Outcomes in Nonmetastatic Hormone Receptor-Positive HER2-Negative Pure Mucinous Breast Cancer: A Multicenter Cohort Study.

BACKGROUND: Although considered a favorable subtype, pure mucinous breast cancer (PMBC) can recur, and evidence for adjuvant therapy is limited. We aimed to compare outcomes of nonmetastatic PMBC with invasive ductal carcinoma (IDC) and invasive lobular carcinoma (ILC) to address these uncertainties. METHODS: Individual patient-level data from 6 centers on stage I-III hormone receptor-positive and HER2-negative PMBC, IDC, and ILC were used to analyze recurrence-free interval (RFI), recurrence-free survival (RFS), and overall survival (OS), and to identify prognostic factors for PMBC. RESULTS: Data from 20,684 IDC cases, 1,475 ILC cases, and 943 PMBC cases were used. Median follow-up was 6.6 years. Five-year RFI, RFS, and OS for PMBC were 96.1%, 94.9%, and 98.1%, respectively. On multivariable Cox regression, PMBC demonstrated superior RFI (hazard ratio [HR], 0.59; 95% CI, 0.43-0.80), RFS (HR, 0.70; 95% CI, 0.56-0.89), and OS (HR, 0.71; 95% CI, 0.53-0.96) compared with IDC. ILC showed comparable outcomes to IDC. Fewer than half (48.7%) of recurrences in PMBC were distant, which was a lower rate than for IDC (67.3%) and ILC (80.6%). In contrast to RFI, RFS events were driven more by non-breast cancer deaths in older patients. Significant prognostic factors for RFI among PMBC included positive lymph node(s) (HR, 2.42; 95% CI, 1.08-5.40), radiotherapy (HR, 0.44; 95% CI, 0.23-0.85), and endocrine therapy (HR, 0.25; 95% CI, 0.09-0.70). No differential chemotherapy associations with outcomes were detected across PMBC subgroups by nodal stage, tumor size, and age. A separate SEER database analysis also did not find any association of improved survival with adjuvant chemotherapy in these subgroups. CONCLUSIONS: Compared with IDC, PMBC demonstrated superior RFI, RFS, and OS. Lymph node negativity, adjuvant radiotherapy, and endocrine therapy were associated with superior RFI. Adjuvant chemotherapy was not associated with better outcomes.

Quantifying payloads of antibody‒drug conjugates using a postcolumn infused-internal standard strategy with LC‒MS.

BACKGROUND: Antibody‒drug conjugates (ADCs) are innovative biopharmaceutics consisting of a monoclonal antibody, linkers, and cytotoxic payloads. Monitoring circulating payload concentrations has the potential to identify ADC toxicity; however, accurate quantification faces challenges, including low plasma concentrations, severe matrix effects, and the absence of stable isotope-labeled internal standards (SIL-IS) for payloads and their derivatives. Previous studies used structural analogs as internal standards, but different retention times between structural analogs and target analytes may hinder effective matrix correction. Therefore, a more flexible approach is required for precise payload quantification. RESULTS: We developed an LC‒MS/MS method incorporating a postcolumn-infused internal standard (PCI-IS) strategy for quantifying payloads and their derivatives of trastuzumab emtansine, trastuzumab deruxtecan, and sacituzumab govitecan, including DM1, MCC-DM1, DXd, SN-38, and SN-38G. Structural analogs (maytansine, Lys-MCC-DM1, and exatecan) were selected as PCI-IS candidates, and their accuracy performance was evaluated based on the percentage of samples within 80%-120% quantification accuracy. Compared to the approach without PCI-IS correction, exatecan enhanced the accuracy performance from 30-40%-100% for SN-38 and DXd, while maytansine and Lys-MCC-DM1 showed comparable accuracy for DM1 and MCC-DM1. This validated PCI-IS analytical method showed superior normalization of matrix effect in all analytes compared to the conventional internal standard approach. The clinical application of this approach showed pronounced differences in DXd and SN-38 concentrations before and after PCI-IS correction. Moreover, only DXd concentrations after PCI-IS correction were significantly higher in patients with thrombocytopenia (p = 0.037). SIGNIFICANCE: This approach effectively addressed the issue of unavailability of SIL-IS for novel ADC payloads and provided more accurate quantification, potentially yielding more robust statistical outcomes for understanding the exposure-toxicity relationship in ADCs. It is anticipated that this PCI-IS strategy may be extrapolated to quantify payloads and derivatives in diverse ADCs, thereby providing invaluable insights into drug toxicity and fortifying patient safety in ADC usage.

