The importance of international collaboration for rare diseases research: a European perspective.

Over the last two decades, important contributions were made at national, European and international levels to foster collaboration into rare diseases research. The European Union (EU) has put much effort into funding rare diseases research, encouraging national funding organizations to collaborate together in the E-Rare program, setting up European Reference Networks for rare diseases and complex conditions, and initiating the International Rare Diseases Research Consortium (IRDiRC) together with the National Institutes of Health in the USA. Co-ordination of the activities of funding agencies, academic researchers, companies, regulatory bodies, and patient advocacy organizations and partnerships with, for example, the European Research Infrastructures maximizes the collective impact of global investments in rare diseases research. This contributes to accelerating progress, for example, in faster diagnosis through enhanced discovery of causative genes, better understanding of natural history of rare diseases through creation of common registries and databases and boosting of innovative therapeutic approaches. Several examples of funded pre-clinical and clinical gene therapy projects show that integration of multinational and multidisciplinary expertize generates new knowledge and can result in multicentre gene therapy trials. International collaboration in rare diseases research is key to improve the life of people living with a rare disease.

Sensitivity of HER-2/neu antibodies in archival tissue samples of invasive breast carcinomas. Correlation with oncogene amplification in 160 cases.

Overexpression and amplification of the HER-2 oncogene in patients with breast cancer has correlated with early onset of metastasis, resistance to hormonal therapy and some forms of chemotherapy, and shortened survival. Therefore, evaluation of this putative prognostic or predictive factor seems critical. Because different antibodies are used for the detection of the 185-kd HER-2 oncoprotein, we studied the sensitivity of 3 frequently used antibodies. Immunohistochemistry results were correlated with gene amplification level as assessed by fluorescence in situ hybridization. Protein overexpression was found in 17.2% and 12.5% of cases using antibodies against the external (TAB250) and internal (CB11) domains of the protein, respectively, and in 38.0% of cases using a rabbit polyclonal antibody. Fluorescence in situ hybridization was successful in all 160 tumors, and amplification was found in 37 tumors (23.1%). The monoclonal antibody TAB250 had the lowest misclassification rate, 9.6% (sensitivity, 67%; specificity, 97.5%).

Reliability of the tissue microarray based FISH for evaluation of the HER-2 oncogene in breast carcinoma.

AIMS: Tumour tissue microarray allows the analysis of hundreds of tumour samples simultaneously on a single microscope slide. However, the extremely small tissue samples taken from the original tissue may not always be representative of the entire tumour. METHODS: The reliability of this new technology was investigated by analysing HER-2 oncogene amplification by fluorescence in situ hybridisation (FISH) from representative slides of the whole tumour and small tissue core biopsies from 29 invasive breast tumours. RESULTS: The tissue microarray method had high accuracy; in only one of 29 cases (3.4%; 95% confidence interval, 0% to 10%) were the results discordant with whole tumour analysis. CONCLUSION: Tumour microarray is a highly reliable method for analysing HER-2 oncogene amplification by FISH in human breast tumours.

Trisomy 21 as the sole abnormality in a refractory anemia with ring sideroblasts.

Numerous chromosome abnormalities have been described in myelodysplastic syndromes, but single karyotypic aberrations are much less frequent. We report the case of a 65-year-old woman who presented a trisomy 21 as the sole karyotypic anomaly for a refractory anemia with ring sideroblasts. The nature of such an anomaly is discussed in regard to pathogenesis and prognosis.

Oncoselective transduction of CD80 and CD86 in tumor cell lines using an autonomous recombinant parvovirus.

BACKGROUND: The aim of this study was to enhance selectively the immunostimulatory properties of tumor cells. Based on their oncotropic properties, we used autonomous recombinant parvoviruses to transduce the genes coding for the constimulatory molecules CD80 (B7-1) or CD86 (B7-2) specifically into tumor cells without transducing normal cells. MATERIALS AND METHODS: After infection of tumor cells by these viruses, surface expression of CD80 and CD86 molecules was assessed by FACS and enhancement of immunostimulatory properties was assessed in alloreactions with G-10 purified T cells. RESULTS: Infection of normal and transformed cells with recombinant MVM- B7-1 or B7-2 viruses leads to expression of costimulatory molecules only by tumor cells and confers on them the capacity to sensitize naive T cells in vitro. CONCLUSION: This approach should ultimately lead to selective expression of costimulatory molecules in tumor tissues in vivo without affecting normal cells.

