Reduced mitochondrial DNA copy number is correlated with tumor progression and prognosis in Chinese breast cancer patients

Somatic mutations and large-scale depletion in mitochondrial DNA (mtDNA) have been extensively detected in various human cancers. However, it still remains unclear whether the alterations in mtDNA content are related to the clinicopathological parameters and patient prognosis in breast cancer. In the present study, we analyzed the copy number of mtDNA in 59 cases of invasive breast tumors and paired nontumorous tissues using quantitative real-time PCR. Our data showed that the level of mtDNA was significantly decreased in tumor tissues as compared to the adjacent nontumorous counterparts (P = 0.001). The reduced copy number in mtDNA was associated with an older onset age (>or=50 years old, P = 0.035) as well as a higher histological grade (P = 0.012). Survival analysis measured by the Kaplan-Meier curves and the log-rank test indicated that patients with reduced mtDNA content had significantly poorer disease-free survival (DFS, P = 0.0079) and overall survival (OS, P = 0.011) rate. In addition, tumors harboring mutations in displacement (D)-loop region, particularly at the polycytidine stretch (T/N ratio = 64.3 +/- 8.2%) or close to the replication origins of the heavy-strand (T/N ratio = 68.7 +/- 5.5%), had a significantly lower copy number of mtDNA than the ones without D-loop alterations. Together, our results suggested that reduced copy number of mtDNA may be involved in breast neoplastic transformation or progression and mtDNA content might be potentially used as a tool to predict prognosis. Somatic mutation in the D-loop region probably is one of key contributing factors leading to decreased mtDNA level in breast tumors.     

Heterogeneous Distribution of Fetal Microchimerism in Local Breast Cancer Environment

Fetal cells enter maternal circulation during pregnancy and persist in the woman’s body for decades, achieving a form of physiological microchimerism. These cells were also evidenced in tumors. We investigated the frequency and concentration of fetal microchimerism in the local breast cancer environment. From 19 patients with confirmed breast neoplasia, after breast surgical resection, we collected three fresh specimens from the tumor core, breast tissue at tumor periphery, and adjacent normal breast tissue. The presence of male DNA was analyzed with a quantitative PCR assay for the sex determining region gene (SRY) gene. In the group of women who had given birth to at least one son, we detected fetal microchimerism in 100% of samples from tumors and their periphery and in 64% (9 of 14) of those from normal breast tissue. The tissues from the tumor and its periphery carry a significantly increased number of SRY copies compared to its neighboring common breast tissue (p = 0.005). The median of the normalized SRY-signal was about 77 (range, 3.2–21467) and 14-fold (range, 1.3–2690) greater in the tumor and respectively in the periphery than in the normal breast tissue. In addition, the relative expression of the SRY gene had a median 5.5 times larger in the tumor than in its periphery (range, 1.1–389.4). We found a heterogeneous distribution of fetal microchimerism in breast cancer environment. In women with sons, breast neoplasia harbors male cells at significantly higher levels than in peripheral and normal breast tissue.     

Less DeltamtDNA4977 than normal in various types of tumors suggests that cancer cells are essentially free of this mutation

Levels of mtDNA(4977) deletions (DeltamtDNA(4977)) have been found to be lower in tumors than in adjacent non-tumoral tissues. In 87 cancer patients, DeltamtDNA(4977) was detected by multiplex polymerase chain reaction (PCR) amplification in 43 (49%) of the tumors and in 74 (85%) of the samples of non-tumoral tissues that were adjacent to the tumors. DeltamtDNA(4977) deletions were detected in 24% of the breast tumors, 52% of the colorectal tumors, 79% of the gastric tumors, and 40% of the head and neck tumors as compared with 77, 83, 100, and 90% of the adjacent respective non-tumoral tissues at the same DNA template dilution. Based on limiting dilution PCR of 16 tumors and their adjacent non-tumoral tissues, it was found that the amount of DeltamtDNA(4977) was 10- to 100-fold lower in the tumor than in the respective control non-tumoral tissues. Real-time PCR experiments were performed to quantify the number of DeltamtDNA(4977) deletions per cell, by determining the mitochondrial-to-nuclear DNA ratio. In all of the cases of breast, colorectal, gastric, and head and neck cancer the proportion of DeltamtDNA(4977) in tumors was lower than that of the respective non-tumoral tissue. Traces of DeltamtDNA(4977) in tumors were apparently due to contamination of tumor tissue with surrounding non-tumoral tissue, as evidenced by tumor microdissection and in situ PCR techniques, suggesting that tumors are essentially free of this mutation. Although the metabolic effect of DeltamtDNA(4977) may be minimal in normal (non-tumor) tissue, in tissue under stress, such as in tumors, even low levels of DeltamtDNA(4977) deletions may be intolerable.     

