The molecular tumor burden index as a response evaluation criterion in breast cancer.

Circulating tumor DNA (ctDNA) is a potential biomarker of prognosis and therapeutic response. We conducted this study to explore the role of the molecular tumor burden index (mTBI) in ctDNA as a therapeutic response and prognostic biomarker in a larger cohort prospective phase III randomized multicenter study. We collected 291 plasma samples from 125 metastatic breast cancer patients from the CAMELLIA study (NCT01917279). Target-capture deep sequencing of 1021 genes was performed to detect somatic variants in ctDNA from the plasma samples. The pretreatment mTBI value was correlated with tumor burden (P = 0.025). Patients with high-level pretreatment mTBI had shorter overall survival than patients with low-level pretreatment mTBI, and the median overall survival was 40.9 months and 68.4 months, respectively (P = 0.011). Patients with mTBI decrease to less than 0.02% at the first tumor evaluation had longer progression-free survival and overall survival (P < 0.001 and P = 0.007, respectively). The mTBI has good sensitivity to identify complete response/partial response and progressive disease based on computed tomography scans (88.5% and 87.5%, respectively). The patients classified as molecular responders had longer progression-free survival and overall survival than the nonmolecular responders in the overall cohort (P < 0.001 and P = 0.036, respectively), as well as in the cohort in which computed tomography scans were defined as representing stable disease (P = 0.027 and P = 0.015, respectively). The mTBI in ctDNA detected in liquid biopsies is a potential biomarker of therapeutic response and prognosis in patients with metastatic breast cancer.

DNA damage response as a prognostic indicator in metastatic breast cancer via mutational analysis.

BACKGROUND: High tumor heterogeneity contributes to breast cancer recurrence and metastasis. However, the lack of indicators to serve as precise and reliable means of predicting breast cancer prognosis has yet to be addressed. This study aims to reveal the prognostic relevance of mutations in metastatic breast cancer (MBC) by large-scale circulating tumor DNA (ctDNA) analysis in China. METHODS: We performed ctDNA panel-captured sequencing of 958 blood samples from MBC patients including 494 hormone receptor (HR)-positive cases, 130 human epidermal growth factor receptor 2-positive cases, and 177 triple-negative breast cancer (TNBC) cases. The somatic mutations and potential targets were assessed. Progression-free survival (PFS) was analyzed using the Kaplan-Meier method. RESULTS: In 801 of the 958 MBC blood samples, 663 mutated genes and 5,829 nonsynonymous alterations were identified. Mutated genes of the highest frequency were tumor protein p53 (TP53, 54%), phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA, 41%), estrogen receptor 1 (ESR1, 12%), myeloid/lymphoid or mixed-lineage leukemia protein 3 (MLL3, 11%), DNA (cytosine-5)-methyltransferase 3A (DNMT3A, 10%), erb-b2 receptor tyrosine kinase 2 (ERBB2, 10%), GATA binding protein 3 (GATA3, 8%), FAT atypical cadherin 1 (FAT1, 7%), phosphatase and tensin homolog (PTEN, 6%), and mitogen-activated protein kinase kinase kinase 1 (MAP3K1, 6%). Enriched mutations and driver genes in MBC varied across stages and in multiple subtypes. Moreover, TP53, ERBB2, or coexisting TP53/PIK3CA mutations in MBC were remarkably related with shorter PFS. Mutated DNA damage response (DDR) genes were significantly associated with tumor mutation burden and mutant-allele tumor heterogeneity score, as well as with worse clinical outcome. CONCLUSIONS: Our findings indicate that the mutations of TP53, PIK3CA, ERBB2, and in particular, DDR genes, in MBC might be relevant indicators of unfavorable prognosis in MBC.

Molecular landscape and efficacy of HER2-targeted therapy in patients with HER2-mutated metastatic breast cancer.

