[Pre-analytical stage for biomarker assessment in breast cancer: 2014 update of the GEFPICS' guidelines in France]. / Recommandations du GEFPICS concernant la phase pré-analytique pour l'évaluation de HER2 et des récepteurs hormonaux dans le cancer du sein : mise à jour 2014.

Biomarker assessment of breast cancer tumor samples is part of the routine workflow of pathology laboratories. International guidelines have recently been updated, with special regards to the pre-analytical steps that are critical for the quality of immunohistochemical and in situ hybridization procedures, whatever the biomarker analyzed. Fixation and specimen handling protocols must be standardized, validated and carefully tracked. Cooperation and training of the personnel involved in the specimen workflow (e.g. radiologists, surgeons, nurses, technicians and pathologists) are of paramount importance. The GEFPICS' update of the recommendations herein details and comments the different steps of the pre-analytical process. Application of these guidelines and participation to quality insurance programs are mandatory to ensure the correct evaluation of oncotheranostic biomarkers.

Semi-quantitative lymph node assessment of (18)F-FDG PET/CT in locally advanced breast cancer: correlation with biological prognostic factors.

PURPOSE: The aim of this study was to analyse the correlation between dual-time-point (18)F-2-deoxy-2-fluoro-D-glucose (FDG) uptakes in lymph nodes assessed by positron emission tomography (PET)/CT and histopathological and immunohistochemical prognostic factors. METHODS: Seventy-five women with locally advanced breast cancer were prospectively evaluated. PET/CT was requested in the initial staging previous to adjuvant chemotherapy (multicentre study). All of the patients underwent (18)F-FDG PET/CT with a dual-time-point acquisition. Both examinations were evaluated qualitatively and semi-quantitatively with calculation of maximum standardized uptake values (SUV(max)) in PET-1 (SUV-1) and in PET-2 (SUV-2) and the percentage variation of the SUV or retention index (RI) between PET-1 and PET-2 in lymph nodes with the greater (18)F-FDG uptake. The biological prognostic parameters such as the steroid receptor status, p53 and HER2 expression, proliferation rate (Ki-67) and grading were determined from tissue of the primary tumour. Metabolic and biological parameters were correlated using Spearman's rank-order correlation coefficient and Mann-Whitney U and Kruskal-Wallis tests. RESULTS: Negative receptor status was correlated with higher SUV-1, SUV-2 and RI in lymph nodes. The results were significant for progesterone receptor status. p53 over-expression and triple-negative status were associated with greater semi-quantitative parameters in lymph nodes. Higher tumoural grades were related with greater semi-quantitative parameters (p > 0.05). CONCLUSION: Biological factors of bad prognosis were correlated with higher semi-quantitative metabolic values in lymph nodes. Therefore these results appear to reveal biological significance of lymph node (18)F-FDG accumulation.

Economic evaluation of the 70-gene prognosis-signature (MammaPrint®) in hormone receptor-positive, lymph node-negative, human epidermal growth factor receptor type 2-negative early stage breast cancer in Japan.

The 70-gene prognosis-signature is validated as a good predictor of recurrence for hormone receptor-positive (ER+), lymph node-negative (LN-), human epidermal growth factor receptor type 2-negative (HER2-) early stage breast cancer (ESBC) in Japanese patient population. Its high cost and potential in avoiding unnecessary adjuvant chemotherapy arouse interest in its economic impact. This study evaluates the cost-effectiveness of including the assay into Japan's social health insurance benefit package. An economic decision tree and Markov model under Japan's health system from the societal perspective is constructed with clinical evidence from the pool analysis of validation studies. One-way sensitivity analyses are also performed. Incremental cost-effectiveness ratio is estimated as ¥3,873,922/quality adjusted life year (QALY) (US$43,044/QALY), which is not more than the suggested social willingness-to-pay for one QALY gain from an innovative medical intervention in Japan, ¥5,000,000/QALY (US$55,556/QALY). However, sensitivity analyses show the instability of this estimation. The introduction of the assay into Japanese practice of ER+, LN-, HER2- ESBC treatment by including it to Japan's social health insurance benefit package has a reasonable chance to be judged as cost-effective and may be justified as an efficient deployment of finite health care resources.