Experimental and analytical pipeline for sub-genomic RNA landscape of coronavirus by Nanopore sequencer.

Coronaviruses (CoVs), including severe acute respiratory syndrome coronavirus 2, can infect a variety of mammalian and avian hosts with significant medical and economic consequences. During the life cycle of CoV, a coordinated series of subgenomic RNAs, including canonical subgenomic messenger RNA and non-canonical defective viral genomes (DVGs), are generated with different biological implications. Studies that adopted the Nanopore sequencer (ONT) to investigate the landscape and dynamics of viral RNA subgenomic transcriptomes applied arbitrary bioinformatics parameters without justification or experimental validation. The current study used bovine coronavirus (BCoV), which can be performed under biosafety level 2 for library construction and experimental validation using traditional colony polymerase chain reaction and Sanger sequencing. Four different ONT protocols, including RNA direct and cDNA direct sequencing with or without exonuclease treatment, were used to generate RNA transcriptomic libraries from BCoV-infected cell lysates. Through rigorously examining the k-mer, gap size, segment size, and bin size, the optimal cutoffs for the bioinformatic pipeline were determined to remove the sequence noise while keeping the informative DVG reads. The sensitivity and specificity of identifying DVG reads using the proposed pipeline can reach 82.6% and 99.6% under the k-mer size cutoff of 15. Exonuclease treatment reduced the abundance of RNA transcripts; however, it was not necessary for future library preparation. Additional recovery of clipped BCoV nucleotide sequences with experimental validation expands the landscape of the CoV discontinuous RNA transcriptome, whose biological function requires future investigation. The results of this study provide the benchmarks for library construction and bioinformatic parameters for studying the discontinuous CoV RNA transcriptome.IMPORTANCEFunctional defective viral genomic RNA, containing all the cis-acting elements required for translation or replication, may play different roles in triggering cell innate immune signaling, interfering with the canonical subgenomic messenger RNA transcription/translation or assisting in establishing persistence infection. This study does not only provide benchmarks for library construction and bioinformatic parameters for studying the discontinuous coronavirus RNA transcriptome but also reveals the complexity of the bovine coronavirus transcriptome, whose functional assays will be critical in future studies.

A novel computer-assisted tool for 3D imaging of programmed death-ligand 1 expression in immunofluorescence-stained and optically cleared breast cancer specimens.

BACKGROUND: Programmed death-1 (PD-1) and programmed death-ligand 1 (PD-L1) are the two most common immune checkpoints targeted in triple-negative breast cancer (BC). Refining patient selection for immunotherapy is non-trivial and finding an appropriate digital pathology framework for spatial analysis of theranostic biomarkers for PD-1/PD-L1 inhibitors remains an unmet clinical need. METHODS: We describe a novel computer-assisted tool for three-dimensional (3D) imaging of PD-L1 expression in immunofluorescence-stained and optically cleared BC specimens (n = 20). The proposed 3D framework appeared to be feasible and showed a high overall agreement with traditional, clinical-grade two-dimensional (2D) staining techniques. Additionally, the results obtained for automated immune cell detection and analysis of PD-L1 expression were satisfactory. RESULTS: The spatial distribution of PD-L1 expression was heterogeneous across various BC tissue layers in the 3D space. Notably, there were six cases (30%) wherein PD-L1 expression levels along different layers crossed the 1% threshold for admitting patients to PD-1/PD-L1 inhibitors. The average PD-L1 expression in 3D space was different from that of traditional immunohistochemistry (IHC) in eight cases (40%). Pending further standardization and optimization, we expect that our technology will become a valuable addition for assessing PD-L1 expression in patients with BC. CONCLUSION: Via a single round of immunofluorescence imaging, our approach may provide a considerable improvement in patient stratification for cancer immunotherapy as compared with standard techniques.

Whole-Brain Radiotherapy Alone vs Preceded by Bevacizumab, Etoposide, and Cisplatin for Untreated Brain Metastases From Breast Cancer: A Randomized Clinical Trial.

