Clasificación molecular del cáncer de mama, obtenida a través de la técnica de hibridación in situ cromogénica (CISH) / Molecular classification of breast cancer patients obtained through the technique of chromogenic in situ hybridization (CISH)

El cáncer de mama es una enfermedad heterogénea compuesta de un número creciente de subtipos biológicos, con una sustancial variabilidad en la evolución de la enfermedad dentro de cada categoría. El objetivo del presente trabajo fue clasificar las muestras objeto a estudio de acuerdo a las clases moleculares de carcinoma de mama: luminal A, luminal B, HER2 y triple negativo, considerando el estado de amplificación de HER2 obtenido a través de la técnica de hibridación in situ cromogénica (CISH). La muestra estuvo constituida por 200 biopsias fijadas en formol al 10%, procesadas por las técnicas habituales hasta la inclusión en parafina, correspondientes a pacientes diagnosticadas con carcinoma ductal infiltrante de la mama, procedentes de consulta privada y del Instituto de Oncología “Dr. Miguel Pérez Carreño”, con estudio inmunohistoquímico (IHQ) para receptores hormonales y HER2 realizado en el Hospital Metropolitano del Norte de Valencia, Venezuela. La clasificación molecular de los tumores de las pacientes, considerando la expresión de los Receptores de Estrógeno (RE) y Receptores de Progesterona (RP) a través de IHQ y la amplificación de HER2 por CISH, permitió agrupar en las diferentes clases moleculares los casos calificados inicialmente como desconocidos, debido a que tenían un resultado indeterminado (2+) para la expresión de HER2 por IHQ; asimismo, esta clasificación ocasionó que algunos casos considerados inicialmente en una clase molecular pasaron a otra clase, posterior a la revaloración del estado de HER2 a través de CISH.

Breast cancer is a heterogeneous disease composed of a growing number of biological subtypes, with substantial variability of the disease progression within each category. The aim of this research was to classify the samples object of study according to the molecular classes of breast cancer: luminal A, luminal B, HER2 and triple negative, as a result of the state of HER2 amplification obtained by the technique of chromogenic in situ hybridization (CISH). The sample consisted of 200 biopsies fixed in 10% formalin, processed by standard techniques up to paraffin embedding, corresponding to patients diagnosed with invasive ductal carcinoma of the breast. These biopsies were obtained from patients from private practice and the Institute of Oncology “Dr. Miguel Pérez Carreño", for immunohistochemistry (IHC) of hormone receptors and HER2 made in the Hospital Metropolitano del Norte, Valencia, Venezuela. The molecular classification of the patient’s tumors considering the expression of estrogen and progesterone receptors by IHC and HER2 amplification by CISH, allowed those cases originally classified as unknown, since they had an indeterminate (2+) outcome for HER2 expression by IHC, to be grouped into the different molecular classes. Also, this classification permitted that some cases, initially considered as belonging to a molecular class, were assigned to another class, after the revaluation of the HER2 status by CISH.

HER 2 status in invasive breast cancer: Immunohistochemistry, fluorescence in-situ hybridization and chromogenic in-situ hybridization.

Introduction : HER2/neu gene status in breast cancers can be evaluated by targeting protein and gene - immunohistochemistry (IHC) and fluorescence in-situ hybridization (FISH). Recent studies have shown chromogenic in-situ hybridization (CISH) as a relatively cheaper alternative. Materials and Methods : Forty-three nonconsecutive, randomly selected primary invasive breast cancer cases were evaluated for c-erbB-2 (HER2 protein) by IHC and gene amplification by FISH and CISH. Results of each of the same were compared. Results : CISH showed approximately 90% and 100% concordance for IHC negative and positive cases, respectively; while approximately 94.4% and 91% concordance with FISH amplified and non-amplified cases, respectively. Conclusion : This study showed feasibility of incorporation of CISH as a low cost option in routine management of breast carcinoma in the Indian setting. Secondly, reconfirmation of IHC negative and positive cases can be done by CISH.

