A six-gene signature predicting breast cancer lung metastasis

The lungs are a frequent target of metastatic breast cancer cells, but the underlying molecular mechanisms are unclear. All existing data were obtained either using statistical association between gene expression measurements found in primary tumors and clinical outcome, or using experimentally derived signatures from mouse tumor models. Here, we describe a distinct approach that consists of using tissue surgically resected from lung metastatic lesions and comparing their gene expression profiles with those from nonpulmonary sites, all coming from breast cancer patients. We show that the gene expression profiles of organ-specific metastatic lesions can be used to predict lung metastasis in breast cancer. We identified a set of 21 lung metastasis-associated genes. Using a cohort of 72 lymph node-negative breast cancer patients, we developed a 6-gene prognostic classifier that discriminated breast primary cancers with a significantly higher risk of lung metastasis. We then validated the predictive ability of the 6-gene signature in 3 independent cohorts of breast cancers consisting of a total of 721 patients. Finally, we show that the signature improves risk stratification independently of known standard clinical variables and a previously established lung metastasis signature based on an experimental breast cancer metastasis model.     

Contralateral breast cancers: Independent cancers or metastases?

A cancer in the contralateral breast in a woman with a previous or synchronous breast cancer is typically considered to be an independent primary tumor. Emerging evidence suggests that in a small subset of these cases the second tumor represents a metastasis. We sought to investigate the issue using massively parallel sequencing targeting 254 genes recurrently mutated in breast cancer. We examined the tumor archives at Memorial Sloan Kettering Cancer Center for the period 1995–2006 to identify cases of contralateral breast cancer where surgery for both tumors was performed at the Center. We report results from 49 patients successfully analyzed by a targeted massively parallel sequencing assay. Somatic mutations and copy number alterations were defined by state‐of‐the‐art algorithms. Clonal relatedness was evaluated by statistical tests specifically designed for this purpose. We found evidence that the tumors in contralateral breasts were clonally related in three cases (6%) on the basis of matching mutations at codons where somatic mutations are rare. Clinical data and the presence of similar patterns of gene copy number alterations were consistent with metastasis for all three cases. In three additional cases, there was a solitary matching mutation at a common PIK3CA locus. The results suggest that a subset of contralateral breast cancers represent metastases rather than independent primary tumors. Massively parallel sequencing analysis can provide important evidence to clarify the diagnosis. However, given the inter‐tumor mutational heterogeneity in breast cancer, sufficiently large gene panels need to be employed to define clonality convincingly in all cases.     

上皮细胞黏附分子表达与乳腺癌淋巴结转移的相关性

目的 探讨上皮细胞黏附分子(Ep-CAM)在乳腺癌中的表达及意义.方法 收集经病理学确诊的乳腺癌新鲜组织标本23例及配对的淋巴结转移灶.应用同位素标记的相对和绝对定量(iTRAQ)蛋白质组学技术筛选及鉴定乳腺癌原发灶和淋巴结转移灶的差异表达蛋白,在4例新鲜乳腺癌原发灶组织和配对淋巴结转移灶中进行Western blotting检测Ep-CAM的表达.采用免疫组织化学法检测252例乳腺病变标本中Ep-CAM的表达.结果 定量蛋白质组学检测结果显示乳腺癌原发灶和淋巴结转移灶存在差异表达蛋白,其中Ep-CAM在转移灶的表达高于原发灶,Western blotting显示Ep-CAM在转移灶的表达(1.46±0.22)高于原发灶(1.16±0.09),变化趋势与蛋白质组学结果一致.免疫组织化学检测结果显示,Ep-CAM在淋巴结转移灶中的阳性表达率(93.16％,109/117)显著高于无淋巴结转移的乳腺癌原发灶(72.73％,64/88),差异有统计学意义(x2=15.921,P=0.000);有淋巴结转移的乳腺癌原发灶Ep-CAM阳性表达率为72.65％(85/117),较配对淋巴结转移灶阳性表达率低,差异有统计学意义(P=0.001).结论 Ep-CAM在乳腺癌原发灶和淋巴结转移灶呈差异表达,该蛋白可能与乳腺癌淋巴结转移有关.     