Importance: The incidence of brain metastasis is increasing in patients with metastatic breast cancer. Treatments to extend the control of brain metastasis are urgently required. Objective: To investigate whether the addition of an induction treatment of bevacizumab, etoposide, and cisplatin (BEEP) improves brain-specific progression-free survival (PFS) after whole-brain radiotherapy (WBRT). Design, Setting, and Participants: This open-label, randomized, multicenter clinical trial assessed patients with brain metastases from breast cancer (BMBC) in Taiwan from September 9, 2014, to December 24, 2018, with survival follow-up until December 31, 2021. Key inclusion criteria included metastatic brain tumors not suitable for focal treatment, WBRT naivety, age 20 to 75 years, and at least 1 measurable brain metastatic lesion. The primary end point was brain-specific PFS, with an expected hazard ratio of 0.60, a 2-sided α ≤ .20, and power of 0.8. Interventions: Eligible patients were randomly assigned at a ratio of 2:1 to the experimental arm, which involved 3 cycles of BEEP followed by WBRT, or the control arm, which involved WBRT alone. Main Outcomes and Measures: The primary end point was the determination of brain-specific PFS by local investigators according to the Response Evaluation Criteria in Solid Tumors, version 1.1, the initiation of other brain-directed treatment after WBRT, or death. Other key end points included brain-specific objective response rate after 8 weeks of BEEP treatment or WBRT and 8-month brain-specific PFS rate, PFS, and overall survival. Results: A total of 118 patients with BMBC were randomized, with the intention-to-treat cohort comprising 112 patients. The median age was 56 years (range, 34-71 years), and 61 patients (54.5%) had ERBB2 (formerly HER2 or HER2/neu)-positive disease. The median (range) brain-specific PFS was 8.1 (0.3-29.5) vs 6.5 (0.9-25.5) months in the experimental and control arms, respectively (hazard ratio, 0.71; 95% CI, 0.44-1.13; P = .15; significant at predefined α ≤ .20). The brain-specific objective response rate at 2 months was not significantly different (BEEP treatment vs WBRT, 41.9% vs 52.6%), but the 8-month brain-specific PFS rate was significantly higher in the experimental group (48.7% vs 26.3%; P = .03). Adverse events were generally manageable with prophylactic granulocyte colony-stimulating factor treatment. Conclusions and Relevance: The findings show that induction BEEP before WBRT may improve the control of BMBC compared with using upfront WBRT, which could address an unmet need for an effective systemic treatment for intractable brain and extracranial metastases from metastatic breast cancer. Trial Registration: ClinicalTrials.gov Identifier: NCT02185352.

Identification of subgenomic mRNAs derived from the coronavirus 1a/1b protein gene: Implications for coronavirus transcription.

Synthesis of coronavirus subgenomic mRNA (sgmRNA) is guided by the transcription regulatory sequence (TRS). sgmRNA derived from the body TRS (TRS-B) located at the 1a/1b protein gene is designated 1ab/sgmRNA. In the current study, we comprehensively identified the 1ab/sgmRNAs synthesized from TRS-Bs located at the 1a/1b protein genes of different coronavirus genera both in vitro and in vivo by RT‒PCR and sequencing. The results suggested that the degree of sequence homology between the leader TRS (TRS-L) and TRS-B may not be a decisive factor for 1ab/sgmRNA synthesis. This observation led us to revisit the coronavirus transcription mechanism and to propose that the disassociation of coronavirus polymerase from the viral genome may be a prerequisite for sgmRNA synthesis. Once the polymerase can disassociate at TRS-B, the sequence homology between TRS-L and TRS-B is important for sgmRNA synthesis. The study therefore extends our understanding of transcription mechanisms.

Identification of the protein coding capability of coronavirus defective viral genomes by mass spectrometry.

During coronavirus infection, in addition to the well-known coronavirus genomes and subgenomic mRNAs, an abundance of defective viral genomes (DVGs) can also be synthesized. In this study, we aimed to examine whether DVGs can encode proteins in infected cells. Nanopore direct RNA sequencing and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis were employed. With the protein databases generated by nanopore direct RNA sequencing and the cell lysates derived from the RNA-protein pull-down assay, six DVG-encoded proteins were identified by LC-MS/MS based on the featured fusion peptides caused by recombination during DVG synthesis. The results suggest that the coronavirus DVGs have the capability to encode proteins. Consequently, future studies determining the biological function of DVG-encoded proteins may contribute to the understanding of their roles in coronavirus pathogenesis and the development of antiviral strategies.

