhibitors with human peptidylglycine α-amidating monooxygenase (PAM) have been investigated. The substrates and inhibitors are the prohormones of calcitonin and oxytocin and their analogues. PAM both secreted into the medium by and extracted from DMS53 small lung carcinoma cells has been studied. The results show that recognition of the prooxytocin and procalcitonin peptide sequences by the enzyme extends more than four and five amino acid residues, respectively, from their C-termini. This substrate sequence recognition is mirrored by increased inhibitor potency with increased peptide length in the glycolate peptidomimetics. Substitution of the C-terminal penultimate glycine and proline residues of prooxytocin and procalcitonin and their analogues with phenylalanine increases the enzyme binding affinity. However, this changes the binding mode from one that depends on peptide sequence recognition to another primarily determined by the phenylalanine moiety, for both the substrates and analogous glycolate inhibitors., (Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.)

Alzheimer's disease involves progressive neuronal loss. Linked to the disease is the amyloid β (Aβ) peptide, a 38-43-amino acid peptide found in extracellular amyloid plaques in the brain. Cyclodextrins are nontoxic, cone-shaped oligosaccharides with a hydrophilic exterior and a hydrophobic cavity making them suitable hosts for aromatic guest molecules in water. β-Cyclodextrin consists of seven α-d-glucopyranoside units and has been shown to reduce the level of fibrillation and neurotoxicity of Aβ. We have studied the interaction between Aβ and a β-cyclodextrin dimer, consisting of two β-cyclodextrin monomers connected by a flexible linker. The β-cyclodextrin monomer has been found to interact with Aβ(1-40) at sites Y10, F19, and/or F20 with a dissociation constant (K(D)) of 3.9 ± 2.0 mM. Here (1)H-(15)N and (1)H-(13)C heteronuclear single-quantum correlation nuclear magnetic resonance (NMR) spectra show that in addition, the β-cyclodextrin monomer and dimer bind to the histidines. NMR translational diffusion experiments reveal the increased affinity of the β-cyclodextrin dimer (apparent K(D) of 1.1 ± 0.5 mM) for Aβ(1-40) compared to that of the β-cyclodextrin monomer. Kinetic aggregation experiments based on thioflavin T fluorescence indicate that the dimer at 0.05-5 mM decreases the lag time of Aβ aggregation, while a concentration of 10 mM increases the lag time. The β-cyclodextrin monomer at a high concentration decreases the lag time of the aggregation. We conclude that cyclodextrin monomers and dimers have specific, modulating effects on the Aβ(1-40) aggregation process. Transmission electron microscopy shows that the regular fibrillar aggregates formed by Aβ(1-40) alone are replaced by a major fraction of amorphous aggregates in the presence of the β-cyclodextrin dimer.

Cyclodextrin [2]rotaxanes have been prepared by coupling dimethylanilines with dicarboxylic acids using DMT-MM, in aqueous solutions of alpha-cyclodextrin, and the example illustrated shows unusual fluorescence emission and other spectroscopic behavior characteristic of the formation of molecular wires in solution, similar to the fibers observed in the solid state.

6A-Deoxy-6A-(N-methyl-3-phenylpropionamido)-beta-cyclodextrin operates as a molecular machine, where the amide group serves as a torsion bar to harness the work output resulting from extraction of 1-adamantanol and consequent complexation of the aryl substituent by the cyclodextrin, when the latter behave as the piston and cylinder, respectively, of a molecular pump. At 25 degrees C, the complexation changes the ratio of the amide (Z)- and (E)-isomers from 2.4:1 to 25:1, on which basis the work performed on the amide bond is calculated to be 1.4 kcal mol-1. trans-6A-Deoxy-6A-(N-methylcinnamido)-beta-cyclodextrin and the cis isomer function as a more advanced version of the machine, with the alkene moiety serving as a photochemical on/off switch. Irradiation at 300 nm converts the trans cinnamide to the cis isomer, while the reverse process occurs at 254 nm. With the cis isomer there is little interaction of the phenyl group with the cyclodextrin cavity, so in that mode the machine is turned off. By contrast, complexation of the aryl substituent by the cyclodextrin occurs with the trans cinnamide and changes the ratio of the amide (Z)- and (E)-isomers from 2.6:1 to 100:1. Consequently, in this mode the machine is turned on, and the work harnessed by the amide bond is 2.1 kcal mol-1.

Fluorescence transfer across a donor-acceptor tagged rotaxane was studied and a small conformational change of the rotaxane observed using fluorescent spectroscopy and ROESY NMR.

The spinning rate of an encapsulated cyclophane guest is affected by the "size" of the coguests.