Human epidermal growth factor receptor 2 (HER2) protein overexpression or gene amplification is an important predictive biomarker for identifying patients with breast cancer, who may benefit from HER2-targeted therapy. However, little is known about the molecular landscape and efficacy of HER2-targeted therapy in patients with HER2-mutated metastatic breast cancer. We analysed the HER2 mutation features of 1184 patients with invasive breast cancer. In addition, a single-arm, prospective, phase-II study (NCT03412383) of pyrotinib was conducted in patient with metastatic HER2 amplification-negative, mutation-positive breast cancer. Peripheral blood was collected from each patient and circulating tumour DNA (ctDNA) sequencing was performed using a 1021 gene panel. HER2 mutations were detected in 8.9% (105/1184) of patients. The HER2 amplification-positive patients had a higher mutation frequency than the HER2 amplification-negative patients (19.5% vs. 4.8%, P < 0.001). A multivariate Cox regression analysis indicated that patients with HER2 mutations had a shorter progression-free survival (PFS) than HER2 wild-type patients (median PFS 4.7 months vs. 11.0 months, hazard ratio 2.65, 95% confidence interval 1.25-5.65, P = 0.011). Ten HER2 amplification-negative, mutation-positive patients who received pyrotinib monotherapy were ultimately included in the efficacy analysis. The median PFS was 4.9 months. The objective response rate (complete response + partial response) was 40.0% and the clinical benefit rate (complete response + partial response + stable disease over 24 weeks) was 60%. In conclusion, a HER2 gene mutation analysis is potentially useful to identify biomarkers of trastuzumab resistance in HER2 amplification-positive patients. Patients with HER2-mutated, non-amplified metastatic breast cancers may benefit from pyrotinib.

Taxotere-induced WNT16 Expression in Carcinoma-Associated Fibroblasts Might Associate with Progression and Chemoresistance of Breast Cancer.

OBJECTIVE: The objective of this study is to investigate the relationship between WNT16 expression in carcinoma-associated fibroblasts and Taxotere treatments in breast cancer. PROCEDURES: WNT16 expression in primarily cultured carcinoma-associated fibroblasts was detected via Real-Time PCR and Western blot. A total of 106 breast cancer cases were prepared for immunohistochemistry. Multiple assays were performed to examine the function of breast cancer cells (MDA-MB-231) co-cultured with carcinoma-associated fibroblasts when WNT16 was inhibited. RESULTS: Enhanced WNT16 expression was verified in carcinoma-associated fibroblasts after Taxotere treatment (p<0.0001). MDA-MB-231 cells co-cultured with carcinoma-associated fibroblasts displayed decreasing invasion, proliferation abilities, and an increasing sensitivity to Taxotere treatment when WNT16 was inhibited. The WNT16 expression of carcinoma-associated fibroblasts in breast cancer was significantly associated with tumor size (p=0.0164), and chemotherapy treatment (p=0.0015). CONCLUSIONS: The results indicate that Taxotere-induced WNT16 expression in carcinoma-associated fibroblasts might contribute to the progression and chemoresistance of breast cancer.

Clinical spectrum and prognostic value of TP53 mutations in circulating tumor DNA from breast cancer patients in China.