Assessment of letrozole and tamoxifen alone and in sequence for postmenopausal women with steroid hormone receptor-positive breast cancer: the BIG 1-98 randomised clinical trial at 8·1 years median follow-up.

BACKGROUND: Postmenopausal women with hormone receptor-positive early breast cancer have persistent, long-term risk of breast-cancer recurrence and death. Therefore, trials assessing endocrine therapies for this patient population need extended follow-up. We present an update of efficacy outcomes in the Breast International Group (BIG) 1-98 study at 8·1 years median follow-up. METHODS: BIG 1-98 is a randomised, phase 3, double-blind trial of postmenopausal women with hormone receptor-positive early breast cancer that compares 5 years of tamoxifen or letrozole monotherapy, or sequential treatment with 2 years of one of these drugs followed by 3 years of the other. Randomisation was done with permuted blocks, and stratified according to the two-arm or four-arm randomisation option, participating institution, and chemotherapy use. Patients, investigators, data managers, and medical reviewers were masked. The primary efficacy endpoint was disease-free survival (events were invasive breast cancer relapse, second primaries [contralateral breast and non-breast], or death without previous cancer event). Secondary endpoints were overall survival, distant recurrence-free interval (DRFI), and breast cancer-free interval (BCFI). The monotherapy comparison included patients randomly assigned to tamoxifen or letrozole for 5 years. In 2005, after a significant disease-free survival benefit was reported for letrozole as compared with tamoxifen, a protocol amendment facilitated the crossover to letrozole of patients who were still receiving tamoxifen alone; Cox models and Kaplan-Meier estimates with inverse probability of censoring weighting (IPCW) are used to account for selective crossover to letrozole of patients (n=619) in the tamoxifen arm. Comparison of sequential treatments to letrozole monotherapy included patients enrolled and randomly assigned to letrozole for 5 years, letrozole for 2 years followed by tamoxifen for 3 years, or tamoxifen for 2 years followed by letrozole for 3 years. Treatment has ended for all patients and detailed safety results for adverse events that occurred during the 5 years of treatment have been reported elsewhere. Follow-up is continuing for those enrolled in the four-arm option. BIG 1-98 is registered at clinicaltrials.govNCT00004205. FINDINGS: 8010 patients were included in the trial, with a median follow-up of 8·1 years (range 0-12·4). 2459 were randomly assigned to monotherapy with tamoxifen for 5 years and 2463 to monotherapy with letrozole for 5 years. In the four-arm option of the trial, 1546 were randomly assigned to letrozole for 5 years, 1548 to tamoxifen for 5 years, 1540 to letrozole for 2 years followed by tamoxifen for 3 years, and 1548 to tamoxifen for 2 years followed by letrozole for 3 years. At a median follow-up of 8·7 years from randomisation (range 0-12·4), letrozole monotherapy was significantly better than tamoxifen, whether by IPCW or intention-to-treat analysis (IPCW disease-free survival HR 0·82 [95% CI 0·74-0·92], overall survival HR 0·79 [0·69-0·90], DRFI HR 0·79 [0·68-0·92], BCFI HR 0·80 [0·70-0·92]; intention-to-treat disease-free survival HR 0·86 [0·78-0·96], overall survival HR 0·87 [0·77-0·999], DRFI HR 0·86 [0·74-0·998], BCFI HR 0·86 [0·76-0·98]). At a median follow-up of 8·0 years from randomisation (range 0-11·2) for the comparison of the sequential groups with letrozole monotherapy, there were no statistically significant differences in any of the four endpoints for either sequence. 8-year intention-to-treat estimates (each with SE ≤1·1%) for letrozole monotherapy, letrozole followed by tamoxifen, and tamoxifen followed by letrozole were 78·6%, 77·8%, 77·3% for disease-free survival; 87·5%, 87·7%, 85·9% for overall survival; 89·9%, 88·7%, 88·1% for DRFI; and 86·1%, 85·3%, 84·3% for BCFI. INTERPRETATION: For postmenopausal women with endocrine-responsive early breast cancer, a reduction in breast cancer recurrence and mortality is obtained by letrozole monotherapy when compared with tamoxifen montherapy. Sequential treatments involving tamoxifen and letrozole do not improve outcome compared with letrozole monotherapy, but might be useful strategies when considering an individual patient's risk of recurrence and treatment tolerability. FUNDING: Novartis, United States National Cancer Institute, International Breast Cancer Study Group.

