Pathological complete response and residual DCIS following neoadjuvant chemotherapy for breast carcinoma

Patients who have no residual invasive cancer following neoadjuvant chemotherapy for breast carcinoma have a better overall survival than those with residual disease. Many classification systems assessing pathological response to neoadjuvant chemotherapy include residual ductal carcinoma in situ (DCIS) only in the definition of pathological complete response. The purpose of this study was to investigate whether patients with residual DCIS only have the same prognosis as those with no residual invasive or in situ disease. A retrospective analysis of a prospectively maintained database identified 435 patients, who received neoadjuvant chemotherapy for operable breast cancer between February 1985 and February 2003. Of these, 30 (7%; 95% CI 5-9%) had no residual invasive disease or DCIS and 20 (5%; CI 3-7%) had residual DCIS only. With a median follow-up of 61 months, there was no statistical difference in disease-free survival, 80% (95% CI 60-90%) in those with no residual invasive or in situ disease and 61% (95% CI 35-80%) in those with DCIS only (P = 0.4). No significant difference in 5-year overall survival was observed, 93% (95% CI 75-98%) in those with no residual invasive or in situ disease and 82% (95% CI 52-94%) in those with DCIS only (P = 0.3). Due to the small number of patients and limited number of events in each group, it is not possible to draw definitive conclusions from this study. Further analyses of other databases are required to confirm our finding of no difference in disease-free and overall survival between patients with residual DCIS and those with no invasive or in situ disease following neoadjuvant chemotherapy for breast cancer.

Heterogeneity of MUC1 expression by human breast carcinoma cell lines in vivo and in vitro

Increased expression of the epithelial mucin MUC1 has been linked to tumor aggressiveness in human breast carcinoma. Recent studies have demonstrated that overexpression of MUC1 interferes with cell-substrate and cell-cell adhesion by masking cell surface integrins and E-cadherin. Additionally, the cytoplasmic tail of MUC1 is involved in signal transduction and interactions with catenins. In the present study, we have examined the in vitro expression of MUC1 mRNA and protein in a panel of 14 human breast cancer cell lines using northern blotting, western blotting, immunocytochemistry, and flow cytometry. Considerable variability of expression was noted not only between cell lines but also within several individual lines. Many cell lines such as BT 20, KPL-1, and T47D expressed abundant MUC1 whilst others such as MDA-MB-231 and MCF-7 showed intermediate expression, and MDA-MB-435 and MDA-MB-453 expressed very low levels. Low levels of MUC1 expression were associated with decreased expression of cytokeratin and increased expression of vimentin. Additionally, 12 of the cell lines were established as xenografts in immunocompromised (SCID) mice, and MUC1 expression in both the primary tumors as well as metastases was assessed immunohistochemically. In general, in vivo expression mirrored in vitro expression, although there was reduced in vivo expression in T47D and ZR-75-1 xenografts. Although we showed no correlation between tumorigenicity or metastasis and MUC1 expression, this study will assist development of experimental models to assess the influence of MUC1 of on breast cancer progression.

Specific morphological features predictive for the basal phenotype in grade 3 invasive ductal carcinoma of breast

Aims: Cytokeratin (CK) 14, a myoepithelial marker, is also expressed in a proportion of breast carcinomas. There is evidence that these tumours show a differing metastatic pattern and clinical outcome from other invasive ductal carcinomas (IDCs) and may need different management. Currently, they are not identified in routine practice and no morphological guidelines exist to aid their identification. The aim of this study was to analyse the histological features of CK14+ IDC. Methods and results: A detailed histological review of 453 grade 3 IDCs revealed 88 (19.4%) that expressed CK14. Assessment was made independently by two pathologists using a standardized 'tick-box' proforma covering grade, architectural and cytological features. The results were analysed using logistic regression to identify features that predicted for basal phenotype. Concordance between the two pathologists was fair to good for most parameters (kappa 0.4-0.6). On multiple logistic regression, the basal phenotype was highly significantly associated with the presence of a central scar (P = 0.005), tumour necrosis (P < 0.0001), presence of spindle cells (P = 0.006) or squamous metaplasia (P < 0.0001), high total mitotic count (> 40 per 10 high-power field) (P = 0.0002) and high nuclear-cytoplasmic ratio (P = 0.0002). Conclusions: Specific morphological features are strongly associated with basal-like breast carcinoma. These could be used in routine diagnostic practice to identify this important subset of tumours.