BACKGROUND: TP53 mutations are common in breast cancer. There is currently no large-scale cohort study to investigate the TP53 landscape in breast cancer patients from China. The predictive value of TP53 mutations for the efficacy of human epidermal growth factor receptor 2 (HER2)-targeted therapy in breast cancer remains controversial. In the present study, we aimed to analyze the clinical spectrum and prognostic value of TP53 mutations in circulating tumor DNA (ctDNA) from breast cancer patients in China. METHODS: We retrospectively analyzed the clinical data and TP53 mutation features in ctDNA samples from 804 patients with metastatic breast cancer. TP53 mutations were detected by target region capture-based next-generation sequencing. The relationship between TP53 mutation status and disease-free survival (DFS) was analyzed in 444 patients with metastatic breast cancer. Moreover, the relationship between TP53 mutation status and progression-free survival (PFS) was analyzed in 55 HER2-positive patients treated with first-line trastuzumab-based therapy. Kaplan-Meier analysis was performed to estimate the survival curves of the different subgroups, and the log-rank test was used to compare the curves. A Cox regression model was used to estimate multivariable-adjusted hazard ratios and their 95% confidence intervals (CIs) associated with the DFS and PFS. RESULTS: Among the 804 investigated patients, 431 (53.6%) patients harbored TP53 mutations. TP53 mutations were differentially distributed among different molecular subtypes of breast cancer (P < 0.05). Patients with TP53 mutations had a shorter DFS than those with wild-type TP53 (hazard ratio = 1.32, 95% CI = 1.09-1.61, P = 0.005). TP53 mutations in exons 5-8 were associated with worse outcome (hazard ratio = 1.50, 95% CI = 1.11-2.03, P = 0.009). However, TP53 mutation status was not significantly associated with PFS in HER2-positive patients who received first-line trastuzumab-based therapy (P = 0.966). Interestingly, in the taxane combination group, patients with TP53 mutations exhibited longer PFS than those without TP53 mutations (hazard ratio = 0.08, 95% CI = 0.02-0.30, P < 0.001). However, in the non-taxane combination group, patients with TP53 mutations displayed shorter PFS than those with wild-type TP53 (hazard ratio = 4.84, 95% CI = 1.60-14.66, P = 0.005). CONCLUSIONS: TP53 mutations in exons 5-8 may be an independent prognostic marker for short DFS in patients with metastatic breast cancer. TP53 mutations had opposite effects on trastuzumab-treated patients treated with and without taxanes.

Safety and efficacy of sirolimus combined with endocrine therapy in patients with advanced hormone receptor-positive breast cancer and the exploration of biomarkers.

BACKGROUND: We performed a retrospective study on the efficacy and safety of sirolimus (an mTOR inhibitor) in hormone receptor (HR)-positive advanced breast cancer and searched for biomarkers to predict its efficacy. METHODS: All patients with HR-positive metastatic breast cancer treated with sirolimus plus endocrine therapy between December 2017 and July 2018 at the Cancer Hospital, Chinese Academy of Medical Sciences were consecutively and retrospectively reviewed. Mutations in circulating tumour DNA (ctDNA) were assayed for 1021 tumour-related genes via gene panel target capture-based next-generation sequencing. RESULTS: Thirty-six patients with metastatic breast cancer treated with sirolimus plus endocrine therapy were included. The progression-free survival (PFS) rates between the sirolimus group and everolimus group were similar, and the median PFS was 4.9 months and 5.5 months, respectively (hazard ratio 1.56, 95% CI 0.86-2.81, P = 0.142). The objective response rate in the 36 patients was 19.4%, and the clinical benefit rate was 41.7%. Lipid metabolism disorder was the most common adverse event (69.4%), and 13.9% of patients had stomatitis. Most (94.4%) adverse events were grade 1-2. Twenty patients (55.6%) underwent ctDNA analysis before receiving sirolimus therapy. For patients who received less than 3 lines of chemotherapy, those with PI3K/Akt/mTOR pathway alterations had a better response to sirolimus than those without alterations, with a median PFS of 7.0 months vs 4.3 months (hazard ratio = 0.01, 95% CI 0.00-0.34, P = 0.010). CONCLUSIONS: Sirolimus is a potentially effective treatment option for patients with HR-positive advanced breast cancer.

Identification of candidate genes associated with tubal origin of high-grade serous ovarian cancer.