原发性双侧乳腺癌预后分析

目的探讨原发性双侧乳腺癌的预后。方法回顾了119例原发性双侧乳腺癌患者的临床病理资料。分析双侧乳腺癌的预后情况及年龄和月经因素对预后的影响。结果双侧乳腺癌占同期可手术乳腺癌的3．67％（119／3239）。双侧乳腺癌患者中位年龄46岁，双侧发病中位间隔时间为48个月。双侧癌发生间隔时间按24个月来划分同时性（43／119）和异时性乳腺癌。从第二侧原发肿瘤手术算起，中位随访48个月，单侧和双侧乳腺癌的总生存率（OS）差异无显著性，但后者的无病生存率（DFS）较低（P=0．0469）；同时性和异时性乳腺癌的DFS（P=0．5399）和OS（P=0．5829）的差异无显著性。单因素分析发现第二原发癌发病≤45岁、双侧均绝经前发病或分别绝经前后发病的患者预后较差；多因素分析发现第二原发癌发生年龄是独立预后因素。结论从第二原发肿瘤手术起随访，单侧和双侧乳腺癌的OS无显著差别，但后者DFS较差；同时性和异时性双侧乳腺癌的预后相似。第二原发癌发病年龄是双侧乳腺癌患者的重要预后指标。     

Breast cancer brain metastases show increased levels of genomic aberration-based homologous recombination deficiency scores relative to their corresponding primary tumors

BackgroundBased on its mechanism of action, PARP inhibitor therapy is expected to benefit mainly tumor cases with homologous recombination deficiency (HRD). Therefore, identification of tumor types with increased HRD is important for the optimal use of this class of therapeutic agents. HRD levels can be estimated using various mutational signatures from next generation sequencing data and we used this approach to determine whether breast cancer brain metastases show altered levels of HRD scores relative to their corresponding primary tumor.Patients and methodsWe used a previously published next generation sequencing dataset of 21 matched primary breast cancer/brain metastasis pairs to derive the various mutational signatures/HRD scores strongly associated with HRD. We also carried out the myChoice HRD analysis on an independent cohort of 17 breast cancer patients with matched primary/brain metastasis pairs.ResultsAll of the mutational signatures indicative of HRD showed a significant increase in the brain metastases relative to their matched primary tumor in the previously published whole exome sequencing dataset. In the independent validation cohort, the myChoice HRD assay showed an increased level in 87.5% of the brain metastases relative to the primary tumor, with 56% of brain metastases being HRD positive according to the myChoice criteria.ConclusionsThe consistent observation that brain metastases of breast cancer tend to have higher HRD measures may raise the possibility that brain metastases may be more sensitive to PARP inhibitor treatment. This observation warrants further investigation to assess whether this increase is common to other metastatic sites as well, and whether clinical trials should adjust their strategy in the application of HRD measures for the prioritization of patients for PARP inhibitor therapy.     

The risk factors and prognosis of bilateral primary breast cancer: a comparative study with unilateral breast cancer

This study was performed to determine the risk factors and evaluate the outcome of bilateral breast cancer (BBC). We reviewed the records of 170 patients with BBC and 1,677 with unilateral breast cancer (UBC), and compared their personal history, histopatholgical characteristics, clinical findings, and treatment, and postoperative follow-up records. The patients with UBC were more likely to develop contralateral cancer with the features including: young age at onset, especially younger than 40, premenopause, late primiparity, breast cancer family history, benign mammary disease history, and a tumor larger than 5 cm (p < 0.05). After adjustment by multivariate analysis, we concluded that breast cancer family history and age at onset younger than 40 years old were the independent risk factors for BBC. There were no significant differences for distant metastasis or overall survival between BBC and UBC (p > 0.05). We observed that 64.1% of the second breast cancer occurred within 5 years after the operation of the first cancer, and medical examination could improve the early diagnosis of the contralateral breast cancer. Contrary to common belief, our study showed that BBC and UBC had similar biological features and prognosis (p > 0.05). The excessive treatment and prophylactic measures may be unnecessary in this seemingly aggressive breast cancer. The patients with UBC younger than 40 or with breast cancer family history should have intensive contralateral breast followup, especially within 5 years after in the initial treatment.     

