A baseline CTC count ≥2 CTCs/7.5 mL was associated with worse prognosis. However, some improvement in PFS and OS was shown with concurrent XT, thus baseline CTC could be a predictive marker. As the current trial was not designed to evaluate a change in chemotherapy according to on-treatment CTC changes, prospective investigation is required.

Objective: The aim of this study was to elucidate the clinicopathological characteristics of breast ductal carcinomas in situ (DCIS) with aldehyde dehydrogenase 1 (ALDH1)-positive cancer stem cells (CSCs). Methods: DCIS (n = 194) were subjected to immunohistochemical staining and results were examined for associations with various clinicopathological parameters. The ALDEFLUOR assay for breast cancer cell lines was also performed to determine the proportion of CSCs according to intrinsic subtype. Results: DCIS with ALDH1-positive CSCs were significantly (p < 0.05) more likely to be estrogen receptor-α (ER)-negative, progesterone receptor (PgR)-negative, Ki67-positive and HER2-positive tumors. Luminal A subtype (8.6%) showed a significantly (p < 0.001) lower ALDH1 positivity than the other subtypes [luminal B (50.0%), luminal HER2 (36.8%), HER2 (35.3%) and triple-negative subtype (26.7%)]. Double immunostaining revealed that ALDH1-positive CSCs did not overlap with ER-, PgR- or Ki67-positive tumor cells but did overlap with HER2-positive tumor cells. The percentage of ALDEFLUOR-positive CSCs was lower in the luminal A cell lines (0.02% for T-47D and 0.4% for MCF7) than in the luminal HER2 (9.1% for BT-474 and 9.5% for MDA-MB-361), HER2 (13.9% for AU565 and 33.2% for SK-BR-3) and triple-negative cell lines (28.4% for MDA-MB-231 and 30.7% for MDA-MB-468). Conclusions: Our results suggest that most CSCs in DCIS are in the G0 phase (Ki67 negative) and do not express ER or PgR, but they do express HER2 in HER2-positive tumors.

