The cancer stem cell (CSC) hypothesis states that tumours consist of a cellular hierarchy with CSCs at the apex driving tumour recurrence and metastasis. Hence, CSCs are potentially of profound ...clinical importance. We set out to establish the clinical relevance of breast CSC markers by profiling a large cohort of breast tumours in tissue microarrays (TMAs) using immunohistochemistry (IHC).
We included 4, 125 patients enrolled in the SEARCH population-based study with tumours represented in TMAs and classified into molecular subtype according to a validated IHC-based five-marker scheme. IHC was used to detect CD44/CD24, ALDH1A1, aldehyde dehydrogenase family 1 member A3 (ALDH1A3) and integrin alpha-6 (ITGA6). A 'Total CSC' score representing expression of all four CSC markers was also investigated. Association with breast cancer specific survival (BCSS) at 10 years was assessed using a Cox proportional-hazards model. This study was complied with REMARK criteria.
In ER negative cases, multivariate analysis showed that ITGA6 was an independent prognostic factor with a time-dependent effect restricted to the first two years of follow-up (hazard ratio (HR) for 0 to 2 years follow-up, 2.4; 95% confidence interval (95% CI), 1.2 to 4.8; P = 0.009). The composite 'Total CSC' score carried independent prognostic significance in ER negative cases for the first four years of follow-up (HR for 0 to 4 years follow-up, 1.3; 95% CI, 1.1 to 1.6; P = 0.006).
Breast CSC markers do not identify identical subpopulations in primary tumours. Both ITGA6 and a composite Total CSC score show independent prognostic significance in ER negative disease. The use of multiple markers to identify tumours enriched for CSCs has the greatest prognostic value. In the absence of more specific markers, we propose that the effective translation of the CSC hypothesis into patient benefit will necessitate the use of a panel of markers to robustly identify tumours enriched for CSCs.
PREDICT (http://www.predict.nhs.uk) is a prognostication and treatment benefit tool for early breast cancer (EBC). The aim of this study was to incorporate the prognostic effect of KI67 status in a ...new version (v3), and compare performance with the Predict model that includes HER2 status (v2).
The validation study was based on 1,726 patients with EBC treated in Nottingham between 1989 and 1998. KI67 positivity for PREDICT is defined as >10% of tumour cells staining positive. ROC curves were constructed for Predict models with (v3) and without (v2) KI67 input. Comparison was made using the method of DeLong.
In 1274 ER+ patients the predicted number of events at 10 years increased from 196 for v2 to 204 for v3 compared to 221 observed. The area under the ROC curve (AUC) improved from 0.7611 to 0.7676 (p=0.005) in ER+ patients and from 0.7546 to 0.7595 (p=0.0008) in all 1726 patients (ER+ and ER-).
Addition of KI67 to PREDICT has led to a statistically significant improvement in the model performance for ER+ patients and will aid clinical decision making in these patients. Further studies should determine whether other markers including gene expression profiling provide additional prognostic information to that provided by PREDICT.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Pathogenic germline variants in ataxia-telangiectasia mutated (ATM), a gene that plays a role in DNA damage response and cell cycle checkpoints, confer an increased breast cancer (BC) risk. Here, we ...investigated the phenotypic characteristics and landscape of somatic genetic alterations in 24 BCs from ATM germline mutation carriers by whole-exome and targeted sequencing. ATM-associated BCs were consistently hormone receptor positive and largely displayed minimal immune infiltrate. Although 79.2% of these tumors exhibited loss of heterozygosity of the ATM wild-type allele, none displayed high activity of mutational signature 3 associated with defective homologous recombination DNA (HRD) repair. No TP53 mutations were found in the ATM-associated BCs. Analysis of an independent data set confirmed that germline ATM variants and TP53 somatic mutations are mutually exclusive. Our findings indicate that ATM-associated BCs often harbor bi-allelic inactivation of ATM, are phenotypically distinct from BRCA1/2-associated BCs, lack HRD-related mutational signatures, and that TP53 and ATM genetic alterations are likely epistatic.
