Age at diagnosis predicts local recurrence in women treated with breast-conserving surgery and postoperative radiation therapy for ductal carcinoma in situ: a population-based outcomes analysis.

PURPOSE: The main goal of treating ductal carcinoma in situ (dcis) is to prevent the development of invasive breast cancer. Most women are treated with breast-conserving surgery (bcs) and radiotherapy. Age at diagnosis may be a risk factor for recurrence, leading to concerns that additional treatment may be necessary for younger women. We report a population-based study of women with dcis treated with bcs and radiotherapy and an evaluation of the effect of age on local recurrence (lr). METHODS: All women diagnosed with dcis in Ontario from 1994 to 2003 were identified. Treatments and outcomes were collected through administrative databases and validated by chart review. Women treated with bcs and radiotherapy were included. Survival analyses were performed to evaluate the effect of age on outcomes. RESULTS: We identified 5752 cases of dcis; 1607 women received bcs and radiotherapy. The median follow-up was 10.0 years. The 10-year cumulative lr rate was 27% for women younger than 45 years, 14% for women 45-50 years, and 11% for women more than 50 years of age (p < 0.0001). The 10-year cumulative invasive lr rate was 22% for women younger than 45 years, 10% for women 45-50 years, and 7% for women more than 50 years of age (p < 0.0001). On multivariate analyses, young age (<45 years) was significantly associated with lr and invasive lr [hazard ratio (hr) for lr: 2.6; 95% confidence interval (ci): 1.9 to 3.7; p < 0.0001; hr for invasive lr: 3.0; 95% ci: 2.0 to 4.4; p < 0.0001]. An age of 45-50 years was also significantly associated with invasive lr (hr: 1.6; 95% ci: 1.0 to 2.4; p = 0.04). CONCLUSIONS: Age at diagnosis is a strong predictor of lr in women with dcis after treatment with bcs and radiotherapy.

Mitochondrial D310 mutations in the early development of breast cancer.

BACKGROUND: The role of mitochondrial DNA (mtDNA) mutations in the development of breast cancer is largely unknown. In this study, we investigated the frequency and pattern of mutations in the D310 region, the most commonly mutated region in mtDNA, in a series of breast lesions. METHODS: Using capillary electrophoresis, we genotyped the D310 sequence of neoplastic epithelial cells from 23 patients with synchronous ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC), 26 patients with IDC only and 29 patients with DCIS only. RESULTS: A majority of DCIS (68.4%) and IDC (71.4%) lesions harbour different D310 sequences compared with their matched normal control. Specific D310 sequences were more frequently identified in tumour samples (77.1% of DCIS and 75.5% of IDC) compared with normal tissues (35.3% of normal; P<0.0001). No difference was identified between DCIS lesions with synchronous IDC and those from pure DCIS cases. In five cases, histologically normal tissue adjacent to tumour was found to share D310 sequences with the tumour, while normal tissue taken further away did not. CONCLUSION: Although D310 alterations do not seem to be related to DCIS progression, they were found in histologically normal cells adjacent to tumour. This suggests a field of genetically altered cells, thus D310 mutations could represent a potential marker for the clonal expansion of premalignant breast cancer cells.

Acquisition of metastatic tissue from patients with bone metastases from breast cancer.