[Detection of extra chromosomes 12 by fluorescent in situ hybridization (FISH) in ovarian stromal tumors. Study of 12 cases and review of the literature]. / Détection de chromosomes 12 surnuméraires par hybridation in situ fluorescente (FISH) dans des tumeurs stromales ovariennes.

Chromosomic aberrations play a major role in the initiation and the progression of benign as well as malignant tumors. In particular, trisomy 12 is frequently observed in female genitourinary tract tumors and constitutes a recurrent and often unique anomaly in stromal ovarian tumors such as fibrothecomas. Today, the genetic analysis of fresh or fixed solid tumors is enabled by the fluorescent in situ hybridization method (FISH). Using FISH and/or conventional cytogenetics, we analysed 12 ovarian stromal tumors (6 fibromas, 3 fibro thecomas and 3 thecomas). All of these tumors were benign and trisomy 12 was observed in all cases. Moreover, 3 cases presented trisomy and tetrasomy for chromosome 12 simultaneously. The high frequency of trisomy 12 in this tumor type suggests that this abnormality might be implicated in ovarian tumorigenesis.

In situ amplification of oestrogen receptor alpha mRNA in breast cancer cell lines and tumours.

The aim of this work was to develop a direct in situ reverse transcription polymerase chain reaction (in situ RT-PCR) assay for the detection of oestrogen receptor alpha (ERalpha) mRNA on in vitro cell lines and breast tumour cell smears. ERalpha mRNA amplification was performed on MCF-7 (ERalpha positive) and MDA-MB-231 (ERalpha negative) cell lines as well as on 51 cytological smears of breast tumour samples from patients. The in situ amplification of mRNA in cell lines and ex vivo breast tumours was successful. However, finding an equilibrium between optimal cell morphology and PCR performance varied with each tumour, leading to difficulty in standardisation for daily practice. Nonetheless, in situ RT-PCR is a useful tool for the detection of ERalpha mRNA in selected cases, both in vitro and ex vivo. J Clin PATHOL: Mol Pathol

Chromogenic in situ hybridization: a practical alternative for fluorescence in situ hybridization to detect HER-2/neu oncogene amplification in archival breast cancer samples.

Determination of HER-2/neu oncogene amplification has become necessary for selection of breast cancer patients for trastuzumab (Herceptin) therapy. Fluorescence in situ hybridization (FISH) is currently regarded as a gold standard method for detecting HER-2/neu amplification, but it is not very practical for routine histopathological laboratories. We evaluated a new modification of in situ hybridization, the chromogenic in situ hybridization (CISH), which enables detection of HER-2/neu gene copies with conventional peroxidase reaction. Archival formalin-fixed paraffin-embedded tumor tissue sections were pretreated (by heating in a microwave oven and using enzyme digestion) and hybridized with a digoxigenin-labeled DNA probe. The probe was detected with anti-digoxigenin fluorescein, anti-fluorescein peroxidase, and diaminobenzidine. Gene copies visualized by CISH could be easily distinguished with a x40 objective in hematoxylin-stained tissue sections. HER-2/neu amplification typically appeared as large peroxidase-positive intranuclear gene copy clusters. CISH and FISH (according to Vysis, made from frozen pulverized tumor samples) correlated well in a series of 157 breast cancers (kappa coefficient, 0.81). The few different classifications were mostly because of low-level amplifications by FISH that were negative by CISH and immunohistochemistry with monoclonal antibody CB-11. We conclude that CISH, using conventional bright-field microscopy in evaluation, is a useful alternative for determination of HER-2/neu amplification in paraffin-embedded tumor samples, especially for confirming the immunohistochemical staining results.

Trisomy 6 in Merkel cell carcinoma: a recurrent chromosomal aberration.

