FXYD3 functionally demarcates an ancestral breast cancer stem cell subpopulation with features of drug-tolerant persisters.

The heterogeneity of cancer stem cells (CSCs) within tumors presents a challenge in therapeutic targeting. To decipher the cellular plasticity that fuels phenotypic heterogeneity, we undertook single-cell transcriptomics analysis in triple-negative breast cancer (TNBC) to identify subpopulations in CSCs. We found a subpopulation of CSCs with ancestral features that is marked by FXYD domain-containing ion transport regulator 3 (FXYD3), a component of the Na+/K+ pump. Accordingly, FXYD3+ CSCs evolve and proliferate, while displaying traits of alveolar progenitors that are normally induced during pregnancy. Clinically, FXYD3+ CSCs were persistent during neoadjuvant chemotherapy, hence linking them to drug-tolerant persisters (DTPs) and identifying them as crucial therapeutic targets. Importantly, FXYD3+ CSCs were sensitive to senolytic Na+/K+ pump inhibitors, such as cardiac glycosides. Together, our data indicate that FXYD3+ CSCs with ancestral features are drivers of plasticity and chemoresistance in TNBC. Targeting the Na+/K+ pump could be an effective strategy to eliminate CSCs with ancestral and DTP features that could improve TNBC prognosis.

Clinical features and morphology of collagen fibrils in patients with vascular Ehlers-Danlos based on electron microscopy.

Background: Vascular-type Ehlers-Danlos syndrome (vEDS) is caused by collagen III deficit resulting from heterogeneous mutations in COL3A1, which occasionally causes sudden death due to arterial/visceral rupture. However, it is difficult to conduct basic research on the pathophysiology of vEDS. Moreover, the number of patients with vEDS is small, limiting the number of available samples. Furthermore, the symptoms of vEDS may vary among family members, even if they share the same mutation. Accordingly, many aspects of the pathology of vEDS remain unknown. Therefore, we investigated the structural abnormalities in collagen fibrils and endoplasmic reticulum (ER) stress in skin samples using electron microscopy as well as their relationship with clinical symptoms in 30 patients with vEDS (vEDS group) and 48 patients without vEDS (disease-negative control group). Methods: Differences between the two groups were evaluated in terms of the sizes of collagen fibrils using coefficient of variation (COV). Results: COV was found to be significantly higher in the vEDS group than in the disease-negative control group, indicating irregularity in the size of collagen fibrils. However, in the vEDS group, some patients had low COV and seldom experienced serious complications and ER stress. Conclusion: ER stress might affect collagen fibril-composing proteins. Moreover, as this stress varies among people based on environmental factors and aging, it may be the underlying cause of varying vEDS symptoms.

Prediction of pathological complete response to neoadjuvant chemotherapy in breast cancer using radiomics of pretreatment dynamic contrast-enhanced MRI.

PURPOSE: To investigate if the pretreatment dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI)-based radiomics machine learning predicts the pathological complete response (pCR) to neoadjuvant chemotherapy (NAC) in breast cancer patients. METHODS: Seventy-eight breast cancer patients who underwent DCE-MRI before NAC and confirmed as pCR or non-pCR were enrolled. Early enhancement mapping images of pretreatment DCE-MRI were created using subtraction formula as follows: Early enhancement mapping = (Signal 1 min - Signal pre)/Signal pre. Images of the whole tumors were manually segmented and radiomics features extracted. Five prediction models were built using five scenarios that included clinical information, subjective radiological findings, first order texture features, second order texture features, and their combinations. In texture analysis workflow, the corresponding variables were identified by mutual information for feature selection and random forest was used for model prediction. In five models, the area under the receiver operating characteristic curves (AUC) to predict the pCR and several metrics for model evaluation were analyzed. RESULTS: The best diagnostic performance based on F-score was achieved when both first and second order texture features with clinical information and subjective radiological findings were used (AUC = 0.77). The second best diagnostic performance was achieved with an AUC of 0.76 for first order texture features followed by an AUC of 0.76 for first and second order texture features. CONCLUSIONS: Pretreatment DCE-MRI can improve the prediction of pCR in breast cancer patients when all texture features with clinical information and subjective radiological findings are input to build the prediction model.