Targeting the conserved coronavirus octamer motif GGAAGAGC is a strategy for the development of coronavirus vaccine.

BACKGROUND: Coronaviruses are pathogens of humans and animals that cause widespread and costly diseases. The development of effective strategies to combat the threat of coronaviruses is therefore a top priority. The conserved coronavirus octamer motif 5'GGAAGAGC3' exists in the 3' untranslated region of all identified coronaviruses. In the current study, we aimed to examine whether targeting the coronavirus octamer motif GGAAGAGC is a promising approach to develop coronavirus vaccine. METHODS: Plaque assays were used to determine the titers of mouse hepatitis virus (MHV)-A59 octamer mutant (MHVoctm) and wild-type (wt) MHV-A59 (MHVwt). Western blotting was used for the determination of translation efficiency of MHVoctm and MHVwt. Plaque assays and RT-qPCR were employed to examine whether MHVoctm was more sensitive to interferon treatment than MHVwt. Weight loss, clinical signs, survival rate, viral RNA detection and histopathological examination were used to evaluate whether MHVoctm was a vaccine candidate against MHVwt infection in BALB/c mice. RESULTS: In this study, we showed that (i) the MHVoctm with mutation of coronavirus octamer was able to grow to high titers but attenuated in mice, (ii) with the reduced multiplicity of infection (MOI), the difference in gene expression between MHVoctm and MHVwt became more evident in cultured cells, (iii) MHVoctm was more sensitive to interferon treatment than MHVwt and (iv) mice inoculated with MHVoctm were protected from MHVwt infection. CONCLUSIONS: Based on the results obtained from cultured cells, it was suggested that the synergistic effects of octamer mutation, multiplicity of infection and immune response may be a mechanism explaining the distinct phenotypes of octamer-mutated coronavirus in cell culture and mice. In addition, targeting the conserved coronavirus octamer motif is a strategy for development of coronavirus vaccine. Since the conserved octamer exists in all coronaviruses, this strategy of targeting the conserved octamer motif can also be applied to other human and animal coronaviruses for the development of coronavirus vaccines, especially the emergence of novel coronaviruses such as SARS-CoV-2, saving time and cost for vaccine development and disease control.

Evolution of the coronavirus spike protein in the full-length genome and defective viral genome under diverse selection pressures.

How coronaviruses evolve by altering the structures of their full-length genome and defective viral genome (DVG) under dynamic selection pressures has not been studied. In this study, we aimed to experimentally identify the dynamic evolutionary patterns of the S protein sequence in the full-length genome and DVG under diverse selection pressures, including persistence, innate immunity and antiviral drugs. The evolutionary features of the S protein sequence in the full-length genome and in the DVG under diverse selection pressures are as follows: (i) the number of nucleotide (nt) mutations does not necessarily increase with the number of selection pressures; (ii) certain types of selection pressure(s) can lead to specific nt mutations; (iii) the mutated nt sequence can be reverted to the wild-type nt sequence under the certain type of selection pressure(s); (iv) the DVG can also undergo mutations and evolve independently of the full-length genome; and (v) DVG species are regulated during evolution under diverse selection pressures. The various evolutionary patterns of the S protein sequence in the full-length genome and DVG identified in this study may contribute to coronaviral fitness under diverse selection pressures.

Biological characterization of coronavirus noncanonical transcripts in vitro and in vivo.