Evidence indicates that high-grade serous ovarian carcinoma arises from the fallopian tube, rather than ovarian surface epithelium. This is termed the 'tubal origin' theory. The aim of the present study was to compare the immunophenotype and gene expression profiling among high-grade serous ovarian carcinoma (HGSOC), fallopian tube epithelium (FTE) and ovarian surface epithelium (OSE) based on tubal origin theory, and identify the differential genes associated with ovarian carcinogenesis. A total of 61 cases of fresh tissue samples including 21 cases of HGSOC, 20 cases of OSE, and 20 cases of FTE were obtained following surgical resection. Immunostaining was performed to detect the expression of PAX8, which has been considered as a potential immunophenotype marker of Müllerian origin. Illumina BeadChip was applied for gene expression profiling. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to confirm the differential expression of candidate genes between HGSOC and FTE. The results of the present study demonstrated that PAX8 was highly expressed in HGSOC (19/21, 90.4%) and FTE (20/20, 100%), but not in OSE (3/20, 14.3%). A dendrogram generated by cluster analysis indicated a higher similarity of gene expression profile between HGSOC and FTE than OSE. A total of 2,412 differentially expressed genes were identified (absolute fold change >2) between HGSOC and FTE, including 822 upregulated genes in cancer and 1,590 downregulated genes. S100 calcium binding protein P, Ras-interacting protein 1, Wnt family member 5A, tumor-associated calcium signal transducer 2, Dickkopf Wnt signaling pathway inhibitor 3 and tumor suppressor candidate 3 genes were identified as candidate markers, of which the differential gene expression in HGSOC and FTE was confirmed by RT-qPCR (P<0.05). The results indicate the presence of a greater similarity in the immunophenotype and gene expression profile of HGSOC and FTE, when compared with OSE, which was consistent with the tubal origin theory of HGSOC.

Study on the influential biochemical indices of Cd(II) on Eisenia fetida in oxidative stress by principal component analysis in the natural soil.

With the aggravation of heavy metal pollution in soil, the individual heavy metal content monitoring cannot predict the true effects of harmful substances on the ecosystems. Thus, the effective biological evaluation system should be established to assess the pollution risk caused by heavy metal. Earthworms are widely distributed in the soil, and at the bottom of the food chain, the changes of biochemical indices play an important role in the early warning for heavy metal pollution. Principal component analysis (PCA) is a statistical method that derives several independent principal components from the original variable based on retaining the information as much as possible. This paper is aimed at finding out and analyzing the key monitoring factors related to Cd2+ on the earthworm Eisenia fetida in oxidative stress. The Cd2+ stress concentrations were set at 0, 1, 10, 20, 100, 200, 400, and 800 mg kg-1, and the post-clitellum segment of earthworm was chosen to determine TP, POD, SOD, GST, GPX, CAT, MDA, VE, and AChE. The results showed that the main bioindicators associated with oxidative stress reaction were GST, POD, and MDA at the exposure time of 10 days; at 20 days GPX, MDA, and AChE; at 30 days CAT, TP, and GPX; CAT, MDA, and SOD at 40th day. These results indicated that PCA can quickly, effectively, directly, and scientifically select biomarkers of oxidative stress induced by Cd and improve the accuracy and scientificity of earthworm as a biomarker in monitoring and early warning for heavy metal-contaminated soil.

Taxotere-induced elevated expression of IL8 in carcinoma-associated fibroblasts of breast invasive ductal cancer.

Breast cancer is the most common malignant tumor in women worldwide, and accounts for an estimated 29% of new cases and 15% of cancer-associated mortalities each year. Invasive ductal carcinoma represents 70-80% of all breast cancer cases, which are responsible for the majority of breast cancer fatalities. Though great progress has been made in understanding the tumorigenesis and development of breast cancer, problems surrounding treatment persist. It was previously reported that carcinoma-associated fibroblasts (CAFs) may be closely associated with chemotherapy resistance. In the present study, primary-cultured CAFs from surgically resected breast invasive ductal cancer tissues were prepared and tested to clarify the change of gene expression profile following treatment with 20 ng/ml Taxotere® for 24 h through microarray analysis. In addition, quantitative polymerase chain reaction and western blotting were performed to compare the gene and protein expression of the candidate gene in CAFs prior to and following Taxotere treatment. Based on the obtained data, 35 differentially expressed genes were identified, including ACTA2, ACTC1, ACTG, ALDH1B1, AMY1A, C5orf13, CNN1, CXCR7, DDAH1, FGF1, PDLIM3, MAMLD1, MYH11, OXTR, PDLIM5, RARRES1, SERPINA3, TRIL, C14orf43, C1orf51, CXCL12, CXCL2, EGR2, EGR3, IER3, interleukin (IL)8, IRF1, JUNB, MMP1, NAV2, NFKBIA, NFKBIZ, TRIB1, WNT16 and ZC3H12A. It was observed that the expression of the candidate gene IL8 in the CAFs of breast invasive cancer following treatment with Taxotere was increased (P<0.05). Overall, elevated expression of IL8 induced by Taxotere in CAFs potentially supports the association between IL8 and chemotherapy response.