Case-based survey: The impact of breast-cancer treatment on fertility in Japan.

AIM: To assess the impact of breast-cancer treatment on fertility. METHODS: We conducted a retrospective, case-based survey of treatments administered for infertility and pregnancy outcomes after patients underwent treatment for breast cancer. Surveys were distributed to breast oncology facilities and reproductive endocrinology and infertility (REI) facilities. RESULTS: As high as 60% of the pregnancies in women under the age of 35 years occurred spontaneously. Additionally, the fertility rates decreased as age increased (under 35 years of age: 40%, 35-39 years of age: 21%, 40-44 years of age: 10%, respectively). In women who became pregnant after treatment for breast cancer, conception was achieved within 1 to 3 years after beginning to try for pregnancy. CONCLUSIONS: After treatment for breast cancer, women can expect spontaneous pregnancy, especially if they are under 35 years of age. It is important for patients 35 years of age and older to commence assisted reproductive technology in a timely manner when pursuing fertility after treatment for breast cancer.

A novel oral inhibitor for one-carbon metabolism and checkpoint kinase 1 inhibitor as a rational combination treatment for breast cancer.

Patients with triple-negative breast cancer have a poor prognosis as only a few efficient targeted therapies are available. Cancer cells are characterized by their unregulated proliferation and require large amounts of nucleotides to replicate their DNA. One-carbon metabolism contributes to purine and pyrimidine nucleotide synthesis by supplying one carbon atom. Although mitochondrial one-carbon metabolism has recently been focused on as an important target for cancer treatment, few specific inhibitors have been reported. In this study, we aimed to examine the effects of DS18561882 (DS18), a novel, orally active, specific inhibitor of methylenetetrahydrofolate dehydrogenase (MTHFD2), a mitochondrial enzyme involved in one-carbon metabolism. Treatment with DS18 led to a marked reduction in cancer-cell proliferation; however, it did not induce cell death. Combinatorial treatment with DS18 and inhibitors of checkpoint kinase 1 (Chk1), an activator of the S phase checkpoint pathway, efficiently induced apoptotic cell death in breast cancer cells and suppressed tumorigenesis in a triple-negative breast cancer patient-derived xenograft model. Mechanistically, MTHFD2 inhibition led to cell cycle arrest and slowed nucleotide synthesis. This finding suggests that DNA replication stress occurs due to nucleotide shortage and that the S-phase checkpoint pathway is activated, leading to cell-cycle arrest. Combinatorial treatment with both inhibitors released cell-cycle arrest, but induced accumulation of DNA double-strand breaks, leading to apoptotic cell death. Collectively, a combination of MTHFD2 and Chk1 inhibitors would be a rational treatment option for patients with triple-negative breast cancer.

Neural EGFL like 2 expressed in myoepithelial cells and suppressed breast cancer cell migration.

Breast tissue has a branching structure that contains double-layered cells, consisting primarily of luminal epithelial cells inside and myoepithelial cells outside. Ductal carcinoma in situ (DCIS) still has myoepithelial cells surrounding the cancer cells. However, myoepithelial cells disappear in invasive ductal carcinoma. In this study, we detected expression of neural EGFL like (NELL) 2 and one of its receptors, roundabout guidance receptor (ROBO) 3, in myoepithelial and luminal epithelial cells (respectively) in normal breast tissue. NELL2 also was expressed in myoepithelial cells surrounding the non-cancerous intraductal proliferative lesions and DCIS. However, the expression level and proportion of NELL2-positive cells in DCIS were lower than those in normal and non-cancerous intraductal proliferative lesions. ROBO3 expression was decreased in invasive ductal carcinoma compared to that in normal and non-cancerous intraductal proliferative lesions. An evaluation of NELL2's function in breast cancer cell lines demonstrated that full-length NELL2 suppressed cell adhesion and migration in vitro. In contrast, the N-terminal domain of NELL2 increased cell adhesion in the early phase and migration in vitro in some breast cancer cells. These results suggested that full-length NELL2 protein, when expressed in myoepithelial cells, might serve as an inhibitor of breast cancer cell migration.