Evaluation of oestrogen and progesterone receptor status in HER-2 positive breast carcinomas and correlation with outcome

Aim: HER-2/neu amplification occurs in 15-25% of breast carcinomas. This oncogene, also referred to as c-erbB-2, encodes a transmembrane tyrosine kinase receptor belonging to the epidermal growth factor receptor family. HER-2 over-expression is reported to be associated with a poor prognosis in breast carcinoma patients and in some studies is associated with a poorer response to anti-oestrogen therapy. These patients are less likely to benefit from CMF (cyclophosphamide, methotrexate, fluorouracil)-based chemotherapy compared with anthracycline-based chemotherapy. The aim of this study was to evaluate breast carcinomas to determine hormone receptor status and if there is a difference in breast cancer specific survival for HER-2 positive patients. Methods: A total of 591 breast carcinomas were evaluated using immunohistochemistry (IHC) for oestrogen receptor (ERp), progesterone receptor (PRp) and three different HER2 antibodies (CB11, A0485 and TAB250). Percentage of tumour cells and intensity of staining for ERp were evaluated using a semiquantitative method. Results: Of the 591 tumours, 91 (15.4%) showed 3+ membrane staining for HER-2 with one or more antibodies. Of these 91 tumours, 41 (45.1%) were ERp+/ PRp+, seven (7.7%) were ERp+/PR-, six (6.6%) were ERp-/PRp+ and 37 (40.7%) were ERp-/PR-. Of HER-2 positive tumours, 5.5% showed > 80% 3+ staining for ERp compared with 31.8% of 0-2+ HER-2 tumours; 24.2% of HER-2-positive tumours showed 60% or more cells with 2+ or 3+ staining for ERp. Treatment data were available for 209 patients and no difference was observed in breast cancer specific survival (BCSS) with HER-2 status and tamoxifen. Conclusion: Oestrogen receptor status cannot be used to select tumours for evaluation of HER-2 status, and oestrogen and progesterone receptor positivity does not preclude a positive HER-2 status. There is a higher proportion of ERp negative tumours associated with HER-2 positivity, however, more than 20% of HER-2 positive tumours show moderate or strong staining for ERp. HER-2 positive patients in this study did not show an adverse BCSS with tamoxifen treatment unlike some previous studies.

Monitoring dendritic cells in clinical practice using a new whole blood single-platform TruCOUNT assay

Dendritic cells (DC) from distinct DC subsets are essential contributors to normal human immune responses. Despite this, reliable assays that enable DC to be counted precisely have been slow to evolve. We have now developed a new single-platform flow cytometric assay based on TruCOUN(TM) beads and the whole blood Lyse/No-Wash protocol that allows precise counting of the CD14(-) blood DC subsets: CD11c(+)CD16(-) DC, CD11c(+)CD16(+) DC, CD123(hi) DC, CD1c(+) DC and BDCA-3(+) DC. This assay requires 50 mul of whole blood; does not rely on a hematology blood analyser for the absolute DC counts; allows DC counting in EDTA samples 24 It after collection; and is suitable for cord blood and peripheral blood. The data is highly reproducible with intra-assay and inter-assay coefficients of variation less than 3% and 11%, respectively. This assay does not produce the DC-T lymphocyte conjugates that result in DC counting abnormalities in conventional gradient-density separation procedures. Using the TruCOUNT assay, we established that absolute blood DC counts reduce with age in healthy individuals. In preliminary studies, we found a significantly lower absolute blood CD11c(+)CD16(+) DC count in stage III/IV versus stage I/II breast carcinoma patients and a lower absolute blood CD123(hi) DC count in multiple myeloma patients, compared to age-matched controls. These data indicate that scientific progress in DC counting technology will lead to the global standardization of DC counting and allow clinically meaningful data to be obtained. (C) 2003 Elsevier B.V. All rights reserved.