Biopsies of metastatic tissue are increasingly being performed. Bone is the most frequent site of metastasis in breast cancer patients, but bone remains technically challenging to biopsy. Difficulties with both tissue acquisition and techniques for analysis of hormone receptor status are well described. Bone biopsies can be carried out by either by standard posterior iliac crest bone marrow trephine/aspiration or CT-guided biopsy of a radiologically evident bone metastasis. The differential yield of these techniques is unknown. Results from three prospective studies of similar methodology were pooled. Patients underwent both an outpatient posterior iliac crest bone marrow trephine/aspiration and a CT-guided biopsy of a radiologically evident bone metastasis. Samples were assessed for the presence of malignant cells and where possible also for estrogen (ER) and progesterone receptor (PgR) expression. 40 patients were enrolled. Bone marrow aspiration/trephine biopsy was completed in 39/40 (97.5%) and CT-guided biopsy was completed in 34/40 (85%) of patients. Sufficient tumor cells for hormone receptor analysis were available in 19/39 (48.8%) and 16/34 (47%) of and bone marrow aspiration/trephine and CT-guided biopsies, respectively. Significant discordance in ER and PgR between the primary and the bone metastasis was also seen. Nine patients had tissue available from both bone marrow and CT-guided bone biopsies. ER and PgR concordance between these sites was 100 and 78%, respectively. Performing studies on human bone metastases is technically challenging, with relatively low yields regardless of technique. Given resource issues and similar success rates when comparing both techniques, bone marrow examination may be utilized first and if inadequate tissue is obtained, CT-guided biopsies can then be used.

Limited tissue fixation times and whole genomic amplification do not impact array CGH profiles.

BACKGROUND: Array comparative genomic hybridisation (CGH) is a powerful method for the genetic analysis of lesional and normal tissues to identify genomic imbalances associated with malignancies. However, the use of this technique with DNA extracted from archival formalin fixed, paraffin embedded (FFPE) tissue specimens, the most widely available resource for retrospective studies, is subject to quantitative and qualitative limitations. In this report, the suitability and integrity of the DNA extracted from FFPE MCF7 breast cancer cells fixed for different periods of time for array CGH applications were examined. RESULTS: Using our established cDNA microarray protocol in conjunction with whole genome amplification methods, the genetic profiles of freshly harvested MCF7 cells and their matched FFPE counterparts were analysed. Congruent profiles between FFPE MCF7 cells and their fresh counterpart and between amplified and non-amplified FFPE MCF7 cells were observed. Our results demonstrate that formalin fixation of <20 hours has no significant adverse effect on the integrity of DNA for array CGH studies. CONCLUSIONS: Our findings attest to the fidelity of our array CGH methods to effectively examine material recovered from FFPE tissue specimens for microarray applications. This in turn has great potential to identify novel diagnostic and prognostic markers for human disease.

p53 missense mutations in microdissected high-grade ductal carcinoma in situ of the breast.

BACKGROUND: To understand the role of sporadic mutations in the tumor suppressor gene p53 (also known as TP53) in the pathogenesis of breast cancer, it is important to identify at which histologic stage such mutations first occur. We previously showed that a p53 mutation present in invasive breast cancer was found in all surrounding areas of ductal carcinoma in situ (DCIS) but not in areas of hyperplasia or normal breast epithelium. In the present investigation, we studied patients with DCIS, but without invasive breast cancer, to determine the spectrum of DCIS types that can harbor a p53 mutation. METHODS: Formalin-fixed, paraffin-embedded tissues from 94 patients with DCIS were evaluated histologically for the predominant cellular architectural pattern, degree of necrosis, and nuclear grade. Each specimen was also assigned an overall histologic grade (with the use of the Van Nuys Prognostic Index pathologic classification). Tissue specimens were stained immunohistochemically with an anti-p53 antibody. Positively stained tissue areas were analyzed for the presence of p53 mutations by single-strand conformation polymorphism and direct sequencing. All statistical tests were two-sided. RESULTS: DCIS from 10 of 94 patients were found to contain p53 missense mutations. All 10 were of a solid or a comedo histologic pattern and contained cells of nuclear grade 2 or 3 (i.e., more abnormal nuclei). The frequency of p53 missense mutations was statistically significantly different among the three overall histologic grade categories (zero [0%] of 49 with low-grade DCIS, one [4.35%] of 23 with intermediate-grade DCIS, and nine [40.9%] of 22 with high-grade DCIS; df = 2 and P<.0001). CONCLUSION: The DCIS types in patients in this series are representative of clinically detected DCIS. Our finding that p53 mutations can occur before the development of invasive breast cancer, particularly in DCIS of high histologic grade, has potentially important implications for prevention and treatment.