UNLABELLED: We retrospectively investigated 17 cases of primary and metastasizing Merkel cell carcinomas (MCC) from 14 patients using chromosomal in-situ hybridization (CISH) to study the occurrence of trisomy 6 in these lesions. METHODS AND RESULTS: Histological diagnosis on all tumour samples was obtained on haematoxylin and eosin stained sections. Immunohistochemistry was performed with antibodies against pancytokeratin (CAM 5.2), cytokeratin 20 (CK20), MIC2 antigen (CD99), neuron-specific enolase (NSE), and chromogranin A (chrA). Sections (4 microm) of the paraffin-embedded tumours were analysed with alpha-satellite centromeric probes for chromosome 6 or 17 using CISH. The signal was amplified by the Tyramide Signal Amplification (TSA) assay. Immunohistochemically, the tumours showed the same general epithelial neuro-endocrine pattern: 11/13 expressed cytokeratin 20, and 47% exhibited trisomy 6, with no significant difference between primary and metastatic lesions. Incomplete follow-up data did not allow us to establish a prognostic value of trisomy 6, however, this aberration might be an additional diagnostic tool in distinguishing MCC from other small round blue cell tumours. CONCLUSIONS: CISH seems to be a promising adjunctive method to diagnose Merkel cell carcinoma. Trisomy 6 should be investigated more closely in these cases, as has been done for chromosomes 1 and 11. Of particular interest would be identification of modifications in proto-oncogene(s) located on chromosome 6.

Comparison of HER-2 status between primary breast cancer and corresponding distant metastatic sites.

BACKGROUND: The humanized anti-HER-2 monoclonal antibody trastuzumab (Herceptin) is a new treatment modality for metastatic breast cancer, the efficacy of which is directly correlated with the HER-2 status of the tumour, evaluated either by immunohistochemistry (IHC) and/or by fluorescence in situ hybridisation (FISH). This analysis is generally performed on the primary tumour. There are few data regarding the HER-2 status in the corresponding distant metastases. METHODS: HER-2 status in 107 patients with a primary breast tumour and at least one distant metastatic lesion was analysed by IHC and FISH. RESULTS: We found similar levels of amplification (25% and 24%) and overexpression (13% and 19%) of HER-2 in primary and metastatic samples, respectively. Among paired primary/metastatic tumours, six (6%) showed discordance by HercepTest(TM) (n = 100): all six cases showed greater Her-2 overexpression in the metastatic tissue. By FISH (n = 68), five (7%) cases were discordant: two cases were amplified in the primary tumour but not in the metastasis, and three samples showed amplification in the metastasis but not in the primary. Finally, we analysed HER-2 status in different metastatic lesions from 17 patients that had at least two distant metastatic sites. Discordance between different sites from the same patient was 18% by IHC and 19% by FISH. CONCLUSIONS: Between the paired primary tumour and distant metastatic lesions, 94% and 93% of samples had concordant HER-2 status when analysed by IHC or FISH, respectively. These results do not support routine determination of HER-2 on metastatic sites, particularly when FISH results from the primary tumour are available.

Evaluation of HER2, p53, bcl-2, topoisomerase II-alpha, heat shock proteins 27 and 70 in primary breast cancer and metastatic ipsilateral axillary lymph nodes.

BACKGROUND: Breast cancer is a heterogeneous disease. Predictive biological markers (BM) of responsiveness to therapy need to be identified. Evaluation of BM is mainly done at the primary site. However, in the adjuvant therapy of breast cancer, the main goal is control of micrometastases. It is still unknown whether heterogeneity in the expression of BM between the primary site and its micrometastases exists. OBJECTIVE: To evaluate the expression of some BM with potential predictive value from the primary breast cancer site and metastatic ipsilateral axillary lymph nodes. PATIENTS AND METHODS: Focality (percentage of positive cells) and intensity staining scores were evaluated for each marker. Freshly cut sections (4 microm) from embedded blocks of breast cancer fixed in formalin or bouin were put onto superfrost slides (Menzel-Gläser). Protein expression was evaluated immunohistochemically (IHC) using monoclonal antibodies against: topo II-alpha (clone KiS1, 1 microg/ml, Roche) with a trypsine pre-treatment (P); HSP27 (clone G3.1, 1/60, Biogenex), HSP70 (clone BRM.22, 1/80, Biogenex) and HER2 (clone CB11, 1/40, Novocastra; without P); p53 (clone D07, 1/750, Dako) and bcl-2 (clone 124, 1/60, Dako) with citrate buffer as P. RESULTS: Overall, the percentage of discordant marker status in the primary tumour and its metastatic lymph nodes was 2% for HER2, 6% for p53, 15% for bcl-2, 19% for topoisomerase II-alpha, 24% for HSP27 and 30% for HSP70. For the subgroup of patients with positive BM in the primary tumour, the percentage of discordance was 6% for HER2, 7% for p53, 14% for bcl-2, 19% for HSP70, 21% for topoisomerase II-alpha and 36% for HSP27. For the subgroup of patients with positive BM in the lymph nodes, the percentage of discordance was 9% for bcl-2, 15% for HER2 and p53, 21% for topoisomerase II-alpha, 22% for HSP27 and 25% for HSP70. CONCLUSIONS: 1) No biological marker had 100% concordant results. 2) Although some discordant cases might be explained by the limitations of the IHC technique, future studies aiming to evaluate the predictive value of BM in the adjuvant therapy of breast cancer should take into account a possible difference in BM expression between the primary and the metastatic sites.