Although most women with luminal breast cancer do well on endocrine therapy alone, some will develop fatal recurrence thereby necessitating the need to prospectively determine those for whom ...additional cytotoxic therapy will be beneficial. Categorical combinations of immunohistochemical measures of ER, PR, HER2, and KI67 are traditionally used to classify patients into luminal A-like and B-like subtypes for chemotherapeutic reasons, but this may lead to the loss of prognostically relevant information. Here, we compared the prognostic value of quantitative measures of these markers, combined in the IHC4-score, to categorical combinations in subtypes. Using image analysis-based scores for all four markers, we computed the IHC4-score for 2498 patients with luminal breast cancer from two European study populations. We defined subtypes (A-like (ER + and PR + : and HER2- and low KI67) and B-like (ER + and/or PR + : and HER2 + or high KI67)) by combining binary categories of these markers. Hazard ratios and 95% confidence intervals for associations with 10-year breast cancer-specific survival were estimated in Cox proportional-hazard models. We accounted for clinical prognostic factors, including grade, tumor size, lymph-nodal involvement, and age, by using the PREDICT-score. Overall, Subtypes hazard ratio (95% confidence interval) B-like vs. A-like = 1.64 (1.25-2.14); P-value < 0.001 and IHC4-score hazard ratio (95% confidence interval)/1 standard deviation = 1.32 (1.20-1.44); P-value < 0.001 were prognostic in univariable models. However, IHC4-score hazard ratio (95% confidence interval)/1 standard deviation = 1.24 (1.11-1.37); P-value < 0.001; likelihood ratio chi-square (LRχ
) = 12.5 provided more prognostic information than Subtype hazard ratio (95% confidence interval) B-like vs. A-like = 1.38 (1.02-1.88); P-value = 0.04; LRχ
= 4.3 in multivariable models. Further, higher values of the IHC4-score were associated with worse prognosis, regardless of subtype (P-heterogeneity = 0.97). These findings enhance the value of the IHC4-score as an adjunct to clinical prognostication tools for aiding chemotherapy decision-making in luminal breast cancer patients, irrespective of subtype.
E-cadherin is involved in cell–cell adhesion and epithelial-to-mesenchymal transitions. In cancers, loss or inactivation of E-cadherin is associated with epithelial cell proliferation and invasion. ...Here, we sought to determine, if risk associations for 18 breast cancer susceptibility single nucleotide polymorphisms (SNPs) differed by E-cadherin tumor tissue expression in the Polish Breast Cancer Study (PBCS), using data on 1,347 invasive breast cancer cases and 2,366 controls. E-cadherin expression (low/high) was assessed using immunohistochemical staining of tumor tissue microarrays. Replication data on 2,006 cases and 6,714 controls from the Study of Epidemiology and Risk Factors in Cancer Heredity was used to follow-up promising findings from PBCS. In PBCS, we found the rs11249433 SNP at the 1p11.2 locus to be more strongly associated with risk of E-cadherin low tumors (OR = 1.30, 95 % CI = 1.08–1.56) than with E-cadherin high tumors OR = 1.06, 95 % CI = 0.95–1.18; case-only
p
-heterogeneity (
p
-het) = 0.05. Findings in PBCS for rs11249433 were replicated in SEARCH. Combined analyses of the two datasets for SNP rs11249433 revealed significant heterogeneity by E-cadherin expression (combined case-only
p
-het = 0.004). Further, among carriers of rs11249433, the highest risk was seen for E-cadherin low tumors that were ER-positive and of lobular histology. Our results in two independent data sets suggest that rs11249433, which is located between the
NOTCH2
and
FCGR1B
genes within the 1p11.2 locus, is more strongly associated with risk of breast tumors with low or absent E-cadherin expression, and suggest that evaluation of E-cadherin tumor tissue expression may be useful in clarifying breast cancer risk factor associations.
The development of molecular pathologic components in epidemiologic studies offers opportunities to relate etiologic factors to specific tumor types, which in turn may allow the development of better ...overall risk prediction and provide clues about mechanisms that mediate risk factors. In addition, this research may help identify or validate tissue biomarkers related to prognosis and prediction of treatment responses. In this mini review, we highlight specific considerations related to the incorporation of pathology in epidemiologic studies, using breast cancer research as a model. Issues related to ensuring the representativeness of cases for which research tissue is available and understanding limitations resulting from variable procedures for tissue collection, fixation, and processing are discussed. The growing importance of molecular pathology in clinical medicine has led to increased emphasis on optimized tissue preparation, which should enhance this type of research. In addition, the availability of new technologies including tissue microarrays, image scanning, and automated analysis to achieve high-throughput standardized assessment of immunohistochemical markers, and potentially other assays, is enabling consistent scoring of a growing list of markers in large studies. Concurrently, methodologic research to extend the range of assays that can be done on fixed tissues is expanding possibilities for molecular pathologic studies in epidemiologic research.
Luminal A breast cancer defined as hormone receptor positive and human epidermal growth factor receptor 2 (HER2) negative is known to be heterogeneous. Previous study showed that luminal A tumours ...with the expression of basal markers ((cytokeratin (CK) 5 or CK5/6) or epidermal growth factor receptor (EGFR)) were associated with poorer prognosis compared with those that stained negative for basal markers. Prompted by this study, we assessed whether tumour characteristics and risk factors differed by basal marker status within luminal A tumours.
We pooled 5040 luminal A cases defined by immunohistochemistry (4490 basal-negative ((CK5 (or CK5/6))- and EGFR-) and 550 basal-positive ((CK5 (or CK5/6+)) or EGFR+)) from eight studies participating in the Breast Cancer Association Consortium. Case-case comparison was performed using unconditional logistic regression.