Histone-deacetylase inhibitors for the treatment of cancer

Histone deacetylase inhibitors (HDACi) are a promising new class of chemotherapeutic drug currently in early phase clinical trials. A large number of structurally diverse HDACi have been purified or synthesised that mostly inhibit the activity of all eleven class I and II HDACs. While these agents demonstrate many features required for anti-cancer activity such as low toxicity against normal cells and an ability to inhibit tumor cell growth and survival at nanomolar concentrations, their mechanisms of action are largely unknown. Initially, a model was proposed whereby HDACi-mediated transactivation of a specific gene or set of genes was responsible for the inhibition of cell cycle progression or induction of apoptosis. Given that HDACs can regulate the activity of a number of nonhistone proteins and that histone acetylation is important for events such as DNA replication and mitosis that do not directly involve gene transcription, it appears that the initial mechanistic model for HDACi may have been too simple. Herein, we provide an update on the transcription-dependent and - independent events that may be important for the anti-tumor activities of HDACi and discuss the use of these compounds in combination with other chemotherapeutic drugs.

Participation in the RACS sentinel node biopsy versus axillary clearance trial

Background: The Royal Australasian College of Surgeons (RACS) SNAC trial is a randomized controlled trial of sentinel node biopsy (SNB) versus axillary clearance (AC). It opened in May 2001 and is recruiting rapidly with good acceptance by consumers. Methods: A study of eligibility and treatment choices was conducted between November 2001 and September 2002 for women presenting with early breast cancer to 10 centres participating in the trial. Results: More than half of the 622 women (54%) were ineligible for trial entry because they had large (> 3 cm) or multicentric cancers. Participation was offered to 92% of eligible women and was taken up by 63%. The commonest reason for not participating was the desire to choose treatment rather than have it randomly allocated. Despite this there is a great acceptance of clinical trials because very few women (4% of those eligible) gave 'lack of interest in clinical trials' as the reason for non-participation. Few women who declined trial participation chose to have SNB alone (4.5% of those eligible). Conclusion: Sentinel node biopsy may become the standard of care for managing small breast cancers, but a significant number of patients will still require or choose axillary dissection. Results from large randomized trials are needed to determine the relative benefits and harms of SNB compared with AC. Surgeons must carefully discuss options for management with their patients.

Unlocking pathology archives for molecular genetic studies: a reliable method to generate probes for chromogenic and fluorescent in situ hybridization

Chromogenic (CISH) and fluorescent ( FISH) in situ hybridization have emerged as reliable techniques to identify amplifications and chromosomal translocations. CISH provides a spatial distribution of gene copy number changes in tumour tissue and allows a direct correlation between copy number changes and the morphological features of neoplastic cells. However, the limited number of commercially available gene probes has hindered the use of this technique. We have devised a protocol to generate probes for CISH that can be applied to formalin-fixed, paraffin-embedded tissue sections (FFPETS). Bacterial artificial chromosomes ( BACs) containing fragments of human DNA which map to specific genomic regions of interest are amplified with phi 29 polymerase and random primer labelled with biotin. The genomic location of these can be readily confirmed by BAC end pair sequencing and FISH mapping on normal lymphocyte metaphase spreads. To demonstrate the reliability of the probes generated with this protocol, four strategies were employed: (i) probes mapping to cyclin D1 (CCND1) were generated and their performance was compared with that of a commercially available probe for the same gene in a series of 10 FFPETS of breast cancer samples of which five harboured CCND1 amplification; (ii) probes targeting cyclin-dependent kinase 4 were used to validate an amplification identified by microarray-based comparative genomic hybridization (aCGH) in a pleomorphic adenoma; (iii) probes targeting fibroblast growth factor receptor 1 and CCND1 were used to validate amplifications mapping to these regions, as defined by aCGH, in an invasive lobular breast carcinoma with FISH and CISH; and (iv) gene-specific probes for ETV6 and NTRK3 were used to demonstrate the presence of t(12; 15)(p12; q25) translocation in a case of breast secretory carcinoma with dual colour FISH. In summary, this protocol enables the generation of probes mapping to any gene of interest that can be applied to FFPETS, allowing correlation of morphological features with gene copy number.