Tumor-infiltrating dendritic cells in adenocarcinomas of the breast: a study of 143 neoplasms with a correlation to usual prognostic factors and to clinical outcome.

Dendritic cells (DC) are the most potent antigen-presenting cells, and induce antigen-specific immune responses. Infiltration of tumors by DC is thought to reflect the interaction between the host immune system and tumor cells. Tumor-infiltrating DC (TIDC) are believed to evolve into tumor-antigen pulsed cells and then to migrate to local lymph nodes, where they activate anti-tumor immune responses. Indirect clinical evidence supporting this theory is provided by studies showing that high TIDC densities are associated with favorable prognosis in some tumor types. In the present study, we evaluated 143 primary breast adenocarcinomas for the presence of DC, using immunohistochemistry with the anti-S100 protein antibody. We analyzed the relationship between the degree of infiltration by S100+ TIDC and the usual prognostic factors and clinical outcome. The results show that 42% of breast adenocarcinomas contain S100 TIDC. The number of S100+ TIDC varies according to the grade of tumors as follows: GRIII > GRII > GRI. A relationship is also found between S100+ TIDC and tumor size, lymph-node involvement, estrogen/progesterone receptor status and age. However, the presence of S100+ TIDC, even at the highest density, was not correlated with metastasis-free survival or overall survival.

HER-2 and topo-isomerase IIalpha as predictive markers in a population of node-positive breast cancer patients randomly treated with adjuvant CMF or epirubicin plus cyclophosphamide.

BACKGROUND: The predictive role of HER-2 in node-positive breast cancer patients receiving CMF or an anthracycline-based adjuvant therapy remains unclear. In addition, topo-isomerase II alpha (topo IIalpha), as the cellular target of anthracyclines, might have value as a predictive marker. PATIENTS AND METHODS: Four hundred eighty-one archival primary tumor samples were collected among 777 patients entered into a multicenter phase III trial comparing classical CMF with epirubicin cyclophosphamide (HEC) as adjuvant therapy of node-positive breast cancer. HER-2 was evaluated by immunohistochemistry (IHC) using different antibodies (Abs). Topo IIalpha was evaluated by IHC using the Ab KiS 1. In each subgroup of patients identified by HER-2 and topo IIalpha, adjusted hazard ratios for event-free survival (EFS) and the corresponding 95% confidence intervals have been calculated for the different study comparisons. An interaction test has been performed to investigate the role of HER-2 and topo IIalpha as predictive markers. RESULTS: When HER-2 was evaluated by CB-11 and 4D5 mAbs, the EFS adjusted hazard ratios (HR) for the main study comparison HEC vs. CMF were: HER-2 positive: 0.33 (95% confidence interval (95% CI): 0.09 1.27, P = 0.08), HER-2 negative: 1.16 (95%, CI: 0.71-1.90, P = 0.56); the P-value for the interaction test was 0.10. When HER-2 was evaluated by TAB-250 + pAbl Abs, the adjusted HR for the same comparison were: HER-2 positive: 1.06 (95% CI: 0.45-2.52, P = 0.90), HER-2 negative: 0.99 (95% CI: 0.58-1.68, P = 0.97); the P-value for the interaction test was 0.84. With regard to topo IIalpha, the adjusted HR for the EFS comparison HEC vs. CMF were: topo IIalpha positive: 0.66 (95% CI: 0.32-1.36, P = 0.25), topo IIalpha negative: 1.26 (95% CI: 0.63-2.50, P = 0.51); the P-value for the interaction test was 0.13. CONCLUSIONS: This study suggests that in node-positive breast cancer patients randomly treated with CMF or an epirubicin-based regimen, the predictive value of HER-2 may vary according to the Abs used in the immunohistochemistry assay. In addition, the study supports the concept that topo IIalpha might be involved in the determination of tumor responsiveness to an anthracycline-based adjuvant therapy.