Endoplasmic reticulum stress and collagenous formation anomalies in vascular-type Ehlers-Danlos syndrome via electron microscopy.

Vascular-type Ehlers-Danlos syndrome (vEDS) is an autosomal-dominant inherited disorder caused by a deficit in collagen III. It results from heterogeneous mutations in the α1 collagen III gene (COL3A1) and is associated with life-threatening complications, even in younger patients. However, the details of the pathogenesis underlying the COL3A1 mutation causing vEDS remain unclear. Here, we focus on anomalies in collagen fiber size and the endoplasmic reticulum (ER) stress response in patients with vEDS using electron microscopy (EM). We discovered that although the infants did not have vEDS, collagenous formations were similar to their samples in vEDS. Moreover, we examined the expression of activating transcription factor 6 (ATF6) as an ER stress marker and cartilage oligomeric matrix protein (COMP) as a binding partner protein for collagen fibrils in the dermis and COL3A1. The expression levels of ATF6 in the vEDS group were significantly higher than in infants and controls; COMP and COL3A1 levels were significantly lower. The fragile collagen fibrils in vEDS might form as a result of ER stress and that small, newly formed collagen fibrils may appear. This research revealed a novel prospect regarding an issue that has been unclear for a long time, which is the reason for the abnormal sizes of collagenous fibrils in vEDS.

MCM10 compensates for Myc-induced DNA replication stress in breast cancer stem-like cells.

Cancer stem-like cells (CSCs) induce drug resistance and recurrence of tumors when they experience DNA replication stress. However, the mechanisms underlying DNA replication stress in CSCs and its compensation remain unclear. Here, we demonstrate that upregulated c-Myc expression induces stronger DNA replication stress in patient-derived breast CSCs than in differentiated cancer cells. Our results suggest critical roles for mini-chromosome maintenance protein 10 (MCM10), a firing (activating) factor of DNA replication origins, to compensate for DNA replication stress in CSCs. MCM10 expression is upregulated in CSCs and is maintained by c-Myc. c-Myc-dependent collisions between RNA transcription and DNA replication machinery may occur in nuclei, thereby causing DNA replication stress. MCM10 may activate dormant replication origins close to these collisions to ensure the progression of replication. Moreover, patient-derived breast CSCs were found to be dependent on MCM10 for their maintenance, even after enrichment for CSCs that were resistant to paclitaxel, the standard chemotherapeutic agent. Further, MCM10 depletion decreased the growth of cancer cells, but not of normal cells. Therefore, MCM10 may robustly compensate for DNA replication stress and facilitate genome duplication in cancer cells in the S-phase, which is more pronounced in CSCs. Overall, we provide a preclinical rationale to target the c-Myc-MCM10 axis for preventing drug resistance and recurrence of tumors.

Nationwide survey of Japanese breast oncology and reproductive endocrinology departments about the impact of breast cancer treatment on fertility.

The purpose of this study is to assess the impact of breast cancer treatment on the reproductive potential. We conducted a nationwide survey of breast oncology and reproductive endocrinology and infertility (REI) departments using a questionnaire designed to assess the impact of breast cancer treatment on fertility. We received responses from 312 breast oncology departments (response rate, 31.9%) and 541 REI departments (response rate, 50.9%). The most common method of achieving pregnancy reported by breast oncology departments was natural insemination (69.6%), followed by assisted reproductive technology ( 15.6%) and intrauterine insemination (IUI; 14.8%). The most common method of achieving pregnancy reported by REI departments was conventional in vitro fertilization and/or intracytoplasmic sperm injection (51.0%), followed by natural insemination with or without ovulation induction (40.0%) and IUI (8.0%). The overall pregnancy rate for patients who underwent treatment for infertility at REI departments after breast cancer treatment was 39.0%. Vast patients who experienced breast cancer treatments conceived mainly by natural insemination based on the data from breast oncology departments. On the other hand, 61.0% of the patients who visited REI departments presumably due to infertility by natural insemination did not conceive even by infertility treatments with exclusive knowledge in REI departments.