Mutations in the MYB intron I regulatory sequence increase transcription in colon cancers

Although MYB overexpression in colorectal cancer (CRC) is known to be a prognostic indicator for poor survival, the basis for this overexpression is unclear. Among multiple levels of MYB regulation, the most dynamic is the control of transcriptional elongation by sequences within intron I. The authors have proposed that this regulatory sequence is transcribed into an RNA stem-loop and 19-residue polyuridine tract, and is subject to mutation in CRC. When this region was examined in colorectal and breast carcinoma cell lines and tissues, the authors found frequent mutations only in CRC. It was determined that these mutations allowed increased transcription compared with the wild type sequence. These data suggest that this MYB regulatory region within intron I is subject to mutations in CRC but not breast cancer, perhaps consistent with the mutagenic insult that occurs within the colon and not mammary tissue. In CRC, these mutations may contribute to MYB overexpression, highlighting the importance of noncoding sequences in the regulation of key cancer genes. (c) 2006 Wiley-Liss, Inc.

Pleomorphic lobular carcinoma of the breast: role of comprehensive molecular pathology in characterization of an entity

Immunohistochemical analysis of E-cadherin has changed the way lobular neoplasia is perceived. It has helped to classify difficult cases of carcinoma in situ with indeterminate features and led to the identification of new variants of lobular carcinoma. Pleomorphic lobular carcinoma (PLC) and pleomorphic lobular carcinoma in situ (PLCIS), recently described variants of invasive and in situ classic lobular carcinoma, are reported to be associated with more aggressive clinical behaviour. Although PLC/PLCIS show morphological features of classic lobular neoplasia and lack E-cadherin expression, it is still unclear whether these lesions evolve through the same genetic pathway as lobular carcinomas or are high-grade ductal neoplasms that have lost E-cadherin. Here we have analysed a case of extensive PLCIS and invasive PLC associated with areas of E-cadherin-negative carcinoma in situ with indeterminate features, using immunohistochemistry, chromogenic in situ hybridization, high-resolution comparative genomic hybridization (CGH) and array-based CGH. We observed that all lesions lacked E-cadherin and beta-catenin and showed gain of 1q and loss of 16q, features that are typical of lobular carcinomas but are not seen in high-grade ductal lesions. In addition, amplifications of c-myc and HER2 were detected in the pleomorphic components, which may account for the high-grade features in this case and the reported aggressive clinical behaviour of these lesions. Taken together, these data suggest that at least some PLCs may evolve from the same precursor or through the same genetic pathway as classic lobular carcinomas. Copyright (c) 2005 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Sentinel node biopsy for breast cancer: Using local results for estimation of risk to the patient

Background: Sentinel node biopsy (SNB) is being increasingly used but its place outside randomized trials has not yet been established. Methods: The first 114 sentinel node (SN) biopsies performed for breast cancer at the Princess Alexandra Hospital from March 1999 to June 2001 are presented. In 111 cases axillary dissection was also performed, allowing the accuracy of the technique to be assessed. A standard combination of preoperative lymphoscintigraphy, intraoperative gamma probe and injection of blue dye was used in most cases. Results are discussed in relation to the risk and potential consequences of understaging. Results: Where both probe and dye were used, the SN was identified in 90% of patients. A significant number of patients were treated in two stages and the technique was no less effective in patients who had SNB performed at a second operation after the primary tumour had already been removed. The interval from radioisotope injection to operation was very wide (between 2 and 22 h) and did not affect the outcome. Nodal metastases were present in 42 patients in whom an SN was found, and in 40 of these the SN was positive, giving a false negative rate of 4.8% (2/42), with the overall percentage of patients understaged being 2%. For this particular group as a whole, the increased risk of death due to systemic therapy being withheld as a consequence of understaging (if SNB alone had been employed) is estimated at less than 1/500. The risk for individuals will vary depending on other features of the particular primary tumour. Conclusion: For patients who elect to have the axilla staged using SNB alone, the risk and consequences of understaging need to be discussed. These risks can be estimated by allowing for the specific surgeon's false negative rate for the technique, and considering the likelihood of nodal metastases for a given tumour. There appears to be no disadvantage with performing SNB at a second operation after the primary tumour has already been removed. Clearly, for a large number of patients, SNB alone will be safe, but ideally participation in randomized trials should continue to be encouraged.