Candidate gene copy number analysis by PCR and multicapillary electrophoresis.

Genetic polymorphisms are often considered as risk factors of complex diseases serving as valuable and easily detectable biomarkers, also stable during the whole lifespan. A novel type of genetic polymorphism has been identified just recently, referred to as gene copy number variation (CNV) or copy number polymorphism. CNV of glycogen synthase kinase 3 beta and its adjacent gene, Nr1i2 (pregnane X receptor isoform), has been reported to associate with bipolar depression. In our study we introduced multicapillary electrophoresis for gene copy number analysis as an affordable alternative to real-time PCR quantification with TaqMan gene probes. Our results show the reliability of the developed method based on conventional PCR followed by separation of products by multicapillary electrophoresis with quantitative evaluation. This method can be readily implemented for the analysis of candidate gene CNVs in high throughput clinical laboratories and also in personalized medicine care of depression-related risk factors.

Assessment of the proliferative status of epithelial cell types in the endometrium of young and menopausal transition women.

BACKGROUND: We determined protein and mRNA expressions of markers of normal human endometrial proliferation and hypothesized that dysregulation of the endometrial response to estradiol (E(2)) and progesterone would be observed in the older menopausal transition (MT) women compared with mid-reproductive age (MRA) controls. METHODS: Endometrial biopsies were prospectively obtained from MRA and MT non-randomized healthy volunteers during proliferative (+/- exogenous E(2)) and secretory (MRA only) menstrual cycle phases. mRNA and/or nuclear protein expressions of proliferative markers (MKI67, PCNA and MCM2), cell-cycle regulators (cyclins A1, E1 and D1 and cyclin dependent kinase Inhibitor B; CCNA1, CCNE1, CCND1 and CDKN1B) and sex-steroid receptors [estrogen receptor (ER) and progesterone receptor (PR)] were assessed in endometrial lumen, gland and stroma. RESULTS: MRA women had significantly higher proliferative than secretory expression of MKI67, PCNA, MCM2, CCNA1, CCNE1, ESR1 and PGR in lumen and gland (minimal stromal changes), whereas CDKN1B protein expression was higher during the secretory phase. E(2)-treatment of MT women led to relatively less MKI67 glandular protein expression compared with MRA women; no other age-related differences were observed. CONCLUSION: Although the MT does not appear to alter the proliferative cell phenotype of endometrial epithelium and stroma, the data suggest that prior to the MT, age is associated with a decrease in some proliferative markers and steroid receptor expression status within different endometrial cell types.

Quality control for biomarker determination in oncology: the experience of the Italian Network for Quality Assessment of Tumor Biomarkers (INQAT).

Biomarker analysis and evaluation in oncology is the product of a number of processes (including managerial, technical and interpretation steps) which need to be monitored and controlled to prevent and correct errors and guarantee a satisfactory level of quality. Several biomarkers have recently moved to clinical validation studies and successively to clinical practice without any definition of standard procedures and/or quality control (QC) schemes necessary to guarantee the reproducibility of the laboratory information. In Italy several national scientific societies and single researchers have activated -- often on a pilot level -- specific external quality assessment protocols, thereby potentially jeopardizing the clinical reality even further. In view of the seriousness of the problem, in 1998 the Italian Ministry of Health sponsored a National Survey Project to coordinate and standardize the procedures and to develop QC programs for the analysis of cancer biomarkers of potential clinical relevance. Twelve QC programs focused on biomarkers and concerning morphological, immunohistochemical, biochemical, molecular, and immunoenzymatic assays were coordinated and implemented. Specifically, external QC programs for the analytical phase of immunohistochemical p53, Bcl-2, c-erb-2/neu/HER2, and microvessel density determination, of morphological evaluation of tumor differentiation grade, and of molecular p53 analysis were activated for the first time within the project. Several hundreds of Italian laboratories took part in these QC programs, the results of which are available on the web site of the Network (www.cqlaboncologico.it). Financial support from the Italian Government and the National Research Council (CNR) will guarantee the pursuit of activities that will be extended to new biomarkers, to preanalytical phases of the assays, and to revision of the criteria of clinical usefulness for evaluating the cost/benefit ratio.