Mitochondrial DNA Copy Number Is Associated with Breast Cancer Risk

Mitochondrial DNA (mtDNA) copy number in peripheral blood is associated with increased risk of several cancers. However, data from prospective studies on mtDNA copy number and breast cancer risk are lacking. We evaluated the association between mtDNA copy number in peripheral blood and breast cancer risk in a nested case-control study of 183 breast cancer cases with pre-diagnostic blood samples and 529 individually matched controls among participants of the Singapore Chinese Health Study. The mtDNA copy number was measured using real time PCR. Conditional logistic regression analyses showed that there was an overall positive association between mtDNA copy number and breast cancer risk (P_trend = 0.01). The elevated risk for higher mtDNA copy numbers was primarily seen for women with <3 years between blood draw and cancer diagnosis; ORs (95% CIs) for 2^nd, 3^rd, 4^th, and 5^th quintile of mtDNA copy number were 1.52 (0.61, 3.82), 2.52 (1.03, 6.12), 3.12 (1.31, 7.43), and 3.06 (1.25, 7.47), respectively, compared with the 1^st quintile (P_trend = 0.004). There was no association between mtDNA copy number and breast cancer risk among women who donated a blood sample ≥3 years before breast cancer diagnosis (P_trend = 0.41). This study supports a prospective association between increased mtDNA copy number and breast cancer risk that is dependent on the time interval between blood collection and breast cancer diagnosis. Future studies are warranted to confirm these findings and to elucidate the biological role of mtDNA copy number in breast cancer risk.     

Selection of DNA aptamers for breast cancer

A method of selection of DNA aptamers to breast tumor tissue based on the use of postoperative material has been developed. Breast cancer tissues were used as the positive target; the negative targets included benign tumor tissue, adjacent healthy tissues, breast tissues from mastopathy patients, and also tissues of other types of malignant tumors. During selection a pool of DNA aptamers demonstrating selective binding to breast cancer cells and tissues and insignificant binding to breast benign tissues has been obtained. These DNA aptamers can be used for identification of protein markers, breast cancer diagnostics, and targeted delivery of anticancer drugs.     

TGF-β polymorphism and its expression correlated with CXCR4 expression in human breast cancer

The role of chemokines and the growth factors has been extensively analyzed both in cancer risk and tumor progression. The transforming growth factor beta (TGF-β) and chemokine (C-X-C motif) receptor 4 (CXCR4) genes are implicated in several diseases, including breast cancer. Genomic DNA was obtained from 21 samples of peripheral blood or from normal tissue, previously fixed in formalin and embedded in paraffin for TGF-β T869C polymorphism analyses. Total cellular RNA was extracted from the same 21 patients, but from fresh tissue (tumor and adjacent healthy from the same breast) for expression analysis by Real Time PCR. No significant differences were observed in genotype distribution according to clinicopathological characteristics. Transforming growth factor beta (TGF-β) mRNA expression was assessed according to T869C polymorphism and CC patients presented a higher TGF-β expression but not significant when compared to other genotypes (p?=?0.064). A positive correlation was observed in relative mRNA expressions of CXCR4 and TGF-β (p?=?0.020). It is known that overexpression of TGF-β by both tumor and stromal tissue can facilitate the development of metastases, mainly by TGF-β stimulated angiogenesis and increased tumor cell motility. Our findings suggested a role of these genes as progression markers for breast carcinoma.     