Columnar cell lesions of the breast: The missing link in breast cancer progression? A morphological and molecular analysis

Columnar cell lesions (CCLs) of the breast are a spectrum of lesions that have posed difficulties to pathologists for many years, prompting discussion concerning their biologic and clinical significance. We present a study of CCL in context with hyperplasia of usual type (HUT) and the more advanced lesions ductal carcinoma in situ (DCIS) and invasive ductal carcinoma. A total of 81 lesions from 18 patients were subjected to a comprehensive morphologic review based upon a modified version of Schnitt's classification system for CCL, immunophenotypic analysis (estrogen receptor [ER], progesterone receptor [PgR], Her2/neu, cytokeratin 5/6 [CK5/6], cytokeratin 14 [CK14], E-cadherin, p53) and for the first time, a whole genome molecular analysis by comparative genomic hybridization. Multiple CCLs from 3 patients were studied in particular detail, with topographic information and/or showing a morphologic spectrum of CCL within individual terminal duct lobular units. CCLs were ER an PgR positive, CK5/6 and CK14 negative, exhibit low numbers of genetic alterations and recurrent 16q loss, features that are similar to those of low grade in situ and invasive carcinoma. The molecular genetic profiles closely reflect the degree of proliferation and atypia in CCL, indicating some of these lesions represent both a morphologic and molecular continuum. In addition, overlapping chromosomal alterations between CCL and more advanced lesions within individual terminal duct lobular units suggest a commonality in molecular evolution. These data further support the hypothesis that CCLs are a nonobligate, intermediary step in the development of some forms of low grade in situ and invasive carcinoma. Copyright: © 2005 Lippincott Williams & Wilkins, Inc.

Molecular evolution of breast cancer

Molecular analysis of invasive breast cancer and its precursors has furthered our understanding of breast cancer progression. In the past few years, new multi-step pathways of breast cancer progression have been delineated through genotypic-phenotypic correlations. Nuclear grade, more than any other pathological feature, is strongly associated with the number and pattern of molecular genetic abnormalities in breast cancer cells. Thus, there are two distinct major pathways to the evolution of low- and high-grade invasive carcinomas: whilst the former consistently show oestrogen receptor (ER) and progesterone receptor (PgR) positivity and 16q loss, the latter are usually ER/PgR-negative and show Her-2 over-expression/amplification and complex karyotypes. The boundaries between the evolutionary pathways of well-differentiated/low-grade ductal and lobular carcinomas have been blurred, with changes in E-cadherin expression being one of the few distinguishing features between the two. In addition, lesions long thought to be precursors of breast carcinomas, such as hyperplasia of usual type, are currently considered mere risk indicators, whilst columnar cell lesions are now implicated as non-obligate precursors of atypical ductal hyperplasia (ADH) and well-differentiated ductal carcinoma in situ (DCIS). However, only through the combination of comprehensive morphological analysis and cutting-edge molecular tools can this knowledge be translated into clinical practice and patient management. Copyright (C) 2005 Pathological Society of Great Britain and Ireland. Published by John Wiley Sons, Ltd.

Typical medullary breast carcinomas have a basal/myoepithelial phenotype

Medullary breast cancer (MBC) is a rare, diagnostically difficult, pathological subtype. Despite being high grade, it has a good prognosis. MBC patients have an excess of BRCA1 germ-fine mutation and reliable identification of MBC could help to identify patients at risk of carrying germline BRCA1 mutations or in whom chemotherapy could be avoided. The aim of this study was therefore to improve diagnosis by establishing an MBC protein expression profile using immunohistochemistry (IHC) on tissue-microarrays (TMA). Using a series of 779 breast carcinomas ('EC' set), diagnosed initially as MBC, a double-reading session was carried out by several pathologists on all of the histological material to establish the diagnosis as firmly as possible using a 'medullary score'. Only MBCs with high scores, i.e. typical MBC (TMBC) (n = 44) and non-TMBC grade III with no or low scores (n = 160), were included in the IHC study. To validate the results obtained on this first set, a control series of TMBC (n = 17) and non-MBC grade III cases (n = 140) ('IPC' set) was studied. The expression of 18 proteins was studied in the 61 TMBCs and 300 grade III cases from the two sets. The global intra-observer concordance of the first reading for the diagnosis of TMBC was 94%, with almost perfect kappa (kappa) of 0.815. TMBC was characterized by a high degree of basal/myoepithelial differentiation. In multivariate analysis with logistic regression, TMBC was defined by the association of P-cadherin (R = 2.29), MIB1 > 50 (R = 3.80), ERBB2 negativity (R = 2.24) and p53 positivity (RR = 1.45). Copyright (c) 2005 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.