Letrozole-induced functional changes in carcinoma-associated fibroblasts and their influence on breast cancer cell biology.

Accumulating evidence suggests that carcinoma-associated fibroblasts (CAFs) influence the efficacy of endocrine therapy. Aromatase inhibitors inhibit the growth of breast tumors by inhibiting the synthesis of estrogen. However, it remains unknown whether the aromatase inhibitor letrozole has an additional impact on CAFs, which further influence the efficacy of endocrine therapy. Primary CAFs were isolated from primary estrogen receptor-positive human breast tumors. Estrogen-deprived culture medium was used to exclude the influence of steroids. In co-culture, primary cultured CAFs increased MCF7 cell adhesion, invasion, migration and proliferation, and letrozole treatment inhibited these increases, except for the increase in proliferation. In total, 258 up-regulated genes and 47 down-regulated genes with an absolute fold change >2 were identified in CAFs co-cultured with MCF7 cell after letrozole treatment. One up-regulated genes (POSTN) and seven down-regulated genes (CCL2, CCL5, CXCL1, IL-8, CXCL5, LEP and NGF) were further validated by real-time PCR. The changes in CCL2 and CXCL1 expression were further confirmed using an automated microscopic imaging-based, high content analysis platform. Although the results need further functional validation, this study is the first to describe the differential tumor-promoting phenotype of CAFs induced by letrozole and the associated gene expression alterations. Most importantly, our data revealed that down-regulation of several secreted factors (CCL2, CCL5, CXCL1 etc.) in CAFs might be partially responsible for the efficacy of letrozole.

Dual inhibition of EGFR at protein and activity level via combinatorial blocking of PI4KIIα as anti-tumor strategy.

Our previous studies indicate that phosphatidylinositol 4-kinase IIα can promote the growth of multi-malignant tumors via HER-2/PI3K and MAPK pathways. However, the molecular mechanisms of this pathway and its potential for clinical application remain unknown. In this study, we found that PI4KIIα could be an ideal combinatorial target for EGFR treatment via regulating EGFR degradation. Results showed that PI4KIIα knockdown reduced EGFR protein level, and the expression of PI4KIIα shows a strong correlation with EGFR in human breast cancer tissues (r = 0.77, P < 0.01). PI4KIIα knockdown greatly prolonged the effects and decreased the effective dosage of AG-1478, a specific inhibitor of EGFR. In addition, it significantly enhanced AG1478-induced inhibition of tumor cell survival and strengthened the effect of the EGFR-targeting anti-cancer drug Iressa in xenograft tumor models. Mechanistically, we found that PI4KIIα suppression increased EGFR ligand-independent degradation. Quantitative proteomic analysis by stable isotope labeling with amino acids in cell culture (SILAC) and LC-MS/MS suggested that HSP90 mediated the effect of PI4KIIα on EGFR. Furthermore, we found that combined inhibition of PI4KIIα and EGFR suppressed both PI3K/AKT and MAPK/ERK pathways, and resulted in downregulation of multiple oncogenes like PRDX2, FASN, MTA2, ultimately leading to suppression of tumor growth. Therefore, we conclude that combined inhibition of PI4KIIα and EGFR exerts a multiple anti-tumor effect. Dual inhibition of EGFR at protein and activity level via combinatorial blocking of PI4KIIα presents a novel strategy to combat EGFR-dependent tumors.

Candidate markers that associate with chemotherapy resistance in breast cancer through the study on Taxotere-induced damage to tumor microenvironment and gene expression profiling of carcinoma-associated fibroblasts (CAFs).