Alteration of mitochondrial DNA sequence and copy number in nasal polyp tissue

This study was designed to investigate the possibility that mtDNA mutations might arise in inflammatory or chronically damaged nasal polyp tissue from 23 patients. Thirteen patients (57%) displayed nasal polyp tissue-specific mtDNA mutations in the hypervariable segment of the control region and cytochrome b gene, which were not found in the corresponding blood cells and/or adjacent normal tissue. Nasal polyp tissue-specific length heteroplasmic mutations were also detected in nucleotide position (np) 303–315 homopolymeric poly C track (39%), np 514–523 CA repeats (17%) and np 16184–16193 poly C track (30%). The average mtDNA copy number was about three times higher in nasal polyp tissue than in the corresponding peripheral blood cells and adjacent non-polyp tissues. The level of reactive oxygen species (ROS) was significantly higher in the nasal polyp tissues compared to those from the corresponding samples. High level of ROS in nasal polyp tissue may contribute to development of mtDNA mutations, which may play a crucial role in the vicious cycle of pathophysiology of nasal polyps.     

Microbial Dysbiosis Is Associated with Human Breast Cancer

Breast cancer affects one in eight women in their lifetime. Though diet, age and genetic predisposition are established risk factors, the majority of breast cancers have unknown etiology. The human microbiota refers to the collection of microbes inhabiting the human body. Imbalance in microbial communities, or microbial dysbiosis, has been implicated in various human diseases including obesity, diabetes, and colon cancer. Therefore, we investigated the potential role of microbiota in breast cancer by next-generation sequencing using breast tumor tissue and paired normal adjacent tissue from the same patient. In a qualitative survey of the breast microbiota DNA, we found that the bacterium Methylobacterium radiotolerans is relatively enriched in tumor tissue, while the bacterium Sphingomonas yanoikuyae is relatively enriched in paired normal tissue. The relative abundances of these two bacterial species were inversely correlated in paired normal breast tissue but not in tumor tissue, indicating that dysbiosis is associated with breast cancer. Furthermore, the total bacterial DNA load was reduced in tumor versus paired normal and healthy breast tissue as determined by quantitative PCR. Interestingly, bacterial DNA load correlated inversely with advanced disease, a finding that could have broad implications in diagnosis and staging of breast cancer. Lastly, we observed lower basal levels of antibacterial response gene expression in tumor versus healthy breast tissue. Taken together, these data indicate that microbial DNA is present in the breast and that bacteria or their components may influence the local immune microenvironment. Our findings suggest a previously unrecognized link between dysbiosis and breast cancer which has potential diagnostic and therapeutic implications.     

DNA Copy Number Aberrations in Breast Cancer by Array Comparative Genomic Hybridization

Array comparative genomic hybridization (CGH) has been popularly used for an-alyzing DNA copy number variations in diseases like cancer. In this study, we investigated 82 sporadic samples from 49 breast cancer patients using 1-Mb reso-lution bacterial artificial chromosome CGH arrays. A number of highly frequent genomic aberrations were discovered, which may act as "drivers" of tumor pro-gression. Meanwhile, the genomic profiles of four "normal" breast tissue samples taken at least 2 cm away from the primary tumor sites were also found to have some genomic aberrations that recurred with high frequency in the primary tu-mors, which may have important implications for clinical therapy. Additionally, we performed class comparison and class prediction for various clinicopathological pa-rameters, and a list of characteristic genomic aberrations associated with different clinicopathological phenotypes was compiled. Our study provides clues for further investigations of the underlying mechanisms of breast carcinogenesis.     

Cell Surface-Specific N-Glycan Profiling in Breast Cancer

Aberrant changes in specific glycans have been shown to be associated with immunosurveillance, tumorigenesis, tumor progression and metastasis. In this study, the N-glycan profiling of membrane proteins from human breast cancer cell lines and tissues was detected using modified DNA sequencer-assisted fluorophore-assisted carbohydrate electrophoresis (DSA-FACE). The N-glycan profiles of membrane proteins were analyzed from 7 breast cancer cell lines and MCF 10A, as well as from 100 pairs of breast cancer and corresponding adjacent tissues. The results showed that, compared with the matched adjacent normal tissue samples, two biantennary N-glycans (NA2 and NA2FB) were significantly decreased (p <0.0001) in the breast cancer tissue samples, while the triantennary glycan (NA3FB) and a high-mannose glycan (M8) were dramatically increased (p = 0.001 and p <0.0001, respectively). Moreover, the alterations in these specific N-glycans occurred through the oncogenesis and progression of breast cancer. These results suggested that the modified method based on DSA-FACE is a high-throughput detection technology that is suited for analyzing cell surface N-glycans. These cell surface-specific N-glycans may be helpful in recognizing the mechanisms of tumor cell immunologic escape and could be potential targets for new breast cancer drugs.     