Establishing the origin of metastatic deposits in the setting of multiple primary malignancies: The role of massively parallel sequencing

In this proof‐of‐principle study, we sought to define whether targeted capture massively parallel sequencing can be employed to determine the origin of metastatic deposits in cases of synchronous primary malignancies and metastases in distinct anatomical sites. DNA samples extracted from synchronous tumor masses in the breast, adnexal, and pelvic‐peritoneal regions from a 62‐year‐old BRCA1 germline mutation carrier were subjected to targeted massively parallel sequencing using a platform comprising 300 cancer genes known to harbor actionable mutations. In addition to BRCA1 germline mutations, all lesions harbored somatic loss of the BRCA1 wild‐type allele and TP53 somatic mutations. The primary breast cancer displayed a TP53 frameshift (p.Q317fs) mutation, whereas and the adnexal lesion harbored a TP53 nonsense (p.R213*) mutation, consistent with a diagnosis of two independent primary tumors (i.e. breast and ovarian cancer). The adnexal tumor and all pelvic‐peritoneal implants harbored identical TP53 (p.R213*) and NCOA2 (p.G952R) somatic mutations. Evidence of genetic heterogeneity within and between lesions was observed, both in terms of somatic mutations and copy number aberrations. The repertoires of somatic genetic aberrations found in the breast, ovarian, and pelvic‐peritoneal lesions provided direct evidence in support of the distinct origin of the breast and ovarian cancers, and established that the pelvic‐peritoneal implants were clonally related to the ovarian lesion. These observations were consistent with those obtained with immunohistochemical analyses employing markers to differentiate between carcinomas of the breast and ovary, including WT1 and PAX8. Our results on this case of a patient with BRCA1‐mutant breast and ovarian cancer demonstrate that massively parallel sequencing may constitute a useful tool to define the relationship, clonality and intra‐tumor genetic heterogeneity between primary tumor masses and their metastatic deposits in patients with multiple primary malignancies and synchronous metastases.     

Metformin and erlotinib synergize to inhibit basal breast cancer

Basal-like breast cancers (BBCs) are enriched for increased EGFR expression and decreased expression of PTEN. We found that treatment with metformin and erlotinib synergistically induced apoptosis in a subset of BBC cell lines. The drug combination led to enhanced reduction of EGFR, AKT, S6 and 4EBP1 phosphorylation, as well as prevented colony formation and inhibited mammosphere outgrowth. Our data with other compounds suggested that biguanides combined with EGFR inhibitors have the potential to outperform other targeted drug combinations and could be employed in other breast cancer subtypes, as well as other tumor types, with activated EGFR and PI3K signaling. Analysis of BBC cell line alterations led to the hypothesis that loss of PTEN sensitized cells to the drug combination which was confirmed using isogenic cell line models with and without PTEN expression. Combined metformin and erlotinib led to partial regression of PTEN-null and EGFR-amplified xenografted MDA-MB-468 BBC tumors with evidence of significant apoptosis, reduction of EGFR and AKT signaling, and lack of altered plasma insulin levels. Combined treatment also inhibited xenografted PTEN null HCC-70 BBC cells. Measurement of trough plasma drug levels in xenografted mice and a separately performed pharmacokinetics modeling study support possible clinical translation.     

Striking similarities in genetic aberrations between a rectal tumor and its lung recurrence

We are reporting on a colorectal cancer patient with the longest disease-free interval ever published, where chromosomal microarray analysis was used to confirm the link between the primary and metastatic lesions. This rare case reports on a patient with late recurrence of colorectal cancer in the lung 19 years after its initial diagnosis. We used high-resolution array CGH (aCGH) to analyze the genetic aberrations of both the primary rectal and the recurrent metastatic lung lesions. Interestingly, we found striking similarities between the two lesions, despite the 19 years disease-free interval. In addition, most of the genes that were previously reported to be associated with a high recurrence score showed copy number gains by aCGH in one or both lesions. Our findings suggest that aCGH may be a helpful tool in analyzing the origin of metastases and underline the need for a better understanding of the characteristics of rectal tumors that have a late recurrence potential.     