Recently, emerging evidence has suggested that carcinoma-associated fibroblasts (CAFs) could contribute to chemotherapy resistances in breast cancer treatment. The aim of this study is to compare the gene expression profiling of CAFs before and after chemotherapy and pick up candidate genes that might associate with chemotherapy resistance and could be used as predictors of treatment response. CAFs were cultured from surgically resected primary breast cancers and identified with immunohistochemistry (IHC) and Flow cytometry (FCM). MDA-MB-231 cells were cultured as the breast cancer cell line. Cell adhesion assay, invasion assay, and proliferation assay (MTT) were performed to compare the function of MDA-MB-231 cells co-cultured with CAFs and MDA-MB-231 cells without co-culture, after chemotherapy. Totally 6 pairs of CAFs were prepared for microarray analysis. Each pair of CAFs were obtained from the same patient and classified into two groups. One group was treated with Taxotere (regarded as after chemotherapy) while the other group was not processed with Taxotere (regarded as before chemotherapy). According to our study, the primary-cultured CAFs exhibited characteristic phenotype. After chemotherapy, MDA-MB-231 cells co-cultured with CAFs displayed increasing adhesion, invasiveness and proliferation abilities, compared with MDA-MB-231 cells without CAFs. Moreover, 35 differentially expressed genes (absolute fold change >2) were identified between CAFs after chemotherapy and before chemotherapy, including 17 up-regulated genes and 18 down-regulated genes. CXCL2, MMP1, IL8, RARRES1, FGF1, and CXCR7 were picked up as the candidate markers, of which the differential expression in CAFs before and after chemotherapy was confirmed. The results indicate the changes of gene expression in CAFs induced by Taxotere treatment and propose the candidate markers that possibly associate with chemotherapy resistance in breast cancer.

Local recurrence involving the sternum and ribs following mastectomy and titanium mesh implants for chest wall reconstruction: A case report.

Approximately 30% of breast carcinoma patients experience local recurrence, which is commonly considered the first sign of treatment failure. Local recurrence involving the deep chest wall may result in thoracic defects and influence normal cardiopulmonary function. Many studies have reported various techniques using different materials for chest wall reconstruction, and titanium mesh has recently received attention as a novel bone substitute. In the present case report, a 46-year-old female who had not yet entered menopause presented for routine follow-up. Her past history was significant for having had a left modified radical mastectomy followed by chemotherapy and tamoxifen treatment for an invasive ductal breast carcinoma. Examination results revealed an invasive ductal carcinoma invading the chest wall. The patient underwent surgical excision and received a titanium mesh implant for chest wall reconstruction. The patient chose to undergo local radiation therapy and endocrine treatment following surgery. Local recurrence of breast cancer involving the deep chest wall is relatively rare. According to the guidelines, surgical excision followed by radiotherapy is the standard treatment and chemotherapy is not recommended. In our case, a titanium mesh was successfully applied for chest wall reconstruction.

Down-regulation of GRIM-19 is associated with STAT3 overexpression in breast carcinomas.

To evaluate the association of gene associated with retinoic-interferon-induced mortality 19 (GRIM-19) with clinicopathologic features as well as its target gene signal transducer and activator of transcription 3 (STAT3) in patients with breast cancer, GRIM-19 and STAT3 expression was measured immunohistochemically in 108 breast samples and by Western blotting in 20 breast cancer tissues and corresponding nontumorous tissues. Expression of GRIM-19 was severely depressed in the carcinomas relative to matched nontumorous tissues (P < .001), and STAT3 was overexpressed in breast cancer tissues (P < .001), conclusions supported by Western blot analysis. Nonexpression of GRIM-19 was significantly associated with lymph node metastasis (P < .001), advanced tumor-node-metastasis stage (P = .02), and triple-negative phenotype (P = .03). Furthermore, down-regulation of GRIM-19 correlated with STAT3 overexpression (r = 0.56; P < .001). Thus, GRIM-19 is suppressed in primary breast carcinomas, with a corresponding increase in STAT3 activity.