Frequency of p53 mutations in breast carcinomas from Ashkenazi Jewish carriers of BRCA1 mutations.

BACKGROUND: Breast carcinomas occurring in carriers of BRCA1 gene mutations may have a distinctly different pathway of molecular pathogenesis from those occurring in noncarriers. Data from murine models implicate loss of p53 (also known as TP53) gene function as a critical early event in the malignant transformation of cells with a BRCA1 mutation. Therefore, breast tumors from BRCA1 mutation carriers might be expected to exhibit a high frequency of p53 mutations. This study examined the frequency of p53 mutations in the breast tumors of Ashkenazi Jewish carriers and noncarriers of BRCA1 mutations. METHODS: Tumor DNA from carriers and noncarriers of BRCA1 mutations was screened for mutations in exons 4 through 10 of the p53 gene by use of the polymerase chain reaction and single-strand conformation polymorphism (SSCP) analysis of the amplified DNA. Direct sequencing was performed on gene fragments that showed altered mobility in SSCP analysis. RESULTS: Mutations in the p53 gene were detected in 10 of 13 tumors from BRCA1 mutation carriers versus 10 of 33 tumors from non-carriers (two-sided P = .007). The p53 mutations were distributed throughout exons 4 through 10 and included both protein-truncating and missense mutations in both groups. CONCLUSIONS: A statistically significantly higher frequency of p53 mutations was found in breast tumors from carriers of BRCA1 mutations than from noncarriers, which adds to the accumulating evidence that loss of p53 function is an important step in the molecular pathogenesis of BRCA1 mutation-associated breast tumors. This finding may have implications for understanding phenotypic differences and potential prognostic differences between BRCA1 mutation-associated hereditary breast cancers and sporadic cancers.

p53 mutations in mammary ductal carcinoma in situ but not in epithelial hyperplasias.

The development and progression of human breast neoplasia is characterized by the accumulation of numerous somatic genetic alterations. Although mutation of the p53 tumor suppressor gene is one of the most common alterations identified in invasive carcinomas, it is not clear whether mutations usually occur in noninvasive lesions before the development of invasion. To investigate the presence and heterogeneity of p53 mutations in breast neoplasia, we studied a morphological spectrum of paired lesions including invasive carcinomas and adjacent noninvasive epithelial lesions. Using 18 invasive ductal carcinomas with known p53 mutations, tissue samples were microdissected from formalin-fixed, paraffin-embedded tissue sections, and mutations in exons 4-8 of the p53 gene were identified by PCR amplification, single-strand conformational polymorphism, and direct sequencing. Multiple geographically distinct foci of invasive carcinoma were microdissected from six different invasive carcinomas, and all samples from each case had the same mutation. The absence of mutation heterogeneity provides evidence that p53 mutations occurred at the time of, or before, the clonal selection of the dominant component of the invasive carcinoma. To delineate the timing of p53 inactivation further in these cases, histological slides were reviewed to identify all noninvasive epithelial lesions. There were eight cases with associated ductal carcinoma in situ (DCIS), and in total, 27 distinct tissue samples representing a spectrum of histological subtypes and grades of DCIS were microdissected. In all 27 samples, the identical p53 mutation was identified in the DCIS as was present in the invasive carcinoma. In contrast, no p53 mutations were identified in any of the 21 microdissected foci of epithelial hyperplasia analyzed, including one sample with atypia. Together, these findings provide support that p53 mutations commonly occur early in breast neoplasia, usually at the stage of DCIS, but are not often identified in foci of hyperplasia. These findings support an important biological role for p53 mutation in progression from noninvasive precursors in breast neoplasia and provide further evidence that p53 mutation could have potential use as a molecular marker.

Somatic mutations in the BRCA1 gene in Chinese sporadic breast and ovarian cancer.