(Background) Cyclin-dependent kinase 4 and 6 inhibitors (CDK4/6i) improve the prognosis of hormone receptor-positive HER2-negative breast cancer (HR+/HER2- BC) by approximately 5 years. However, some patients show resistance to CDK4/6i and have poor prognosis. Thus, predicting resistance in patients is important. Although PAM50 is a strong tool for predicting late recurrence risk in HR+/HER2- BC by analyzing gene expression signatures, it is not always available. The non-luminal disease score (NOLUS), developed as an approximate formula for PAM50, is a pathology-based subtyping assay used to predict non-luminal disease using immunohistochemical analysis (Pascual et al. Front Oncol, 2021). (Materials & Methods) This multicenter, retrospective observational study was approved by the central ethics committee of Gifu University. From December 2017 to December 2021, real-world data of patients with metastatic HR+/HER2- BC who received CDK4/6i therapy were collected from 11 institutes in Japan. Data were obtained for patients who received CDK4/6i, such as palbociclib (PAL) or abemaciclib (ABE), as the first- or second-line endocrine therapy. The association between the efficacy of CDK4/6i and NOLUS was investigated by evaluating pathological and clinical data, including progression-free survival (PFS) and overall survival (OS). Pathological data, including the expression levels of ER, PgR, HER2, and Ki67, were evaluated according to the ASCO/CAP guidelines by experienced pathologists in each institute using either primary or metastatic tumors. PFS was defined as the period from the 1) starting date of combination therapy to progressive disease (PD); 2) the starting date of combination therapy to PD when CDK4/6i was interrupted due to adverse events or patients’ preference; and 3) the starting date of endocrine monotherapy to PD when CDK4/6i was added. NOLUS was calculated using the formula: NOLUS (0-100) = -0.45*ER% − 0.28*PR% + 0.27*Ki67% + 73, and the patients were divided into two groups, NOLUS(+) [≥ 51.38, non-luminal disease] and NOLUS(−) [&lt; 51.38, luminal disease]. The expression levels of ER, PgR, HER2, and Ki67 in each group were compared using Wilcoxon rank-sum and Fisher’s exact tests. Next, prognosis, including survival rate, PFS, and OS, was evaluated with a 95% confidence interval (CI) using the Kaplan–Meier method with the log-rank test. Statistical significance was set at p &lt; 0.05. (Results) Of the 300 patients, 28 (9.3%) were NOLUS(+) and 272 (90.7%) were NOLUS(−). The expression rates (%) in NOLUS(+) and NOLUS(−) were, respectively, 28.2 ± 19.4 and 89.0 ± 11.3 for ER (p &lt; 0.001); 6.3 ± 15.9 and 44.3 ± 37.9 for PgR (p &lt; 0.001); and 42.5 ± 23.8 and 26.9 ± 19.1 for Ki67 (p &lt; 0.001). The expressions of HER2 (score 0, 1, 2, and ISH-negative, 3) were 42.9%, 28.6%, 28.6%, and 0% for NOLUS(+); and 30.8%, 51.7%, 17.5%, and 0.4% for NOLUS(−) (p = 0.086). There were apparent statistical differences in prognosis between the NOLUS(+) and NOLUS(−) groups. PFSs for 6M and 1y were 71.4% and 30.5% for NOLUS(+), and 85.2% and 66.6% for NOLUS(−) (HR, 3.15; 95%CI: 2.02-4.93; p &lt; 0.001). OS for 6M and 1y were 92.6% and 92.6% for NOLUS(+), and 97.7% and 93.8% for NOLUS(−) (HR, 3.01; 95%CI: 1.48-6.09, p = 0.001). NOLUS(−) patients showed statistically better PFS with first-line therapy than with second-line therapy. However, NOLUS(+) patients showed no prognostic difference between the first and second therapeutic lines, suggesting CDK4/6i inefficacy. (Conclusion) CDK4/6i efficacy and prognosis were significantly different between NOLUS(+) and NOLUS(−) patients. This feasible method can predict patients with CDK4/6i-resistance and help select a better therapeutic approach to overcome resistance. Citation Format: Manabu Futamura, Takahiro Nakayama, Tersuhiro Yoshinami, Chiya Oshiro, Mikiya Ishihara, Midori Morita, Akira Watanabe, Azusa Taniguchi, Masami Tsukabe, Masafumi Shimoda, Kanae Mitta, Yoko Chihara, Hiroyuki Yasojima, Yoshimi Ouchi, Yoshihisa Tokumaru, Takuma Ishihara, Norikazu Masuda. Detection of high-risk patients resistant to CDK4/6 inhibitors with hormone receptor-positive HER2-negative breast cancer in Japan [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-02-05.

ESR1 mutations in breast cancer are one of the mechanisms of resistance to aromatase inhibitors. These mutations are common in metastatic breast cancer; however, these are rare in primary breast cancer. However, these data have been analyzed mainly in formalin-fixed, paraffin-embedded tissue; thus, rare mutations that may be present in primary breast cancer may be overlooked. In this study, we developed a highly sensitive mutation detection method called locked nucleic acid (LNA)-clamp droplet digital PCR (ddPCR) and validated it. The mutation detection sensitivity was substantiated to 0.003%. Then, we used this method to analyze ESR1 mutations in fresh-frozen (FF) tissues of primary breast cancer. cDNA extracted from the FF tissues of 212 patients with primary breast cancers were measured. Twenty-eight ESR1 mutations were found in twenty-seven (12.7%) patients. Sixteen (7.5%) patients had Y537S mutations and twelve (5.7%) had D538G mutations. Two mutations with a variant allele frequency (VAF) of ≥0.1% and twenty-six mutations with a VAF of <0.1% were found. By using this LNA-clamp ddPCR, this study demonstrated the presence of minor clones with a VAF of <0.1% in primary breast cancer.