BACKGROUND: In addition to the well-known coronavirus genomes and subgenomic mRNAs, the existence of other coronavirus RNA species, which are collectively referred to as noncanonical transcripts, has been suggested; however, their biological characteristics have not yet been experimentally validated in vitro and in vivo. METHODS: To comprehensively determine the amounts, species and structures of noncanonical transcripts for bovine coronavirus in HRT-18 cells and mouse hepatitis virus A59, a mouse coronavirus, in mouse L cells and mice, nanopore direct RNA sequencing was employed. To experimentally validate the synthesis of noncanonical transcripts under regular infection, Northern blotting was performed. Both Northern blotting and nanopore direct RNA sequencing were also applied to examine the reproducibility of noncanonical transcripts. In addition, Northern blotting was also employed to determine the regulatory features of noncanonical transcripts under different infection conditions, including different cells, multiplicities of infection (MOIs) and coronavirus strains. RESULTS: In the current study, we (i) experimentally determined that coronavirus noncanonical transcripts were abundantly synthesized, (ii) classified the noncanonical transcripts into seven populations based on their structures and potential synthesis mechanisms, (iii) showed that the species and amounts of the noncanonical transcripts were reproducible during regular infection but regulated in altered infection environments, (iv) revealed that coronaviruses may employ various mechanisms to synthesize noncanonical transcripts, and (v) found that the biological characteristics of coronavirus noncanonical transcripts were similar between in vitro and in vivo conditions. CONCLUSIONS: The biological characteristics of noncanonical coronavirus transcripts were experimentally validated for the first time. The identified features of noncanonical transcripts in terms of abundance, reproducibility and variety extend the current model for coronavirus gene expression. The capability of coronaviruses to regulate the species and amounts of noncanonical transcripts may contribute to the pathogenesis of coronaviruses during infection, posing potential challenges in disease control. Thus, the biology of noncanonical transcripts both in vitro and in vivo revealed here can provide a database for biological research, contributing to the development of antiviral strategies.

Unveiling the biology of defective viral genomes in vitro and in vivo: implications for gene expression and pathogenesis of coronavirus.

BACKGROUND: Defective viral genome (DVG) is a truncated version of the full-length virus genome identified in most RNA viruses during infection. The synthesis of DVGs in coronavirus has been suggested; however, the fundamental characteristics of coronavirus DVGs in gene expression and pathogenesis have not been systematically analyzed. METHODS: Nanopore direct RNA sequencing was used to investigate the characteristics of coronavirus DVGs in gene expression including reproducibility, abundance, species and genome structures for bovine coronavirus in cells, and for mouse hepatitis virus (MHV)-A59 (a mouse coronavirus) in cells and in mice. The MHV-A59 full-length genomic cDNAs (~ 31 kilobases) were in vitro constructed to experimentally validate the origin of coronavirus DVG. The synthesis of DVGs was also experimentally identified by RT-PCR followed by sequencing. In addition, the alterations of DVGs in amounts and species under different infection environments and selection pressures including the treatment of antiviral remdesivir and interferon were evaluated based on the banding patterns by RT-PCR. RESULTS: The results are as follows: (i) the structures of DVGs are with diversity, (ii) DVGs are overall synthesized with moderate (MHV-A59 in cells) to high (BCoV in cells and MHV-A59 in mice) reproducibility under regular infection with the same virus inoculum, (iii) DVGs can be synthesized from the full-length coronavirus genome, (iv) the sequences flanking the recombination point of DVGs are AU-rich and thus may contribute to the recombination events during gene expression, (v) the species and amounts of DVG are altered under different infection environments, and (vi) the biological nature of DVGs between in vitro and in vivo is similar. CONCLUSIONS: The identified biological characteristics of coronavirus DVGs in terms of abundance, reproducibility, and variety extend the current model for coronavirus gene expression. In addition, the biological features of alterations in amounts and species of coronavirus DVGs under different infection environments may assist the coronavirus to adapt to the altered environments for virus fitness and may contribute to the coronavirus pathogenesis. Consequently, the unveiled biological features may assist the community to study the gene expression mechanisms of DVGs and their roles in pathogenesis, contributing to the development of antiviral strategy and public health.

Disparity in survival benefits of pembrolizumab between Asian and non-Asian patients with advanced cancers: A systematic review and meta-regression analysis.

BACKGROUND: Immune checkpoint inhibitors have revolutionized the treatment of malignancies. However, disproportionate enrollment among races and ethnicities places the generalizability of global trial results in doubt. METHODS: In this systematic review, phase 3 randomized controlled trials investigating pembrolizumab in advanced cancers and providing subgroup analyses of Asian and non-Asian participants were included. The primary and secondary effect measures were the mean differences (MDs) in the natural logarithms of the hazard ratios (HRs) for overall survival (OS) and progression-free survival (PFS) between these two subgroups, respectively. We used random-effects meta-analysis to calculate the pooled ratios of HRs (i.e., exp(MD)) and implemented a meta-regression analysis to identify significant covariates. RESULTS: A total of 17 and 11 trials were included in the meta-analyses of OS and PFS, respectively. These trials included 2732 (25.49%) Asian and 7000 (65.32%) non-Asian participants in the OS analysis and 1438 (22.5%) Asian and 4129 (64.61%) non-Asian participants in the PFS analysis. The pooled ratio of HRs for OS was 0.87 (95% CI: 0.76-0.99; p = 0.0391), favoring Asian participants, but no significant difference was found in PFS (pooled ratio of HRs: 0.93; 95% CI: 0.82-1.07; p = 0.2391). Both linear meta-regression analyses revealed an open-label design as a crucial covariate, which indicated more benefits for non-Asian participants. CONCLUSIONS: Compared with non-Asian patients, Asian patients with advanced cancers may derive superior OS benefits from pembrolizumab. Although the results warrant further exploration, this meta-analysis provides insight into clinical research design.