Clinical and genomic characterization of mutational signatures across human cancers

Mutational signatures, the generic patterns of mutations, are the footprints of both endogenous and exogenous factors that have influenced cancer development. To date, dozens of mutational signatures have been discerned through computational methods. However, the etiology, mutational properties, clonality, immunology and prognostic value of mutation signatures across cancer types are poorly understood. To address this, we extensively characterized mutational signatures across 8836 cancer samples spanning 42 cancer types. We confirmed and extended clinical and genomic features associated with mutation signatures. Mutation distribution analysis showed that most mutation processes were depleted in exons and APOBEC signatures (SBS2 and SBS13), the Pol-η related signature (SBS9) and SBS40 tended to contribute clustered mutations. We observed that age-related signatures (SBS1 and SBS5) and SBS40 tended to induce mutations affecting cancer genes and subclonal drivers posted by specific signatures (eg, mismatch repair deficiency-related signature SBS44) were unlikely subjected to positive selection. We also revealed early mutation signatures (eg, UV light exposure-related signature SBS7a) and signatures (eg, reactive oxygen species-related signature SBS18) predominated in the late stage of tumorigenesis. Comprehensive association analysis of mutation processes with microenvironment revealed that APOBEC- and mismatch repair deficiency-related signatures were positively associated with immune parameters, while age-related signatures showed negative correlations. In addition, prognostic association analysis showed that many signatures were favorable (eg, SBS9) or adverse factors (eg, SBS18) of patient survival. Our findings enhance appreciation of the role of mutational signatures in tumor evolution and underline their potential in immunotherapy guidance and prognostic prediction.     

Mutational Landscape and Evolutionary Pattern of Liver and Brain Metastasis in Lung Adenocarcinoma

IntroductionA comprehensive genomic analysis of paired primary tumors and their metastatic lesions may provide new insights into the biology of metastatic processes and therefore guide the development of novel strategies for intervention. To date, our knowledge of the genetic divergence and phylogenetic relationships among diverse metastatic lesions from cancer remains limited.MethodsWe performed whole-exome sequencing in 84 tissue and blood samples from 26 patients with lung adenocarcinoma having liver metastases (LiM) or brain metastases (BrM) before any systemic therapy, with the goal to molecularly characterize the metastatic process. Mutational landscape and evolutionary patterns were compared between paired primary lesions (primary lesion of LiM or BrM) and metastases (metastatic site of LiM or BrM).ResultsWe found that common driver mutations, including TP53 and EGFR, were highly consistent between paired primary and metastatic tumors. Although tumor mutational burden was comparable among groups, the LiM group had significantly higher mutational and copy number variational similarity than the BrM group between paired primary lesions and metastases (p = 0.019 and p = 0.035, respectively). Phylogenetic analysis further revealed that LiM-competent disseminations had a higher level of genetic similarity to their paired primary lesions and were genetically diverged from their primary tumors at a relatively later stage than those of BrM. These results suggest that LiM favorably followed the linear progression model, whereas BrM was more consistent with the parallel progression model.ConclusionsThis study suggests that the mutational landscape and evolutionary pattern was distinctly different between the LiM and BrM of lung adenocarcinoma.     

Evaluation of malignancy-risk gene signature in breast cancer patients

We recently developed a malignancy-risk gene signature that was shown to identify histologically-normal tissues with a cancer-like profile. Because the signature was rich with proliferative genes, we postulated it might also be prognostic for existing breast cancers. We evaluated the malignancy risk gene signature to see its clinical association with cancer relapse/progression, and cancer prognosis using six independent external datasets. Six independent external breast cancer datasets were collected and analyzed using the malignancy risk gene signature designed to assess normal breast tissues. Evaluation of the signature in external datasets suggested a strong clinical association with cancer relapse/progression, and prognosis with minimal overlap of signature gene sets. These results suggest a prognostic role for the malignancy risk gene signature in the assessment of existing cancer. Proliferative biology dominates not only the earliest stages of tumor development but also later stages of tumor progression and metastasis.     