Extravasated platelet aggregation contributes to tumor progression via the accumulation of myeloid-derived suppressor cells in gastric cancer with peritoneal metastasis.

Extravasated platelet aggregation (EPA) serves an important role in the cancer microenvironment during cancer progression, and has been demonstrated to interact with tumor cells in several types of cancer. EPA induces epithelial-mesenchymal transition (EMT) via transforming growth factor-ß, and also recruits immunosuppressive cells, including regulatory T (Treg) cells and myeloid-derived suppressor cells (MDSCs). However, the role of EPA in gastric cancer with peritoneal metastasis remains unknown. The present study analyzed the association between EPA and prognosis in patients with gastric cancer with peritoneal metastasis. The present study evaluated 62 patients diagnosed with advanced gastric cancer with peritoneal metastasis between 2001 and 2016. EPA, EMT, Treg cells and MDSCs in peritoneal metastatic lesions were detected by immunohistochemical evaluation of CD42b, SNAIL, FOXP3 and CD33, respectively. CD42b expression was observed in 56.5% (35/62) of peritoneal metastatic lesions. CD42b expression in peritoneal metastatic lesions was associated with poor overall survival compared with lower frequencies (hazard ratio, 2.03; 95% confidence interval, 1.12-3.69; P=0.018). SNAIL, FOXP3 and CD33 expression were not associated with overall survival, but CD33 expression was markedly higher in CD42b-positive patients (P=0.022). These results indicated that EPA affects immunosuppression by recruiting MDSCs in the tumor microenvironment via the secretion of soluble factors, resulting in tumor progression. EPA may be a novel therapeutic target for gastric cancer with peritoneal metastasis.

Anti-Inflammatory Effects of Potassium Iodide on SDS-Induced Murine Skin Inflammation.

Potassium iodide (KI), initially derived from seaweed in the early 19th century, is used for treating sporotrichosis in dermatological practice. KI has also been used to treat several noninfectious inflammatory skin diseases. However, the mechanisms underlying the improvement in such skin diseases remain unknown, and KI is not used widely. Thus, although KI is an old drug, physicians may not prescribe it frequently because they lack knowledge about it. Although KI is very inexpensive and causes few side effects, it has been superseded by new powerful and expensive drugs, such as biological agents. We applied 3% KI topically to areas of inflammation induced by SDS in mice. The levels of IL-1 and TNF-α gene expression were reduced, whereas that of IL-10 gene expression was increased. Small interfering RNA that was designed to reduce IL-10 gene expression levels was injected into the same mice, and the anti-inflammatory effects of KI were not observed. Thus, the pharmacologic action of KI is based on its anti-inflammatory effects caused by the increase in IL-10 levels. This information would increase dermatologists' awareness of KI as an efficacious and cost-effective treatment.

The relation between anti-TGBFR1 immunohistochemical reaction and low Ki67, small tumor size and high estrogen receptor expression in invasive breast cancer.

Most breast cancers are derived from the luminal epithelium, which composes the inside of the breast ductal structure. Ductal carcinoma in situ (DCIS) leads to invasive ductal carcinoma, but noncancerous intraductal proliferative lesions are also a risk factor for ductal carcinoma. The transforming growth factor beta (TGFB) signaling pathway behaves as a tumor suppressor in the early stage of cancer, and conversely as a tumor growth factor in invasive stages in several cancers. In this study, we performed immunohistochemistry with an antibody that detects the cytoplasmic region of TGFB receptor 1 (TGFBR1) and elucidated TGFBR1 protein expression in luminal epithelial cells of noncancerous breast ducts and in several cases of DCIS and invasive carcinoma. TGFBR1 expression was higher in noncancerous breast tissue than in cancerous tissue, and a difference in expression was also seen among histological subtypes. Comparing the expression level of TGFBR1 in cancer cells and clinico-pathological parameters, cases expressing low TGFBR1 tended to show low estrogen receptor expression, large tumor size (≥10 mm), and a high Ki67 labeling index. These data suggested that TGFBR1 protein expression may be related to the suppression of breast cancer cell growth.