A method for detection of HER2 gene amplification in breast cancer tissues: Chromogenic in situ hybridization method and its modification / 第二军医大学学报

Objective: To apply chromogenic in situ hybridization (CISH for detection of HER2 gene amplification in breast cancer tissues and to discuss some modifications of the CISH method. Methods: HER2 gene amplification was detected by CISH in 60 breast cancer specimens with an immunohistochemical score over 2+. The correlation between the results of IHC and CISH was analyzed. Our experience in CISH manipulation was summarized and optimization to CISH was discussed. Results: CISH identified gene amplification in 91% (40/44) specimens with an IHC score of 3+ and in 50% (8/16) specimens with an IHC score of 2+. The total concordance rate between IHC and CISH was 80% (48/60, P<0.01). The thickness of sections should be controlled within 4-5 μm; the denaturation should be complete; and the post-hybridization washing temperature and time were also very important and the temperature should be controlled at 70-75 °C. The dyeing time of hematoxylin should also be restrictedly controlled. Positive control should be set up in the experiment for high quality of the experiment. Conclusion: CISH has high concordance rate with IHC in examining HER2 amplification and it may be a new method for detection of HER2 gene. The thickness of the sections, the post hybridization washing temperature and time, and the time of hematoxylin dyeing should be strictly controlled.

The Prognostic and Predictive Value of EGFR and HER-2 in Advanced Non-small Cell Lung Cancer Patients Who Are Treated with Cisplatin and Paclitaxel

PURPOSE: Although both platinum-based drugs and third-generation drugs are commonly used as first-line therapy for patients with advanced, unresectable non-small cell lung cancer, their effectiveness and clinical outcomes vary. We investigated whether epidermal growth factor receptor (EGFR) and HER-2 were correlated with the chemoresponse and survival after treatment with a cisplatin plus paclitaxel regimen. MATERIALS AND METHODS: Forty-nine tumors were analyzed by chromogenic in situ hybridization (CISH) for EGFR and HER-2 gene amplification. RESULTS: Twenty-eight patients (57%) achieved a partial response (PR), 13 (27%) showed stable disease (SD) and 8 (16%) had progressive disease (PD). EGFR and HER-2 amplification was identified in 43% and 57% of the tumors, respectively. EGFR amplification revealed no association with either a chemoresponse or survival, whereas HER-2 was amplified more frequently in the patients with PD (88% vs. 54%, respectively, p=0.06) and in the patients with shorter survival (12 months vs. 20 months respectively, p=0.027). CONCLUSION: The evaluation of HER-2 gene amplification is a promising approach for identifying those patients who are most likely to benefit from combination chemotherapy with cisplatin and paclitaxel

Chromogenic In Situ Hybridization Analysis to Determinate HER-2/neu Status in Breast Carcinoma

PURPOSE: The determination of HER-2/neu gene amplification has become necessary for the selection of breast cancer patients to undergo anti-HER-2/neu therapy, using a humanized monoclonal antibody. Chromogenic in situ hybridization (CISH) detection of the HER-2/neu gene, a newly developed method, utilizes a robust and unique-sequence DNA probe labeled with digoxygenin, which is sequentially incubated with antidigoxygenin fluorescein, antifluorescein peroxidase and diaminobenzidine. The aim of this study was to establish a CISH assay for the detection of HER-2/neu amplification. The results were compared with those of the immunohistochemistry (IHC) methods, most frequently used for detecting HER-2/neu alteration. METHODS: CISH was performed in 4 groups of infiltrating breast carcinomas. Each group was comprised of 20 cases in which the HER-2/neu stati had previously been scored on a four value scale: 0, 1+, 2+ and 3+ by IHC. The results of CISH and IHC were compared for each tumor group. The HER-2/neu gene amplification detected by CISH was thpically visualized as large DAB-stained clusters or by many dots in the nucleus. RESULTS: The concordance between the CISH and IHC was 95% (kappa=0.901). Three IHC-positive cases (score 2+) showed no gene amplification and one IHC-negative case (score 1+) showed gene amplification by CISH. CONCLUSION: The current study showed excellent agreement between the CISH and IHC methods. CISH is an accurate, practical and economical approach for determining the HER-2/neu stati in breast carcinomas. It is also a useful methodology for confirming the IHC results in paraffin- embedded tumor samples, so offers a promising alternative to IHC in a routine diagnostic setting.

A method for detection of HER2 gene amplification in breast cancer tissues:chromogenic in situ hybridization method and its modification / 第二军医大学学报

Objective:To apply chromogenic in situ hybridization(CISH)for detection of HER2 gene amplification in breast cancer tissues and to discuss some modifications of the CISH method.Methods:HER2 gene amplification was detected by CISH in 60 breast cancer specimens with an immunohistochemical score over 2+.The correlation between the results of IHC and CISH was analyzed.Our experience in CISH manipulation was summarized and optimization to CISH was discussed.Results:CISH identified gene amplification in 91%(40/44)specimens with an IHC score of 3+ and in 50%(8/16)specimens with an IHC score of 2+.The total concordance rate between IHC and CISH was 80%(48/60,P