Properties of Synchronous Versus Metachronous Bilateral Breast Carcinoma with Long Time Follow Up

Background: Breast cancer is the most common cancer type among women with increasing incidence rates,improved prognosis and survival. According to the localization of the tumor, breast cancer is designated asunilateral (UBC) or bilateral (BBC). BBC can be classified as synchronous (SBBC) or metachronous (MBBC)based on the time interval between the diagnosis of the first and the secondary tumors. According to the guidelineof WHO 2012, BBC is generally defined as SBBC when contralateral breast carcinoma is diagnosed within 3months. The aim of this study was to compare the characteristics and patterns of metastasis of BBC patientswith UBC. Materials and Methods: A cohort of 768 patients with breast cancer treated at the Turkish Ministryof Health-Izmir Bozyaka Research and Training Hospital between 1976 and 2012 were studied. Survival analysiswas performed comparing UBC and BBC patients. In addition, evaluations were performed in patients withSBBC and MBBC sub-groups. We used a 3-months interval to distinguish metachronous from synchronous.Results: When clinical and histopathological parameters were statistically evaluated, ER status, event-free andoverall survival were found to be significant between UBC and BBC patients. In comparison of SBBC and MBBCpatients, age, histological type of tumor, event-free and overall survival were found to be significant. Conclusions:BBC cases were found to show worse prognosis than UBC cases. Among BBC, SBBC had the worst prognosisbased on overall survival rates.     

Quantification of multicellular colonization in tumor metastasis using exome-sequencing data

Metastasis is a major cause of cancer-related mortality, and it is essential to understand how metastasis occurs in order to overcome it. One relevant question is the origin of a metastatic tumor cell population. Although the hypothesis of a single-cell origin for metastasis from a primary tumor has long been prevalent, several recent studies using mouse models have supported a multicellular origin of metastasis. Human bulk whole-exome sequencing (WES) studies also have demonstrated a multiple “clonal” origin of metastasis, with different mutational compositions. Specifically, there has not yet been strong research to determine how many founder cells colonize a metastatic tumor. To address this question, under the metastatic model of “single bottleneck followed by rapid growth,” we developed a method to quantify the “founder cell population size” in a metastasis using paired WES data from primary and metachronous metastatic tumors. Simulation studies demonstrated the proposed method gives unbiased results with sufficient accuracy in the range of realistic settings. Applying the proposed method to real WES data from four colorectal cancer patients, all samples supported a multicellular origin of metastasis and the founder size was quantified, ranging from 3 to 17 cells. Such a wide-range of founder sizes estimated by the proposed method suggests that there are large variations in genetic similarity between primary and metastatic tumors in the same subjects, which may explain the observed (dis)similarity of drug responses between tumors.     

Quantitative DNA fingerprinting may distinguish new primary breast cancer from disease recurrence.

PURPOSE: Approximately 10% of women with breast cancer develop a second breast tumor, either a new primary or a recurrence. Differentiating between these entities using standard clinical and pathologic criteria remains challenging. Ambiguous cases arise, and misclassifications may occur. We investigated whether quantitative DNA fingerprinting, based on allele imbalance (AI) or loss of heterozygosity (LOH), could evaluate clonality and distinguish second primary breast cancer from recurrence. METHODS: We developed a scoring system based on the AI/LOH fingerprints of 20 independent breast tumors and generated a decision rule to classify any breast tumor pair as related or unrelated. We validated this approach on eight related tumors (cancers and synchronous positive lymph nodes). Finally, we analyzed paired tumors from 13 women (bilateral cancers, primary tumors and contralateral positive axillary lymph nodes, or two ipsilateral tumors). Each pair's genetic classification was compared with their clinical diagnosis and outcome. RESULTS: Each independent cancer had a unique fingerprint. Every tumor pair's relationship was quantifiable. Six of eight related tumor pairs were genetically classified correctly, two were indeterminate, and none were misclassified. Among the 13 women with two cancers, four of five clinically indeterminate pairs could be classified genetically. In three of 13 women, the pair's classification contradicted the clinical diagnosis. These women had bilateral cancers genetically classified as related and disease progression. This challenges the paradigm that bilateral cancers represent independent tumors. Overall, women with tumors genetically classified as related had poorer outcomes. CONCLUSION: Quantitative AI/LOH fingerprinting is a potentially valuable tool to improve diagnosis and optimize treatment for the growing number of second breast malignancies.     