Inherited mutations in the BRCA1 gene confer increased susceptibility to breast and ovarian cancer. Its role in sporadic carcinogenesis is not well defined. Somatic mutations in breast cancers have not been reported and to date there are only three reports of somatic mutations in sporadic ovarian cancers. To investigate the contribution of BRCA1 mutations to sporadic breast and ovarian cancer in the Chinese population, we analysed 62 samples from Chinese women using the protein truncation test. There were 40 cases of breast cancer under age 50 and 22 cases of ovarian cancer, all unselected for family history. There was no age selection for the ovarian cancers. We found two somatic BRCA1 mutations in exon 11, one in a breast cancer and the other in an ovarian cancer, both of which result in truncated proteins. Our results indicate that somatic BRCA1 mutations, like somatic mutations in the BRCA2 gene, though very rare, can be found in both breast and ovarian cancers and support a tumor suppressor function for BRCA1 in sporadic tumors.

Role of Nek2 on centrosome duplication and aneuploidy in breast cancer cells.

Breast cancer is the most common solid tumor and the second most common cause of death in women. Despite a large body of literature and progress in breast cancer research, many molecular aspects of this complex disease are still poorly understood, hindering the design of specific and effective therapeutic strategies. To identify the molecules important in breast cancer progression and metastasis, we tested the in vivo effects of inhibiting the functions of various kinases and genes involved in the regulation/modulation of the cytoskeleton by downregulating them in mouse PyMT mammary tumor cells and human breast cancer cell lines. These kinases and cytoskeletal regulators were selected based on their prognostic values for breast cancer patient survival. PyMT tumor cells, in which a selected gene was stably knocked down were injected into the tail veins of mice, and the formation of tumors in the lungs was monitored. One of the several genes found to be important for tumor growth in the lungs was NIMA-related kinases 2 (Nek2), a cell cycle-related protein kinase. Furthermore, Nek2 was also important for tumor growth in the mammary fat pad. In various human breast cancer cell lines, Nek2 knockdown induced aneuploidy and cell cycle arrest that led to cell death. Significantly, the breast cancer cell line most sensitive to Nek2 depletion was of the triple negative breast cancer subtype. Our data indicate that Nek2 has a pivotal role in breast cancer growth at primary and secondary sites, and thus may be an attractive and novel therapeutic target for this disease.

Mechanisms and pathways of bone metastasis: challenges and pitfalls of performing molecular research on patient samples.

The molecular mechanisms underlying the development of bone metastases in breast cancer remain unclear. Disseminated tumour cells (DTCs) in the bone marrow of breast cancer patients are commonly identified, even in early stage disease, but their potential to initiate metastases is not known. The mechanism whereby DTCs become overt metastatic tumour cells (MTCs) is therefore, an area of considerable interest. This study explored the analysable yield of genetic material from human biopsy samples in order to describe differences in gene expression between DTCs and bone MTCs. Thirteen breast cancer patients with bone metastases underwent a CT-guided bone metastasis biopsy and a bone marrow biopsy. Tumour cells were enriched and gene expression profiling was conducted to identify differentially expressed genes. The analysable yield of sufficient RNA for microarray analysis was 60% from bone metastasis biopsies and 80% from bone marrow biopsies. A signature of 133 candidate genes differentially expressed between DTCs and MTCs was identified. Several genes relevant to breast cancer metastasis to bone (osteopontin, CTGF, parathyroid hormone receptor, EGFR) were significantly overexpressed in MTCs as compared to DTCs. Biopsies of bone metastases and bone marrow rarely yield enough tissue for robust molecular biology studies using clinical samples. The findings obtained however are interesting and seem to overlap with the bone metastasis gene expression signature described in murine xenograft models. Larger biopsy specimens or improved RNA extraction techniques may improve analysable yield and feasibility of these techniques.