Identification of novel breast tumor-specific mutation(s) in the q11.2 region of chromosome 17 by RAPD/AP-PCR fingerprinting

Analysis of genetic instability in breast cancer tissues compared to uninvolved breast tissues from the same individuals by RAPD (random amplified polymorphic DNA)/AP-PCR (arbitrarily primed PCR) fingerprinting using 30 arbitrary primers revealed 190 amplified DNA fragments. Presumably, each of these represents a gene locus in a different region of the genome of breast cancer tissues. Among these amplified DNA fragments, 65 (34.2%) exhibited presence and absence or reductions and enhancements in the intensity in breast cancer tissues compared to uninvolved breast tissues from the same individuals, and 11 amplified DNA fragments (5.7%) represented polymorphisms in the uninvolved human breast tissues. Reductions and enhancements in the intensity of some of the amplified fragments were observed indicating allelic gains or losses in the breast tumor genome compared to the matched uninvolved tissue genome. The presence or absence of some of the amplified DNA fragments were observed in this study indicating homozygous deletions or insertions in the breast tumor DNA compared to the matched uninvolved tissue DNA. Notably, an insertion of a 1270 bp amplified fragment was observed in 81% (17 of 21) of the tumor samples using the primer, OPC04. This amplified fragment resolved into two, 1200 and 1300 bp, single-stranded amplified fragments on the denaturing sequencing gel. This separation into single-stranded fragments suggests that the amplified fragment contains a conformation that is semistable. The 1270 bp amplified fragment localizes to the q11.2 region of chromosome 17. Sequence analysis of this fragment showed a significant DNA base sequence similarity (93%) with one of the breast tumor-specific human EST. The similarity with EST sequences and RT-PCR analysis showed that a part of this amplified fragment is from the coding region of the genome. Any one of the events observed in this study could play an important role in the development of breast cancer or could occur during the clonal expansion of the genetically unstable breast cells.     

Feasibility of MR Metabolomics for Immediate Analysis of Resection Margins during Breast Cancer Surgery

In this study, the feasibility of high resolution magic angle spinning (HR MAS) magnetic resonance spectroscopy (MRS) of small tissue biopsies to distinguish between tumor and non-involved adjacent tissue was investigated. With the current methods, delineation of the tumor borders during breast cancer surgery is a challenging task for the surgeon, and a significant number of re-surgeries occur. We analyzed 328 tissue samples from 228 breast cancer patients using HR MAS MRS. Partial least squares discriminant analysis (PLS-DA) was applied to discriminate between tumor and non-involved adjacent tissue. Using proper double cross validation, high sensitivity and specificity of 91% and 93%, respectively was achieved. Analysis of the loading profiles from both principal component analysis (PCA) and PLS-DA showed the choline-containing metabolites as main biomarkers for tumor content, with phosphocholine being especially high in tumor tissue. Other indicative metabolites include glycine, taurine and glucose. We conclude that metabolic profiling by HR MAS MRS may be a potential method for on-line analysis of resection margins during breast cancer surgery to reduce the number of re-surgeries and risk of local recurrence.     

Epigenetic and genetic burden measures are associated with tumor characteristics in invasive breast carcinoma

The development and progression of invasive breast cancer is characterized by alterations to the genome and epigenome. However, the relationship between breast tumor characteristics, disease subtypes, and patient outcomes with the cumulative burden of these molecular alterations are not well characterized. We determined the average departure of tumor DNA methylation from adjacent normal breast DNA methylation using Illumina 450K methylation data from 700 invasive breast tumors and 90 adjacent normal breast tissues in The Cancer Genome Atlas. From this we generated a novel summary measure of altered DNA methylation, the DNA methylation dysregulation index (MDI), and examined the relation of MDI with tumor characteristics and summary measures that quantify cumulative burden of genetic mutation and copy number alterations. Our analysis revealed that MDI was significantly associated with tumor stage (P = 0.017). Across invasive breast tumor subtypes we observed significant differences in genome-wide DNA MDIs (P = 4.9E–09) and in a fraction of the genome with copy number alterations (FGA) (P = 4.6E–03). Results from a linear regression adjusted for subject age, tumor stage, and estimated tumor purity indicated a positive significant association of MDI with both MCB and FGA (P = 0.036 and P < 2.2E–16). A recursively partitioned mixture model of all 3 somatic alteration burden measures resulted in classes of tumors whose epigenetic and genetic burden profile were associated with the PAM50 subtype and mutations in TP53, PIK3CA, and CDH1. Together, our work presents a novel framework for characterizing the epigenetic burden and adds to the understanding of the aggregate impact of epigenetic and genetic alterations in breast cancer.     