Ovarian hyperstimulation closely associated with resumption of follicular growth after chemotherapy during tamoxifen treatment in premenopausal women with breast cancer: a multicenter retrospective cohort study.

BACKGROUND: We previously reported that tamoxifen (TAM)-induced ovarian hyperstimulation (OHS) is associated with high serum concentrations of estradiol in premenopausal women with breast cancer. To investigate risk factors for TAM-induced OHS, we performed a retrospective multicenter study. METHODS: Premenopausal patients who received surgical therapy for endocrine-dependent breast cancer (n = 235) were recruited in this study and classified into 4 groups: group A, treated with TAM alone; group B, TAM treatment after 2-year-combined therapy with a gonadotropin-releasing hormone (Gn-RH) agonist; group C, TAM treatment after chemotherapy; group D, 5-year-combined therapy with TAM and a Gn-RH agonist. A serum estradiol value of more than 300 pg/mL or mean follicular diameter of more than 30 mm was defined as OHS. RESULTS: The incidence of OHS in group A (n = 13/26, 50.0%) was significantly higher than those in group B (n = 17/63, 27.0%), group C (n = 20/110, 18.2%), and group D (n = 0/36, 0%). The incidence of OHS was significantly correlated with aging, and the median serum concentration of estradiol in the presence of OHS was 823.0 pg/mL. The incidence of OHS (less than 47 years old) was 62.5% in group A, 48.6% in group B, and 28.2% in group C, respectively. Notably, the incidence rate of OHS following amenorrhea in group C (n = 13/20, 65.0%) was significantly higher than that in group B (n = 1/17, 5.9%). CONCLUSIONS: These findings indicate that the onset of OHS following amenorrhea was common in the post-chemotherapeutic group, while its ratio was low in the group after Gn-RH analog treatment, suggesting that combined treatment-based management involving TAM therapy is necessary for premenopausal patients with breast cancer.

[A Case of Chemotherapy with Low-Dose Paclitaxel and Bevacizumab for Elderly Recurrent Breast Cancer Patient].

An 86-year-old woman underwent mastectomy with sentinel lymph node biopsy for cStage ⅡA breast cancer. The subtype of tumor was triple negative breast cancer. Pulmonary metastasis was found 1 month after surgery. Chemotherapy was done because of her good performance status(PS)and her hope. Administration of S-1 produced SD status of tumor for 8 months. However, NCC-ST-439 was increased and tumor size was enlarged. Therefore, the second line of chemotherapy by low-dose- biweekly paclitaxel and bevacizumab was planned because of her high age and good PS. Thereafter, tumor maker levels dramatically decreased and lung metastasis turned to be small. This therapy had been continued without any severe adverse events for 9 months. Unfortunately, this therapy was failed because of proteinuria, but pulmonary metastasis kept favorable efficacy during administration. Biweekly low-dose paclitaxel and bevacizumab therapy can be safe and effective therapy even for elderly patient with recurrent and metastatic breast cancer.

Diagnostic value of a nested polymerase chain reaction for diagnosing cutaneous sporotrichosis from paraffin-embedded skin tissue.

BACKGROUND: The gold standard for diagnosis of cutaneous sporotrichosis involves the isolation of the fungus, Sporothrix, by a culture test. Generally, the sampling for the culture test is performed at the same time as skin biopsy under local anaesthesia. However, the culture test may occasionally return a false negative result. OBJECTIVE: The aim of our study was to investigate the diagnostic value of a molecular method for diagnosing cutaneous sporotrichosis from formalin-fixed and paraffin-embedded (FFPE) tissues. METHODS: Over a 30-year period, we collected 52 cases of cutaneous sporotrichosis from biopsied specimens that had been positively diagnosed by a culture test. A nested PCR specific for Sporothrix detection was applied using FFPE tissue as template. The results were compared with control samples from 79 patients diagnosed with other cutaneous diseases according to histopathological, clinical findings and a cutler test. RESULTS: Of the 52 patients who were tested positive on the culture test, all cutaneous diseases were detected by PCR. Of the 59 patients in the control group, 58 tested negative by PCR. Under our conditions, the calculated sensitivity of this method was 100%, the specificity was 98.7% and the kappa coefficient was 0.984 (95% CI: 0.953-1.000). CONCLUSIONS: The specific PCR assay used appears to be a useful tool for the prompt and accurate diagnosis of sporotrichosis. Using this method, it would be possible to diagnose cutaneous sporotrichosis for patients who were suspected of cutaneous sporotrichosis but tested negative on culturing, and for pathologically suspected cutaneous sporotrichosis patients for whom the culture test was not undertaken.

