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Breast cancer resistance protein (BCRP), also known as ATP-binding cassette transporter G2 (ABCG2), is associated with chemotherapy resistance. BCRP is also implicated in breast cancer stem cells and has been reported as a poor prognostic factor. However, the relationship of BCRP levels in breast cancer tissues with chemotherapy resistance and prognosis has not been clarified to date. Our laboratory recently developed nanoparticles suitable for the quantitative immunohistochemical (IHC) method called phosphor-integrated dots (PIDs). Using PID-based IHC (IHC-PIDs), many biomarker proteins can be visualized and quantitatively analyzed at the single-particle level in paraffine-embedded, formalin-fixed tumor samples. We aimed to evaluate the correlation between quantitatively analyzed BCRP expression and prognosis in breast cancer tissue samples using immunohistochemistry with IHC-PIDs. Thirty-seven Japanese patients diagnosed with invasive ductal carcinoma of the breast who underwent mastectomy and lymph node dissection at Tohoku University Hospital (Sendai, Japan) between 2004 and 2010 were included. All patients had undergone NAC and were pathologically diagnosed with non-pathological complete response or progressive disease and lymph node metastasis postoperatively. The patients were followed up within a median of 10.1 years (range, 1.1–18.2 years). Among the thirty-seven patients, twenty-eight patients had core needle biopsy specimens taken before NAC. BCRP levels in breast cancer tissue and metastatic lymph nodes were quantitatively detected after preoperative chemotherapy and core needle biopsy. Biomarker assay with IHC-PIDs showed high accuracy for quantitative assessment of BCRP with low expression. The optimal PT+LN BCRP cut-off score for predicting survival was 27.4, with a sensitivity of 60.6% and a specificity of 77.3%. BCRP expression in PT+LN samples was dichotomized into high and low groups at this cutoff value, and OS was compared between these group. The results showed that OS was significantly worse in the high BCRP-expression group (log-rank p=0.0089). In conclusion, high BCRP levels are associated with poor prognosis in patients with breast cancer with residual tumors within the primary tumor and lymph nodes after preoperative chemotherapy. These findings provide a basis for further appropriate adjuvant therapy in these patients. Citation Format: Hiroshi Tada, Kohsuke Gonda, Narufumi Kitamura, Minoru Miyashita, Narumi Harada-Shoji, Yohei Hamanaka, Akiko Ebata, Takanori Ishida. Clinical significance of breast cancer resistance protein (BCRP) quantified by fluorescence nanoparticle in breast cancer patients undergoing neoadjuvant chemotherapy [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-05-15.

Background Triple-negative breast cancer (TNBC), the most heterogeneous type of breast cancer, has a high risk of recurrence and a shorter overall survival compared to the other subtypes. The ability of mitochondria to flexibly adapt to their microenvironment makes them crucial tumorigenesis mediators. in this study, we explored the role of mitochondrial dynamics in TNBC drug resistance and tumorigenesis.Methods The doxorubicin sensitivity of TNBC cell lines HCC38 and MDA-MB-231, as well as luminal cell line MCF-7, was determined using the Wst-8 test. Confocal microscopy and FACS were used to measure the mitochondrial membrane potential (ΔφM), mitophagy, and ROS generation of HCC38 and MDA-MB-231. Agilent Seahorse XF Analyzers measured metabolic characteristics of HCC38, MDA-MB 231and MCF-7. DRP1, Parkin, and p62 immunohistochemistry staining were performed by using both core needle biopsy samples and the samples removed at the surgery of 108 primary breast cancer patients who underwent surgery after neoadjuvant chemotherapy (NAC).Results MDA-MB-231, a doxorubicin resistant TNBC cell line, reduced ΔφM and enhanced mitophagy to maintain ROS production with OXPHOS-based metabolism. HCC38, a cell sensitive to doxorubicin, showed no change in ΔφM and mitophagy, with glycolysis as the primary metabolic characteristic and an uncontrolled rise in ROS production. Clinicopathological studies have found that DRP1 before NAC (pre-DRP1) was highly expressed in TNBC and groups with KI67 greater than 20% (p = 0.009, 0.016). Parkin was also highly expressed in HR- and high KI67 groups (p = 0.013 and 0.005) and the expression of p62 was higher only in high Ki67 groups (p = 0.003). There was a trend in the non-pCR group for DRP1 to decrease and p62 to increase after treatment, although the difference was not statistically significant.Conclusion Our findings showed that mitophagy, in association with OXPHOS metabolism, promoted chemotherapy resistance and tumor growth in TNBC. The lack of effective therapeutic options for patients with TNBC remains a major challenge, therefore, targeting unique mitochondria metabolic signatures within tumor cells can potentially offer another therapeutic strategy for this highly aggressive disease.