脂联素及其受体在乳腺癌中的表达及其与临床病理特征的相关性分析

目的:观察和分析脂联素(ADPN)及脂联素受体(adipo R)在乳腺癌中的表达及其与临床病理特征的相关性。方法:选取60例乳腺癌患者作为病例组,选取30例良性乳腺疾病患者作为对照组,对两组患者血清ADPN水平进行检测和比较。对病例组患者肿瘤组织及对照组患者病变组织中的ADPN、adipo R1、adipo R2表达水平进行检测和比较。对病例组患者的肿瘤原发部位、肿瘤结节数量、病理类型、肿瘤局部浸润情况、淋巴结转移情况、T分期、TNM分期情况及其与ADPN、adipo R1、adipo R2水平的关联性进行观察和分析。结果:病例组患者的血清ADPN水平及肿瘤乳腺组织中ADPN、adipo R1、adipo R2表达水平均显著低于对照组,两组之间的差异均有统计学意义(P0.05)。乳腺癌患者血清ADPN水平和乳腺组织中ADPN表达水平与肿瘤的局部浸润情况、T分期、淋巴结转移情况、TNM分期具有关联性(P0.05),乳腺癌患者乳腺组织中adipo R1和adipo R2表达水平与肿瘤的病理类型、局部浸润情况、T分期、淋巴结转移情况、TNM分期具有关联性(P0.05)。结论:乳腺癌患者外周血中的ADPN及肿瘤组织中的ADPN及其受体均呈现低表达,而且其表达水平与肿瘤的病理类型、浸润和转移情况及临床分期具有关联性,有望作为乳腺癌诊断和治疗的新型靶点。     

Tumor-infiltrating lymphocytes in colorectal tumors display a diversity of T cell receptor sequences that differ from the T cells in adjacent mucosal tissue

Tumors from colorectal cancer (CRC) are generally immunogenic and commonly infiltrated with T lymphocytes. However, the details of the adaptive immune reaction to these tumors are poorly understood. We have accrued both colon tumor samples and adjacent healthy mucosal samples from 15 CRC patients to study lymphocytes infiltrating these tissues. We apply a method for detailed sequencing of T-cell receptor (TCR) sequences from tumor-infiltrating lymphocytes (TILs) in CRC tumors at high throughput to probe T-cell clones in comparison with the TCRs from adjacent healthy mucosal tissue. In parallel, we captured TIL counts using standard immunohistochemistry. The variation in diversity of the TIL repertoire was far wider than the variation of T-cell clones in the healthy mucosa, and the oligoclonality was higher on average in the tumors. However, the diversity of the T-cell repertoire in both CRC tumors and healthy mucosa was on average 100-fold lower than in peripheral blood. Using the TCR sequences to identify and track clones between mucosal and tumor samples, we determined that the immune response in the tumor is different than in the adjacent mucosal tissue, and the number of shared clones is not dependent on distance between the samples. Together, these data imply that CRC tumors induce a specific adaptive immune response, but that this response differs widely in strength and breadth between patients.     

Detection of Human Papillomavirus DNA in Patients with Breast Tumor in China

The presence of HPV in breast tissue and the potential causal association between human papillomavirus (HPV) and breast cancer (BC) remains controversial. The aim of the present study was to compare the HPV prevalence in BC tissues, adjacent normal breast tissues and breast benign disease tissues and to investigate the possible association between HPV and breast tumor development in Chinese women. Paraffin-embedded specimens from 187 pairs of BCs including tumor and normal breast tissue adjacent to tumors and 92 breast benign lesions between June 2009 and July 2014 were investigated by nested polymerase chain reaction (PCR) and type-specific PCR, respectively. With strictly quality control, HPV positive infection was detected in three BC tissues. No HPV positive infection was detected in all normal breast tissue adjacent to tumors and benign breast tissues. Through our detailed analysis, rare HPV infection in this study suggests that HPV might not be associated with BC progression.     