Lifestyle Factors and Energy Intakes with Risks of Breast Cancer among Pre- and Post- Menopausal Women in Taiwan.

Although the incidence of invasive breast cancer (BC) among women in Asian is generally lower than that in Western countries, the incidence of BC has been on the rise in the past three decades in Asian countries. This hospital-based case-control study aimed to explore the relationship between dietary and metabolic factors and BC risk in pre- and post-menopausal women. We enrolled 285 patients with newly diagnosed BC at the National Taiwan University Hospital and 297 controls from the local community and hospital staff. Before receiving anticancer therapy, all patients with BC and control participants completed a 57-question semi-quantitative Food Frequency Questionnaire. For pre-menopausal women, plant-based factor scores rich in seeds and nuts, soy, fruits, and seaweeds correlated significantly with reduced BC risks, whereas menarche occurring at <12 years of age, reduced physical activity, and high-density lipoprotein <40 mg/dL were associated with increased BC risks. For post-menopausal women, plant-based dietary factor scores were also associated with reduced risks, whereas increased body mass index and energy intake levels correlated with increased BC risks. Diets rich in plant-based dietary patterns are protective against BC risk, regardless of menopausal status. Habitual physical activity is protective against BC risk among pre-menopausal Taiwanese women. Maintaining optimal weight and caloric intake is beneficial for reducing post-menopausal BC risk.

Clinical Guidance on the Monitoring and Management of Trastuzumab Deruxtecan (T-DXd)-Related Adverse Events: Insights from an Asia-Pacific Multidisciplinary Panel.

Trastuzumab deruxtecan (T-DXd)-an antibody-drug conjugate targeting the human epidermal growth factor receptor 2 (HER2)-improved outcomes of patients with HER2-positive and HER2-low metastatic breast cancer. Guidance on monitoring and managing T-DXd-related adverse events (AEs) is an emerging unmet need as translating clinical trial experience into real-world practice may be difficult due to practical and cultural considerations and differences in health care infrastructure. Thus, 13 experts including oncologists, pulmonologists and a radiologist from the Asia-Pacific region gathered to provide recommendations for T-DXd-related AE monitoring and management by using the latest evidence from the DESTINY-Breast trials, our own clinical trial experience and loco-regional health care considerations. While subgroup analysis of Asian (excluding Japanese) versus overall population in the DESTINY-Breast03 uncovered no major differences in the AE profile, we concluded that proactive monitoring and management are essential in maximising the benefits with T-DXd. As interstitial lung disease (ILD)/pneumonitis is a serious AE, patients should undergo regular computed tomography scans, but the frequency may have to account for the median time of ILD/pneumonitis onset and access. Trastuzumab deruxtecan appears to be a highly emetic regimen, and prophylaxis with serotonin receptor antagonists and dexamethasone (with or without neurokinin-1 receptor antagonist) should be considered. Health care professionals should be vigilant for treatable causes of fatigue, and patients should be encouraged to use support groups and practice low-intensity exercises. To increase treatment acceptance, patients should be made aware of alopecia risk prior to starting T-DXd. Detailed monitoring and management recommendations for T-DXd-related AEs are discussed further.

Plasma cell-free tumor DNA, PIK3CA and TP53 mutations predicted inferior endocrine-based treatment outcome in endocrine receptor-positive metastatic breast cancer.