P53 protein accumulation in non-invasive lesions surrounding p53 mutation positive invasive breast cancers.

p53 mutation is a common event in sporadic breast cancer being found in 15-50% of invasive carcinomas. The purpose of this study was to determine the earliest histologic stage at which p53 mutation could be detected with a widely used anti-p53 antibody (DO7, Novocastra) which recognizes both wild type and mutant forms. p53 expression was assessed immunohistochemically in 12 primary breast carcinomas with known p53 mutations and in all pre-malignant epithelial lesions surrounding these invasive cancers. Strong p53 nuclear staining was found in all of the tumors known to have missense mutations and none of the tumors with truncation mutations. In cases with intense staining in the invasive carcinoma, a similar quality of staining was also seen in all areas of DCIS (ductal carcinoma in situ) and was representative of missense p53 mutations. Lighter nuclear staining intensity was observed in up to 40% of cells in areas of hyperplasia and in up to 30% of normal breast lobules irrespective of the type of mutation found in the invasive carcinoma. This weak staining was not specific to mutated p53 and may indicate increased amounts of normal p53 protein. We conclude that p53 inactivation occurs prior to invasion in breast carcinogenesis, with mutations being uniformly identified in DCIS associated with p53-mutated invasive carcinomas. In contrast, there is no evidence that epithelial hyperplasia or epithelial cells of the terminal duct lobular unit harbor the same mutations as their associated invasive carcinoma.

Partial characterization of the enhanced survival of female NZB/W mice treated with lithium chloride.

Previous investigations have indicated that initiation of LiCl treatment (4 mg/day) of female NZB/W mice at 10 weeks of age led to enhanced survival of 50% of the mice to > 11 months of age (Lithium, 3, 61-67, 1992). The present results indicate that this enhancement of survival is dose dependent in that 2 mg 7LiCl/day was less effective than 4 mg 7LiCl/day. Daily treatment of groups of mice with 2 or 4 mg LiCl per day starting at 8 weeks of age led to the survival of 40% and 70%, respectively, of the mice at 40 weeks of age, a time when only 10% of the untreated mice remained alive. Initiation of treatment with 2 mg 7LiCl/day after the disease process was evident (24 weeks of age) led to diminished effectiveness. Parallel experiments with mice treated with 4 mg 7LiCl/day revealed 60% long-term survivors in the early treatment groups and 33% in the delayed treatment groups. Cessation of treatment in both groups led to some additional deaths, but 20-27% of the mice remained alive at 60 weeks of age, even though animals had detectable levels of anti-ssDNA antibodies in their serum. Additional experiments with 2 and 4 mg 7LiCl/day in NZB/W mice pretreated with C. parvum-PER, a treatment previously shown to enhance survival (Int. J. Immunopharmac., 14, 35-41, 1992), indicated that the effect of the two modalities was not additive. The results presented indicate that treatment of NZB/W mice with LiCl leads to very effective prolongation of survival by unique mechanisms, possibly involving alteration of the effector phase of autoimmune damage to the kidney or the susceptibility of kidney elements to immune-mediated damage leading to renal failure.

Analysis of mRNA from microdissected frozen tissue sections without RNA isolation.

Molecular study of gene expression in solid tumors is based largely on mRNA extracted from crushed frozen tumor samples. As most tumors are heterogeneous in composition, molecular alterations acquired by neoplastic cells may be masked by normal epithelial, stromal, and inflammatory cells, which may make up a significant volume of many tumors. We have developed a technique whereby reverse transcription polymerase chain reaction (RT-PCR) can be performed on lesions microdissected directly from frozen tumor sections. This allows for molecular analysis of mRNA from histologically homogeneous cell populations. Cryostat sections are placed onto a thin layer of 2% agarose on a glass slide and stained briefly. Microdissected tissue is immersed in a freezing solution to lyse the cells; aliquots are used directly in RT-PCR reactions without further purification. We successfully amplified cDNA fragments of the beta2-microglobulin, p21Waf1, and BRCA1 genes from small microdissected lesions. Also, we examined the effect of varying thickness of cryostat sections (20 versus 40 microm) and several tissue staining dyes. We estimate that a small microdissected region, containing no more than 200 cells, can provide enough mRNA to make cDNA for 80 to 100 PCR reactions. We believe that this technique will be a useful tool to study gene expression in histologically defined tissues.