[A Case of Histiocytoid Breast Carcinoma Accompanied by Multiple Axillary Lymph Node Metastases].

According to previous reports in our country, histiocytoid breast carcinoma is a very rare case of its tissue type, accounting for only 0.3% of all breast cancer cases. We have neither established a diagnostic standard nor a general idea for these tumors. Previously, its inherited pathology was assumed to be a sub-form of invasive lobular carcinoma. However, some researchers indicate the presence of components that are assumed to originate from milk ducts or differentiation in the apocrine gland. In this way, origins of pathologies for these tumors seem to be complex. Previous reports suggest cases of relatively long survival times without spreading to distant sites. Recently, we encountered one case of histiocytoid breast carcinoma accompanied by multiple axillary lymph node metastases.

[A Case of Thrombotic Microangiopathy Arising from Breast Cancer Treated with Chemotherapy].

The present study reports the case of a 49-year-old woman who was diagnosed with cancer of the left breast at the age of 43 years.Following chemotherapy, the patient had undergone partial mastectomy and axillary lymphadenectomy.Postoperatively, she underwent radiotherapy and hormone therapy.Five years and 4 months after the operation, the patient developed pain in the cervical vertebrae and was diagnosed with spinal metastasis.During the period, she began experiencing fatigue and hematological investigations indicated anemia, as well as thrombocytopenia, jaundice, and schistocytes.The patient was referred to our facility for further examination and treatment.On investigation, she was diagnosed with cancer-related thrombotic microangiopathy(TMA).The patient was advised to undergo chemotherapy due to which symptoms of TMA were relieved.She continued to receive chemotherapy for the following 3 years and 2 months until her death.

Amplicons in breast cancers analyzed by multiplex ligation-dependent probe amplification and fluorescence in situ hybridization.

Gene amplification is a common event in breast cancer, and identifies actual and potential targets of molecular therapy. The aim of the present study was to determine the amplification status of 22 genes that are reportedly frequently amplified in breast cancers. An archive of 322 formalin-fixed and paraffin-embedded invasive breast cancer tissues were screened by multiple ligation-dependent probe amplification (MLPA) and a total of 906 gene loci judged as 'gain' or 'amplified' was further confirmed to have been amplified based on fluorescence in situ hybridization (FISH). The results showed that 109 of 322 tumors (34%) displayed gene amplification of at least one of the 22 genes. The frequencies of the amplification of four regions containing known driver oncogenes were as follows: 8p11 (ZNF703, FGFR1, ADAM9, IKBKB), 8q24 (MYC), 11q13 (CCND1, C11ORF30), and 17q11-21 (CPD, MED1, ERBB2, CDC6, TOP2A, MAPT) exhibited amplification in 9.6%, 9.6%, 12.4%, and 12.1% of the tumors, respectively. In addition to homogeneously staining region- or double-minute chromosome-type amplifications, a centromere-associated-type amplification was found in nine tumors. Co-localization of the amplicon on 8p11 and the amplicon on 11q13 in single cells was found in 10 tumors, and in six of those tumors the two amplicons constituted single amplification units. Similarly, an amplicon consisting of ERBB2 and its flanking genes on 17q12-21 co-localized with an amplicon on 8p11 in 10 tumors and with the amplicon on 11q13 in five tumors. Thus, precise and feasible analysis of gene amplification status can be obtained using a combination of MLPA and FISH.