PURPOSE: How to factor both tumor burden and oncogenic genomic mutations as variables to predict the outcome of endocrine-based therapy (ET) in ER-positive/HER2-negative metastatic breast cancer patients (MBC) remains to be explored. METHOD: Blood samples prospectively collected from 163 ER-positive/HER2-negative female MBC patients, before ET, were used for cell-free tumor DNA (cfDNA) analysis. cfDNA was subjected to next-generation sequencing (NGS) to interrogate oncogenic PIK3CA hotspot and TP53 DNA-binding domain (DBD) mutations, including single nucleotide variants (SNVs) or small insertions and deletions (InDels). The variant calling threshold was set at 0.5%. Progression-free survival (PFS) was measured from the start of the ET treatment to the time of disease progression of the same treatment regimen. RESULTS: Overall, the median PFS was 8.3 months (95% CI 5.7-11.1 months). The median cfDNA was 38.5 ng (range 4.4-1935 ng). The proportion of patients with PIK3CA and TP53 alterations were 25.1 and 15.3%, respectively. Patients with high total cfDNA (HR 1.74, p = 0.003), PIK3CA mutation (HR 1.74, p = 0.007), and TP53 mutation (HR 1.64, p = 0.047) in liquid biopsy conferred worse outcome after ET. Even for patients with low tumor burden, the detrimental effect of PIK3CA or TP53 mutation remained significant (p < 0.001). For patients with either PIK3CA (p < 0.001) or TP53 mutation (p = 0.004), there was significant positive correlation between allele frequency (AF) and total cfDNA. CONCLUSION: After adjustment of cfDNA level, PIK3CA and TP53 mutations observed in liquid biopsy exerted detrimental effects on the outcome of ET-based regimens. The AF of PIK3CA or TP53 may be a surrogate marker for PFS.

Deep-learning based breast cancer detection for cross-staining histopathology images.

Hematoxylin and eosin (H&E) staining is the gold standard for tissue characterization in routine pathological diagnoses. However, these visible light dyes do not exclusively label the nuclei and cytoplasm, making clear-cut segmentation of staining signals challenging. Currently, fluorescent staining technology is much more common in clinical research for analyzing tissue morphology and protein distribution owing to its advantages of channel independence, multiplex labeling, and the possibility of enabling 3D tissue labeling. Although both H&E and fluorescent dyes can stain the nucleus and cytoplasm for representative tissue morphology, color variation between these two staining technologies makes cross-analysis difficult, especially with computer-assisted artificial intelligence (AI) algorithms. In this study, we applied color normalization and nucleus extraction methods to overcome the variation between staining technologies. We also developed an available workflow for using an H&E-stained segmentation AI model in the analysis of fluorescent nucleic acid staining images in breast cancer tumor recognition, resulting in 89.6% and 80.5% accuracy in recognizing specific tumor features in H&E- and fluorescent-stained pathological images, respectively. The results show that the cross-staining inference maintained the same precision level as the proposed workflow, providing an opportunity for an expansion of the application of current pathology AI models.

Transcriptomic alterations underlying metaplasia into specific metaplastic components in metaplastic breast carcinoma.

BACKGROUND: Metaplastic breast carcinoma (MpBC) typically consists of carcinoma of no special type (NST) with various metaplastic components. Although previous transcriptomic and proteomic studies have reported subtype-related heterogeneity, the intracase transcriptomic alterations between metaplastic components and paired NST components, which are critical for understanding the pathogenesis underlying the metaplastic processes, remain unclear. METHODS: Fifty-nine NST components and paired metaplastic components (spindle carcinomatous [SPS], matrix-producing, rhabdoid [RHA], and squamous carcinomatous [SQC] components) were microdissected from specimens obtained from 27 patients with MpBC for gene expression profiling using the NanoString Breast Cancer 360 Panel on a NanoString nCounter FLEX platform. BC360-defined signatures were scored using nSolver software. RESULTS: Hierarchical clustering and principal component analysis revealed a heterogeneous gene expression profile (GEP) corresponding to the NST components, but the GEP of metaplastic components exhibited subtype dependence. Compared with the paired NST components, the SPS components demonstrated the upregulation of genes related to stem cells and epithelial-mesenchymal transition and displayed enrichment in claudin-low and macrophage signatures. Despite certain overlaps in the enriched functions and signatures between the RHA and SPS components, the specific differentially expressed genes differed. We observed the RHA-specific upregulation of genes associated with vascular endothelial growth factor signaling. The chondroid matrix-producing components demonstrated the upregulation of hypoxia-related genes and the downregulation of the immune-related MHC2 signature and the TIGIT gene. In the SQC components, TGF-ß and genes associated with cell adhesion were upregulated. The differentially expressed genes among metaplastic components in the 22 MpBC cases with one or predominantly one metaplastic component clustered paired NST samples into clusters with correlation with their associated metaplastic types. These genes could be used to separate the 31 metaplastic components according to respective metaplastic types with an accuracy of 74.2%, suggesting that intrinsic signatures of NST may determine paired metaplastic type. Finally, the EMT activity and stem cell traits in the NST components were correlated with specimens displaying lymph node metastasis. CONCLUSIONS: We presented the distinct transcriptomic alterations underlying metaplasia into specific metaplastic components in MpBCs, which contributes to the understanding of the pathogenesis underlying morphologically distinct metaplasia in MpBCs.