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D-2-hydroxyglutarate dehydrogenase (D2HGDH) catalyzes D-2-hydroxyglutarate to α-ketoglutarate and is involved in the regulation of cellular energy and biosynthetic intermediates. Previously, D2HGDH was reported to decrease 2-hydroxyglutarate level in breast carcinoma cells, but no other report has examined D2HGDH in breast carcinoma, and its significance remains unknown.We first immunolocalized D2HGDH in 224 invasive breast carcinomas and evaluated its clinicopathological significance. We next examined associations between gene expression of D2HGDH and α-ketoglutarate-dependent dioxygenases in 23 breast carcinoma tissues using the gene expression profile data. Finally, we examined the effects of D2HGDH on the proliferation in three breast carcinoma cells.D2HGDH immunoreactivity was detected in 49% of invasive breast carcinomas, and the immunohistochemical D2HGDH status was positively associated with histological grade, HER2 and Ki-67, while it was inversely associated with estrogen receptor. Moreover, it was significantly associated with worse prognosis of the breast cancer patients, and it turned out to be an independent prognostic factor for both the disease-free and breast cancer-specific survival in these patients. Gene expression profile data revealed that D2HGDH expression was positively associated with the expression of 6 α-ketoglutarate-dependent dioxygenases (KDM3A, PLOD1, EGLN2, ALKBH1, ASPH and ALKBH7). Consequent in vitro experiments demonstrated that D2HGDH overexpression significantly increased the cell proliferation activity of MCF-7, T47D and MDA-MB-231 cells.These results suggest that D2HGDH plays an important role in the growth of breast carcinoma, possibly through regulating functions of α-ketoglutarate-dependent dioxygenases, and that D2HGDH status is a potent worse prognostic factor in breast cancer patients.

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薬物性歯肉増殖症に対して原因薬物を変更し，歯周基本治療を行っていたところ，全顎的に著明な歯肉腫脹が出現した症例の紹介を受け，摂食困難な状況から抜歯および義歯装着により良好な機能回復が得られたので，その概要を報告する． 患者は82歳，女性で十数年前より歯肉腫脹を自覚し，年に数回近医歯科で歯周基本治療を受けていた．３年前，近医内科にて降圧薬をCa拮抗薬（アムロジピン）からアンジオテンシンⅡ受容体拮抗薬（オルメサルタン）へ変更したが症状の改善は認められなかった．その約３年後，全顎的に著明な歯肉腫脹が出現し，摂食困難となったため当科を受診した．初診時の口腔内所見では，全顎の歯槽部頬舌側に結節状で分葉状の著明な歯肉腫脹が認められ，左側上下顎臼歯部ではほぼ歯冠全体を被覆していた．歯肉表面は平滑で弾性やや硬であった．現在歯の動揺度は２～３度で，歯周ポケットは前歯部を除き８mm以上であった．血液検査と病理検査で腫瘍性病変は否定されたため，全身麻酔下にて現在歯すべての抜歯と，全顎にわたる歯肉切除術と歯肉整形術を併せて施行した．術後１か月から上下顎全部床義歯作製を開始し，その１か月後に完成した．術後１年以上経過しているが経過良好である．, In this study we report on a case of drug-induced gingival overgrowth exacerbated after changing thecausative drug, which was successfully treated. An 82-year-old woman was aware of gingival swelling ten years before and had been treated by periodontal initial therapy at a dental clinic several times a year. She changed from a calcium channel blocker (amlodipine) to an angiotensin II receptor blocker (olmesartan) by an internal medicine doctor three after, but there were no improvements of the symptoms. Three years later, she complained of an eating disorder caused by severe exacerbated gingival overgrowth and was referred to our department. During the first examination, marked nodular pedunculated gingival swelling of the whole alveolar region in both the labial and lingual sides of her lower and upper teeth was observed. Swollen gingiva had covered almost the entire crown of the left maxillary and mandibular molars. The gingival surface was smooth and its elasticity was relatively hard. Periodontal examination revealed that tooth mobility was within the 2-3 class and the probing depth was greater than 8 mm in all sites but the anterior teeth. As a malignant lesion was ruled out by a blood test and pathological examination, she underwent an operation to extract all of her teeth, gingivectomy, and gingivoplasty under general anesthesia. We began to fabricate maxillary and mandibular complete dentures one month after the operation. The dentures were fitted one month after the start of fabrication. There was no recurrence more than one year after operation.