Molecular reactors are miniature vessels for the assembly of reactants at the molecular level, in order to change the nature of chemical transformations. It seems probable that those that will find most immediate applications are those that change product ratios or give products which would not readily form in the absence of the reactors, and thereby afford easy access to materials that are otherwise difficult to obtain. Molecular machines consist of interrelated parts with separate functions and perform some kind of work, at the molecular level. Practical examples are likely to be relatively uncomplicated and not based on individual functions of single-molecule devices. Instead they will probably rely on extensive redundancy of the molecular components and their interactions and reactions, as well as of the machines themselves.

The conformations of two [2]rotaxanes, each comprising alpha-cyclodextrin as the rotor, a stilbene as the axle and 2,4,6-trinitrophenyl substituents as the capping groups, have been examined in solution and in the solid state, using (1)H NMR spectroscopy and X-ray crystallography, respectively. In solution, introducing substituents onto the stilbene prevents the cyclodextrin from being localized over one end of the axle. Instead the cyclodextrin moves back and forth along the substituted stilbene. In the solid state, the axles of the rotaxanes form extended molecular fibres that are separated from each other and aligned along a single axis. The molecular fibres are strikingly similar to those formed by the axle component of one of the rotaxanes in the absence of the cyclodextrin, but in the latter case they are neither separated nor all aligned.

Eight new [2]rotaxanes have been prepared, incorporating an alpha-cyclodextrin as the rotor, a stilbene as the axle, and trinitrophenyl substituents as capping groups. Strategies have been devised to elaborate these by linking the rotor to the axle, to produce two new [1]rotaxanes. Rotational motion in a selection of these rotaxanes has been investigated through the application of two-dimensional NMR spectroscopy by performing TOCSY, DQF-COSY, ROESY and HMQC experiments. This has shown that a methoxyl group incorporated on the stilbene and a succinamide joining the stilbene and the cyclodextrin behave analogously to a ratchet tooth and pawl, respectively, to restrict rotation.

Human pepsinogen (PG) A and C were cloned in Escherichia coli, but the levels of expression were low and unstable. When there were fused to maltose-binding protein (MBP), the fusion proteins (MBP-PGA and MBP-PGC) were expressed as the major products. Although these fused products were almost totally recovered from the insoluble fraction, the renaturation and purification procedures were easy and simple. MBP-PGA and the PGA segment obtained by factor Xa digestion (designated as r-PGA) possessed proteolytic activities equivalent to native PGA purified from gastric tissue (t-PGA). For PGCs (MBP-PGC, r-PGC and t-PGC) also, the specific activities were almost the same. However, the activities of PGCs were about 3- to 4-hold higher than those of PGAs. In PGA and PGC immunoassay systems, r-PGs (r-PGA and r-PGC) and the EIA kit standard PGs (gastric mucosal PGs) exhibited a good correlation. From these results, r-PGs would seem to be applicable as assay standards without compromising the sensitivity of the immunoassay systems.

A fusion protein between green fluorescent protein (GFP) and neuron-specific enolase (NSE) was expressed in Escherichia coli. The GFP-NSE fusion protein migrated at 62 kDa in SDS-PAGE and retained the fluorescence under non-heating conditions. However, heat-denatured GFP-NSE was non-fluorescent and migrated at 74 kDa corresponding to the theoretical value. This suggests that the special structure of GFP, which is not denatured by SDS, influences its mobility in SDS-PAGE under non-heating conditions. The fluorescence intensity of GFP-NSE was measurable over a wide range by spectrophotometry or densitometry. The competitive immunoassay for NSE was performed using GFP-NSE as labeled antigen. Under our assay conditions, the working range of this system was about 2 -60 ng. This simple and rapid fluorescence immunoassay (FIA) using GFP-tagged antigen may be applicable to many protein markers.

125I-Labeled recombinant human neuron-specific enolase (R-NSE) was inadequate for RIA as a labeled antigen. The binding activity of labeled R-NSE to the antibody was markedly decreased. To supplement this defect and facilitate purification, we constructed two R-NSE derivatives, Y-NSE (one tyrosine residue was added at the N-terminal of R-NSE) and Y-NSE.H6 (six histidine residues were further added at the C-terminal of Y-NSE). The biochemical and immunochemical characteristics of these R-NSE derivatives were essentially the same to those of R-NSE. These derivatives were useful not only as standards for enzyme immunoassay (EIA), but also as labeled antigens for RIA. These results clearly indicate that the reactivity of these modified NSEs to anti-NSE antibody is almost equivalent to that of human brain gammagamma-enolase (B-NSE), and that even if the modified NSEs are labeled, they retain their binding affinities to antibodies in contrast to R-NSE.