Mitochondrial copy number and risk of breast cancer: A pilot study

It has been proposed that the copy number of mitochondria DNA (mtDNA) per cell reflects gene–environment interactions between unknown hereditary factors and exposures affecting levels of oxidative stress. However, whether copy number of mtDNA could be a risk predictor of oxidative stress-related human cancers, such as breast cancer, remains to be determined. To explore the role of mtDNA copy number in breast cancer etiology, we analyzed mtDNA copy number in whole blood from 103 patients with breast cancer and 103 matched control subjects and examined in relation to endogenous antioxidants. Case patients with breast cancer had a statistically significantly higher mtDNA copy number than control subjects (median: 1.29 vs. 0.80, P < 0.01). High mtDNA copy number (above the median in controls) was associated with a statistically significantly increased risk of breast cancer, compared with low copy number (Odds ratio (OR) = 4.67, 95% CI: 2.45–8.92), with a statistically significant dose–response relationship in trend analysis (P < 0.01). Moreover, mtDNA copy number was significantly inversely associated with several important endogenous oxidants and antioxidants in blood in either the cases (total glutathione, CuZn-SOD activity and myeloperoxidase (MPO)) or the controls (catalase (CAT) activity). These results suggest the mtDNA copy number could be associated with risk of breast cancer, perhaps through an oxidative stress mechanism.     

Estrogen and progesterone receptors in colorectal cancer and surrounding mucosa

In this prospective study we have quantified by means of ELISA-methods the cytosolic content of estrogen (ER) and progesterone receptors (PgR) in tumoral tissue and paired normal mucosa from 163 patients with resectable colorectal cancer. Survival analysis was performed in a subgroup of 120 patients and the mean follow-up period was 24.9 months. The cutoff for ER and PgR levels was set at 1 fmol/mg protein. On the basis of this cutoff 20.9% of the cancers were ER positive and 25.8% were PgR positive; normal adjacent tissue presented ER in 18.4% and PgR in 24.5%. Our results did not show any significant correlation between ER and PgR levels in neoplastic tissues. Howewer, a correlation was found in normal mucosa samples (p=0.02). Statistical analysis showed that there was no correlation between tumor ER and PgR content and patient age or sex, tumor location, Dukes' stage, histological differentiation, DNA ploidy status and S-phase fraction. Furthermore, the results did not show any statistical differences in relapse-free and overall survival curves calculated for patients classified according to the hormone receptor content of their tumors. ER and PgR were detected at low levels in normal and neoplastic colorectal tissues without any significant relationship to either clinicopathological tumor characteristics or patient outcome. Their possible role in colorectal cancer remains to be elucidated.     

The MiAge Calculator: a DNA methylation-based mitotic age calculator of human tissue types

Cell division is important in human aging and cancer. The estimation of the number of cell divisions (mitotic age) of a given tissue type in individuals is of great interest as it allows not only the study of biological aging (using a new molecular aging target) but also the stratification of prospective cancer risk. Here, we introduce the MiAge Calculator, a mitotic age calculator based on a novel statistical framework, the MiAge model. MiAge is designed to quantitatively estimate mitotic age (total number of lifetime cell divisions) of a tissue using the stochastic replication errors accumulated in the epigenetic inheritance process during cell divisions. With the MiAge model, the MiAge Calculator was built using the training data of DNA methylation measures of 4,020 tumor and adjacent normal tissue samples from eight TCGA cancer types and was tested using the testing data of DNA methylation measures of 2,221 tumor and adjacent normal tissue samples of five other TCGA cancer types. We showed that within each of the thirteen cancer types studied, the estimated mitotic age is universally accelerated in tumor tissues compared to adjacent normal tissues. Across the thirteen cancer types, we showed that worse cancer survivals are associated with more accelerated mitotic age in tumor tissues. Importantly, we demonstrated the utility of mitotic age by showing that the integration of mitotic age and clinical information leads to improved survival prediction in six out of the thirteen cancer types studied. The MiAge Calculator is available at http://www.columbia.edu/～sw2206/softwares.htm.