In addition to genomic signaling, Estrogen receptor alpha (ERα) is associated with cell proliferation and survival through extranuclear signaling contributing to endocrine therapy (ET) resistance. However, the relationship between extranuclear ERα and ET resistance has not been extensively studied. We sought to measure extranuclear ERα expression by immunohistochemistry using phosphor-integrated dots (IHC-PIDs) and to assess its predictive value for ET resistance. After quantitative detection of ERα by IHC-PIDs in vitro, we developed “the nearest-neighbor method” to calculate the extranuclear ERα. Furthermore, tissue sections from 65 patients with HR+/HER2- BC were examined by IHC-PIDs, and the total ERα, nuclear ERα, extranuclear ERα PIDs score, and ratio of extranuclear-to-nuclear ERα (ENR) were measured using the novel method. We demonstrate that quantification of ERα using IHC-PIDs exhibited strong correlations to real-time qRT-PCR (r2 = 0.94) and flow cytometry (r2 = 0.98). High ERα ENR was significantly associated with poor overall survival (p = 0.048) and disease-free survival (DFS) (p = 0.007). Multivariate analysis revealed that the ERα ENR was an independent prognostic factor for DFS [hazard ratio, 3.8; 95% CI, 1.4–11.8; p = 0.006]. Our automated measurement has high accuracy to localize and assess extranuclear ERα. A high ERα ENR in HR+/HER2− BC indicates decreased likelihood of benefiting from ET.

Simple Summary L-type amino acid transporters such as LAT1 and LAT3 are associated with the uptake of essential amino acids. In particular, LAT1 regulates mammalian target of rapamycin complex 1 (mTORC1) signaling and cell proliferation by regulating leucine uptake. The purpose of this study is to clarify amino acid metabolism via LAT1 and LAT3 in breast cancer and the potential roles of LAT1 in the development of therapeutic resistance and clinical outcome of the patients. Results demonstrated that high LAT1 status was associated with tumor progression in breast cancer patients who received neoadjuvant hormone therapy (NAH), and LAT1 expression in the estrogen deprivation-resistant (EDR) breast carcinoma cell lines were upregulated. JPH203, a selective LAT1 inhibitor, demonstrated inhibitory effects on cell proliferation in the EDR cells. Therefore, LAT1 could serve not only as a prognosis biomarker but also a therapeutic target in estrogen receptor (ER)-positive breast cancer patients. Abstract The PI3K/Akt/mTOR pathway has been well known to interact with the estrogen receptor (ER)-pathway and to be also frequently upregulated in aromatase inhibitor (AI)-resistant breast cancer patients. Intracellular levels of free amino acids, especially leucine, regulate the mammalian target of rapamycin complex 1 (mTORC1) activation. L-type amino acid transporters such as LAT1 and LAT3 are associated with the uptake of essential amino acids. LAT1 expression could mediate leucine uptake, mTORC1 signaling, and cell proliferation. Therefore, in this study, we explored amino acid metabolism, including LAT1, in breast cancer and clarified the potential roles of LAT1 in the development of therapeutic resistance and the eventual clinical outcome of the patients. We evaluated LAT1 and LAT3 expression before and after neoadjuvant hormone therapy (NAH) and examined LAT1 function and expression in estrogen deprivation-resistant (EDR) breast carcinoma cell lines. Tumors tended to be in advanced stages in the cases whose LAT1 expression was high. LAT1 expression in the EDR cell lines was upregulated. JPH203, a selective LAT1 inhibitor, demonstrated inhibitory effects on cell proliferation in EDR cells. Hormone therapy changed the tumor microenvironment and resulted in metabolic reprogramming through inducing LAT1 expression. LAT1 expression then mediated leucine uptake, enhanced mTORC1 signaling, and eventually resulted in AI resistance. Therefore, LAT1 could be the potential therapeutic target in AI-resistant breast cancer patients.