Estrogen-DNA Adducts and Breast Cancer Risk in Premenopausal Asian Women.

The incidence of breast cancer among premenopausal women has been increasing rapidly in recent decades in East Asia. This case-control study investigated whether estrogen-DNA adducts were associated with breast cancer risk in Taiwan. The control group (n = 146) comprised healthy female volunteers and women with non-proliferative breast disease. The case group (n = 221) comprised women either with proliferative benign breast disease or breast cancer. The ratios of estrogen-DNA adducts to their respective metabolites and conjugates in plasma were analyzed using ultraperformance LC/MS-MS. The SNPs of CYP1A1, CYP1B1, and COMT were genotyped. Logistic regression model was used to compare the estrogen-DNA adduct ratios between the two groups. The estrogen-DNA adduct ratio in the case group was significantly higher than that in the control group (median ratio: 58.52 vs. 29.36, P = 0.004). A multiple logistic regression model demonstrated that a unit increase in the natural log of the estrogen-DNA adduct ratio in premenopausal women was a significant predictor of breast cancer risk, with an estimated hazard ratio of 1.718 (1.444-2.046, P < 0.001). However, the CYP1A1, CYP1B1, and COMT SNPs were not associated with the estrogen-DNA adduct ratios. In conclusion, plasma estrogen-DNA adduct ratio was associated with the presence of breast cancer or proliferating benign breast disease in premenopausal women in Taiwan. PREVENTION RELEVANCE: This study provides evidence that endogenous estrogen-induced genotoxicity may contribute to the carcinogenesis of breast cancer in premenopausal Asian women. This work could have important preventive implication for the emerging disease in East Asia.

Clinical Outcomes of Metastatic Breast Cancer in Patients Having Imaging Liver Pseudocirrhosis with or without Evident Varices.

BACKGROUND: Pseudocirrhosis is an imaging finding of malignancies with liver metastasis with or without clinical liver cirrhosis-related portal hypertension (pHTN). This study defined evident pHTN by the presence of esophageal or gastric varices and compared patients' outcomes of metastatic breast cancer with imaging-diagnosed pseudocirrhosis with or without varices. METHODS: The medical records from patients with metastatic breast cancer and pseudocirrhosis between 2005 and 2017 were retrospectively analyzed. Survival outcomes were compared based on endoscopic evidence of esophageal or gastric varices. RESULTS: Among 106 patients with pseudocirrhosis, 33 (31%) had de novo stage IV disease, and 66 (62%) had hormone receptor (HR)-positive and human epidermal growth factor receptor 2 (HER2)-negative breast cancer. Eighty-one (76%) had initial metastases in both hepatic lobes, and 32 (30%) had esophageal or gastric varices. The median overall survival (OS) was 5 and 13 months in patients with and without varices (P = .002). The median OS in patients with HER2-positive, HR-positive/HER2-negative, and triple-negative subtype was 16, 9, and 2 months, respectively (P = .001). Patients with varices usually had cirrhotic complications, including gastrointestinal bleeding, hyperbilirubinemia, hyperammonemia, and coagulopathy. Despite their challenging clinical conditions, 7 patients with varices had OS exceeding 1 year. In multivariate analysis, evident varices (P = .007) and triple-negative subtype (P = .013) were associated with poor OS. CONCLUSIONS: Patients with pseudocirrhosis and evident varices had a significantly shorter median OS, and were usually associated with clinical cirrhosis-related complications. To maximize OS, early identification and meticulous supportive care are warranted.