Clonal analysis of human breast cancer by means of the polymerase chain reaction.

Clonality of human breast cancer was analyzed in small DNA samples prepared from cryostat sections, by means of the polymerase chain reaction (PCR). The method used for clonal analysis was based on restriction fragment length polymorphism of X-chromosome-linked phosphoglycerokinase (PGK) gene and on the differential methylation of the PGK gene due to random inactivation of one of two X-chromosomes by methylation in females. All the 20 breast cancer samples analyzed by the PCR-based method were monoclonal in origin and adjacent normal breast tissues were polyclonal. When DNA samples were prepared from widely separated sites of cancers, every sample was found to be monoclonal, always exhibiting inactivation of the same X-chromosome in each tumor. The study on sensitivity showed that the PCR-based method for clonal analysis can detect the presence of monoclonal cells against a polyclonal background when the monoclonal cell population is 50% or more. These results demonstrate that clonal analysis by means of PCR offers a good method for studying the clonality in small DNA samples prepared from cryostat sections of tumors. This method could be applied to distinguish between benign (polyclonal) and malignant (monoclonal) breast lesions.     

Breast carcinoma with brain metastases: clinical analysis and immunoprofile on tissue microarrays

BackgroundDevelopment of brain metastasis in patients with breast carcinoma correlates with poor outcome. Identification of tumor characteristics associated with breast cancer brain metastases (BCBM) could help identify patients at risk.Patients and methodsWe studied 209 patients with BCBM. We evaluated a panel of proteins relevant to the biology of breast carcinoma on tissue microarrays of 133 primary tumors and 56 BCBM, including paired samples from 43 patients, and correlated the findings with the clinical outcome.ResultsThe median survival after BCBM diagnosis was 19 months (95% confidence interval, 13–23 months). Patients presenting with solitary metastasis had a significantly longer median survival than those with multiple lesions (25 versus 11 months, P ≤ 0.0001). We found no significant discordance in the expression of tested markers, but identified a possible association between the expression of basal cytokeratin CK5/6 in the primary carcinoma and the development of multiple rather than solitary brain metastases.ConclusionsExpression of antigens commonly associated with breast carcinoma does not differ significantly between the primary tumor and the corresponding brain metastases. Although no specific immunoprofile identifies breast carcinomas that develop brain metastases, we observed a possible association between CK5/6 expression in the primary tumor and multiple versus solitary BCBM.     

Expression of the breast cancer metastasis suppressor gene, BRMS1, in human breast carcinoma: lack of correlation with metastasis to axillary lymph nodes.

The BRMS1 (breast cancer metastasis suppressor 1) gene has been found to suppress metastasis in animal models without inhibiting primary tumor growth. The aim of this study was to measure expression of BRMS1 mRNA in a panel of human breast carcinomas and compare its expression with parameters of local dissemination such as tumor size and lymph node metastasis. We also compared expression of BRMS1 mRNA in normal breast tissue, fibroadenomas, primary breast cancers and axillary nodal metastases from primary breast cancers. BRMS1 mRNA was detected in 10/11 (90%) specimens of normal breast tissue, 12/16 (75%) fibroadenomas, 64/82 (78%) primary breast cancer and 11/15 (64%) lymph node metastases (p, NS). In the primary cancer, expression was independent of tumor size, tumor grade, metastasis to axillary nodes and hormone receptor status. Furthermore, similar levels of BRMS1 were found in normal breast tissue, primary breast carcinomas and lymph node metastases from primary breast cancer. Our results do not suggest a role for BRMS1 in suppressing metastasis to local lymph nodes in patients with breast cancer.