Key Points Question Does the performance of adjunctive ultrasonography for breast cancer detection among women undergoing screening mammography change according to breast tissue density? Findings In this secondary analysis of a randomized clinical trial, screening mammography alone demonstrated low sensitivity, whereas adjunctive ultrasonography improved sensitivity both in dense and nondense breasts. Meaning These findings suggest that adjunctive ultrasonography has the potential to improve the detection of early-stage and invasive cancers across both dense and nondense breasts and to mitigate the flaws of mammographic screening., This secondary analysis of a randomized clinical trial evaluates the performance of adjunctive ultrasonography for breast cancer screening in asymptomatic women aged 40 to 49 years undergoing screening mammography according to differences in breast density., Importance Mammography has limited accuracy in breast cancer screening. Ultrasonography, when used in conjunction with mammography screening, is helpful to detect early-stage and invasive cancers for asymptomatic women with dense and nondense breasts. Objective To evaluate the performance of adjunctive ultrasonography with mammography for breast cancer screening, according to differences in breast density. Design, Setting, and Participants This study is a secondary analysis of the Japan Strategic Anti-cancer Randomized Trial. Between July 2007 and March 2011, asymptomatic women aged 40 to 49 years were enrolled in Japan. The present study used data from cases enrolled from the screening center in Miyagi prefecture during 2007 to 2020. Participants were randomly assigned in a 1:1 ratio to undergo either mammography with ultrasonography (intervention group) or mammography alone (control group). Data analysis was performed from February to March 2020. Exposures Ultrasonography adjunctive to mammography for breast cancer screening regardless of breast density. Main Outcomes and Measures Sensitivity, specificity, recall rates, biopsy rates, and characteristics of screen-detected cancers and interval breast cancers were evaluated between study groups and for each modality according to breast density. Results A total of 76 119 women were enrolled, and data for 19 213 women (mean [SD] age, 44.5 [2.8] years) from the Miyagi prefecture were analyzed; 9705 were randomized to the intervention group and 9508 were randomized to the control group. A total of 11 390 women (59.3%) had heterogeneously or extremely dense breasts. Among the overall group, 130 cancers were found. Sensitivity was significantly higher in the intervention group than the control group (93.2% [95% CI, 87.4%-99.0%] vs 66.7% [95% CI, 54.4%-78.9%]; P

Resistance to 5-Fluoruracil (5-FU) has been linked to elevated expression of the main target, thymidylate synthase (TYMS), which catalyses the de novo pathway for production of deoxythymidine monophosphate. The potent oncogenic forkhead box transcription factor, FOXM1 is is regulated by E2F1 which also controls TYMS. This study reveals a significant role of FOXM1 in 5-FU resistance. Overexpression and knock-down studies of FOXM1 in colon cancer cells suggest the importance of FOXM1 in TYMS regulation. ChIP and global ChIP-seq data also confirms that FOXM1 can also potentially regulate other 5-FU targets, such as TYMS, thymidine kinase 1 (TK-1) and thymidine phosphorylase (TYMP). In human colorectal cancer tissue specimens, a strong correlation of FOXM1 and TYMS staining was observed. Elevated FOXM1 and TYMS expression was also observed in acquired 5-FU resistant colon cancer cells (HCT116 5-FU Res). A synergistic effect was observed following treatment of CRC cells with an inhibitor of FOXM1, thiostrepton, in combination with 5-FU. The combination treatment decreased colony formation and migration, and induced cell cycle arrest, DNA damage, and apoptosis in CRC cell lines. In summary, this research demonstrated that FOXM1 plays a pivotal role in 5-FU resistance at least partially through the regulation of TYMS.