Tumour characteristics and risk factors did not vary significantly by the expression of basal markers, although results suggested that basal-positive luminal tumours tended to be smaller and node negative, and were more common in women with a positive family history and lower body mass index.
Most established breast cancer risk factors were similar in basal-positive and basal-negative luminal A tumours. The non-significant but suggestive differences in tumour features and family history warrant further investigations.
Sectioning a whole tissue microarrray (TMA block) and storing the sections maximizes the number of sections obtained, but may impair the antigenicity of the stored sections. We have investigated the ...impact of TMA section storage on antigenicity. First, we reexamined existing TMA data to determine whether antigenicity in stored sections changes over time. Component scores for each marker, based on cellular compartment of staining and score-type, were evaluated separately. Residual components scores adjusted for grade, tumor size, and node positivity, were regressed on the number of days storage to evaluate the effect of storage time. Storage time ranged from 2 to 1897 days, and the mean change in antigenicity per year ranged from -0.88 (95% confidence interval, -1.11 to -0.65) to 0.035 (95% confidence interval, 0.016-0.054). Further analysis showed no significant improvement in the fit of survival models if storage time adjusted scores were included in the models rather than unadjusted scores. We then compared 3 ways of processing TMA sections after cutting-immediate staining, staining after 1 year, and staining after 1 year coated in wax-on the immunohistochemistry results for: progesterone receptor, a routinely used, robust antibody, and MKI67, which is generally considered less robust. The progesterone receptor scores for stored sections were similar to those for unstored sections, whereas the MKI67 scores for stored sections were substantially different to those for unstored sections. Wax coating made little difference to the results. Biomarker antigenicity shows a small decline over time that is unlikely to have an important effect on studies of prognostic biomarkers.
Regional Differences in the Response of Human Pre-Adipocytes to PPARγ and RXRα Agonists
Ciaran P. Sewter ,
Fiona Blows ,
Antonio Vidal-Puig and
Stephen O’Rahilly
From the University of Cambridge, ...Departments of Medicine and Clinical Biochemistry, Addenbrooke’s Hospital, Cambridge, United
Kingdom
Abstract
We have previously reported that omental (OM) preadipocytes respond less well to the prodifferentiating effects of thiazolidinediones
than do preadipocytes from subcutaneous (SC) depots. This finding is consistent with in vivo alterations in fat distribution
that occur in humans treated with thiazolidinediones. To explore these site-related differences further, we used real-time
RT-PCR to quantify the specific mRNAs encoding peroxisome proliferator-activated receptor (PPAR) γ1 and γ2 and found that
both isoforms were more highly expressed in SC than in OM preadipocytes. After 10 days of thiazolidinedione treatment, preadipocytes
from both depots showed a small and comparable increase in expression of PPARγ1 mRNA (1.7 ± 0.2-fold P = 0.007) and 1.3 ± 0.1-fold P = 0.008 increase for SC and OM, respectively). There was a much larger increase in PPARγ2 expression, which was significantly
greater in SC compared with OM preadipocytes (11.1 ± 2.8-fold P = 0.0003 and 5.5 ± 1.7-fold P = 0.0003, respectively; P = 0.014 for SC versus OM). To establish whether the refractoriness of OM preadipocytes to differentiation was unique to activators
of the PPARγ pathway, we examined the effects of the retinoid X receptor (RXR) ligand LG100268. As assessed by glycerol-3-phosphate
dehydrogenase activity, LG100268 had a greater effect on the differentiation of SC compared with OM preadipocytes when examined
alone (SC = 5.7 ± 1.7-fold vs. OM = 1.9 ± 0.6-fold; P < 0.05) or in combination with rosiglitazone (SC = 27.0 ± 7.5 vs. OM = 10.6 ± 3.6-fold; P < 0.05). Consistent with this, RXRα mRNA levels were also higher in SC than in OM preadipocytes. In summary, the previously
reported insensitivity of OM preadipocytes to the differentiating effects of thiazolidinediones may relate to their lower
basal levels of PPARγ1 and γ2 mRNA and their diminished capacity to upregulate PPARγ2 expression in response to ligand. That
omentally derived cells also show reduced responsiveness to the prodifferentiating actions of an RXR ligand and a lower expression
of RXRα in the undifferentiated state suggests that they may have a more generalized resistance to differentiation.
Footnotes
Address correspondence and reprint requests to Stephen O’Rahilly, University of Cambridge, Departments of Medicine and Clinical
Biochemistry, Addenbrooke’s Hospital, Hills Rd., Cambridge, CB2 2QR, U.K. E-mail: sorahill{at}hgmp.mrc.ac.uk .
Received for publication 30 April 2001 and accepted in revised form 15 November 2001.
DM, differentiating medium; OM, omental; PPAR, peroxisome proliferator-activated receptor; PPRE, peroxisome proliferator response
element; RXR, retinoid X receptor; SC, subcutaneous.
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