[Receptor assessment of primary breast carcinoma and synchronous metastatic lymph nodes: histochemical study]. / Assetto recettoriale del carcinoma mammario primitivo e della metastasi linfonodale sincrona: studio istochimico.

Several AA, utilizing the biochemical method (DCC) showed the presence or absence of hormonal receptor in either synchronous or metachronous metastatic nodes (N) can reproduce the receptor assessment (AR) of the primary breast tumor (T). We evaluated the AR in T and in synchronous N, by using two morphological methods, immunocytochemical and histofluorescent, to detect the nuclear Estrogen Receptor (ER1) and cytoplasmic sites of the II type (ER2 and PgR2). We studied 50 patients who underwent radical mastectomy for breast cancer N+. In this series we founded a high correlation between AR in T and in N: 82% for ER1, 94% for ER2, and 92% for PgR2. Also we observed 4 cases (2 ER1 and 2 PgR2) which presented the receptor in N only. These data are comparable to results obtained with the DCC method and confirm the opportunity to perform also morphological techniques to evaluate AR in breast cancer to take into consideration also cellularity and tumor heterogeneity informations.

Assessment of nucleolar organizer regions by automatic image analysis in breast cancer: correlation with DNA content, proliferation rate, receptor status and histopathological grading.

The value of automatic image analysis in the investigation of nucleolus regions (AgNOR) has been examined in tissue sections of 52 malignant and 30 benign breast lesions. Determination of the AgNOR number per cell alone revealed a considerable overlap between benign (range 1.2-3.8) and malignant specimens (range 1.5-16.2). They differed however, highly significantly (P less than 0.001) in their AgNOR sizes. In benign breast disorders the mean AgNOR area per tumour ranged from 0.22 microns2 to 1.07 microns2 (mean 0.39 microns2), whereas in carcinomas AgNOR sites ranged from 0.05 microns2 to 0.22 microns2 (mean 0.09 microns2). AgNOR counts showed a good correlation with histopathological grade (P less than 0.05), aneuploidy (P less than 0.01), proliferation rate as determined by Ki67 immunostaining (P less than 0.01), as well as oestrogen and progesterone receptor content (P less than 0.01). Image analysis proved to be advantageous over AgNOR counting alone as it facilitated the standardization of the AgNOR technique itself and thus, significantly improved its diagnostic specifity.

A simple, low-cost computer program for Scatchard plot analysis of binding data in steroid hormone receptor assays.

This paper presents a relatively simple computer program for Scatchard plot analysis of binding data in steroid hormone receptor determination. The program is written in BASIC and adapted to a low-cost desktop calculator. The program is designed to permit data input without demanding extensive technical knowledge of either computer systems or mathematical background of receptor binding assays.

Quantitative assessment of steroid hormone binding sites by thaw-mount autoradiography.

A procedure for the quantitative assessment of nuclear receptors for steroid hormones--and other substances--in individual cells is presented. Thaw-mount autoradiography, a procedure developed earlier in our laboratory, is utilized. The silver grain yield (specific activity) is 16.6 disintegrations per silver as determined fo tritium in guinea pig uterine tissues. An integrated formula is presented and applied for 3H-estradiol, 3H-diethylstilbestrol, and 3H-aldosterone in sampled tissue. A comparison with data derived from the literature that are based on the homogenization of whole uteri and biochemical analysis shows comparable values wtih the autoradiographic data if the latter are pooled. The pooled ata indicated 12-14,00 molecules of 3H-estradiol per uterine nucleus, while subpopulations of target cells vary between 5,000 and 28,000 per nucleus.