Cristiana Buzelin Nunes

Rabbit monoclonal antibodies show higher sensitivity than mouse monoclonals for estrogen and progesterone receptor evaluation in breast cancer by immunohistochemistry

A novel generation of rabbit monoclonal antibodies for estrogen (ER) and progesterone (PR) receptor evaluation in breast cancer by immunohistochemistry has been released recently. We compared the novel RabMab anti-ER and anti-PR antibodies with the mouse monoclonal antibodies using a tissue microarray of breast carcinomas. Two cylinders (2mm diameter) of formalin-fixed, paraffin-embedded tissue were obtained from 24 invasive breast cancers and were immunostained using anti-ER mouse (1D5 and 6F11) and rabbit antibodies (SP1 and B644), and anti-PR mouse (PgR312 and PgR636) and rabbit antibodies (SP2 and B645). The immunohistochemistry was evaluated by considering positive those tumors in which more than 10% of the tumor cell nuclei stained independently on the staining intensity. Our results demonstrated that rabbit antibodies against ER have a similar staining pattern compared to the 6F11, but better than 1D5 from three different suppliers. The rabbit antibodies against PR (SP2 and B645) provide a stronger and sharper immunohistochemical signal compared to mouse antibodies (PgR636 and PgR312). Both ER and PR rabbit antibodies allow a lower cost per test because of higher working dilutions compared to mouse antibodies using the same procedure. The novel rabbit antibodies against ER and PR are highly sensitive for immunohistochemical testing of breast carcinomas.

Comparative analysis of six different antibodies against Her2 including the novel rabbit monoclonal antibody (SP3) and chromogenic in situ hybridisation in breast carcinomas

Aims: To compare the sensitivity and specificity of new rabbit monoclonal antibody SP3 with those of mouse monoclonal and rabbit polyclonal antibodies using HER2 amplification defined by chromogenic in situ hybridisation (CISH) as the gold standard. Methods: Serial sections from tissue microarrays (TMAs) containing 84 breast carcinomas were submitted to CISH (Zymed HER2 Spot-Light kit) and immunohistochemistry, using NeoMarkers SP3 (rabbit monoclonal), DAKO A0485 and DAKO HercepTest (polyclonal), Novocastra NCL-CB11, Cell Marque CM-CB11, and Genentech 4D5 (mouse monoclonal). Results: The best antibody concordance was between SP3 and HercepTest (κ = 0.74). SP3, A0485 and HercepTest detected all HER2 amplified tumours, but were less specific than mouse monoclonal antibodies. 3/38 (7.9%) and 8/38 (21.0%) non-amplified tumours were scored as 3+ using SP3 and A0485, respectively. 3/46 (6.5%) amplified tumours were negative for NCL-CB11. SP3, HercepTest and A0485 showed no gene amplification on 55%, 62.5% and 92.3% of the 2+ scored tumours, but most of the 2+ scored tumours using monoclonal antibodies were amplified by CISH (80–92.3%). Conclusions: SP3 is more sensitive than mouse monoclonal antibodies for Her2 assessment. However, HercepTest, CB11 and 4D5 show higher specificity than SP3 for the identification of HER2 gene amplification. Mouse monoclonal antibodies show less Her2 2+ tumours; most are amplified by CISH.

Construção de arrays de tecido com equipamento alternativo e de baixo custo para estudo imuno-histoquímico de tumores mamários

BACKGROUND: Tissue microarrays (TMA) are blocks containing numerous cylinders of paraffinized tissue organized in lines and columns allowing analysis of numerous samples in one slide. Commercially available equipment is imported and have high cost (US

False positivity in HER2 testing of breast cancer: novel paths for approaching an old dilemma

11,000.00 to 24,000.00). AIM: we describe a low cost breast-tumor TMA and our experience in its use for immunohistochemistry (IHC). MATERIAL AND METHODS: A model that consists of a work station (Dremel) to which a liver biopsy needle of 2mm of diameter was connected. A receptor block was prepared perforating it until the desired number of rows (55) was reached. Then, the cylinders of tissue were obtained using the same equipment and included in the holes of the receptor block. Two samples were obtained from different tumor areas of each donor block. Cylinders of previously tested positive control tumors for each antibody and one marker (liver sample) that indicated the beginning of slide reading were also included. IHC was performed in sequential 4µm sections from the same array using antibodies against estrogen and progesterone receptors, Ki67, p53 and Her2. The first and the last slides were stained by hematoxylin and eosin to evaluate: number of tissue discs, tissue preservation, and adequacy of the tissue sample. RESULTS: The equipment total cost was US

Efeito da hidrocortisona sobre a resistência cicatricial da pele em camundongos

180,00. The slides showed fine tissue preservation, adequate for morphologic evaluation, and sufficient to confirm diagnosis. The IHC quality was similar to the donor blocks. CONCLUSION: This equipment and technique represent an economical alternative when compared to commercial equipments.

High agreement between whole slide imaging and optical microscopy for assessment of HER2 expression in breast cancer: Whole slide imaging for the assessment of HER2 expression

Aims Variability in determining HER2 status has been reported, especially, differences in sensitivity and specificity among commercially available antibodies, with false positive and false negative results. We compared the sensitivity and specificity of five anti-HER2 antibodies by immunohistochemistry (IHC), using the new dual colour brightfield in situ hybridisation (DDISH) as the gold standard, on invasive breast carcinomas (IBC) arrays. Material and methods Serial sections from tissue microarrays (TMA) containing 200 preselected primary IBC were submitted to DDISH (VENTANA INFORM HER2 Dual ISH assay), and immunohistochemistry, using Dako A0485 and HercepTest (polyclonal), Novocastra CB11 (mouse monoclonal), NeoMarkers SP3 and Ventana 4B5 (rabbit monoclonal). Results From the initial 200 cases, 184 were assessed by DDISH and IHC. The concordance among the antibodies was considered very good (kappa statistics varied from 0.82 to 0.9). The overall concordance between IHC and DDISH ranged from 94.1% for CB11 to 96.6% for A0485. The antibodies A0485, HercepTest, SP3 and 4B5 were over 95% sensitive and specific. CB11 was the most specific antibody (97.1%). 60% (CB11) to 83.3% (SP3) of the 2+ cases showed no gene amplification by DDISH. False negative cases varied from 0.5% (A0485) to 3.8% (CB11) of the cases, and false positive from 1.6% (CB11) to 2.7% (HercepTest, SP3 and 4B5) of the 184 cases. Conclusions There was very good agreement among the five anti-HER2 antibodies. CB11 was the most specific antibody, but showed more false negative cases. A0485, SP3, 4B5 and HercepTest were highly sensitive and specific, but showed more false positive cases.

Predictive factors of breast cancer evaluated by immunohistochemistry

OBJETIVO: O efeito da corticoterapia sobre a cicatrizacao de feridas cirurgicas vem apresentando resultados conflitantes na literatura, principalmente quando usada por tempo prolongado. O objetivo do presente trabalho foi comparar a resistencia cicatricial da pele de camundongos submetidos a administracao de hidrocortisona, em distintos periodos pos-operatorios. METODO: Foram estudados 150 camundongos machos submetidos a incisao e sutura da pele dorsal, divididos em cinco grupos. No Grupo 1 (n=6) avaliou-se apenas a resistencia da pele integra. Nos demais grupos (n = 36) realizaram-se incisao e sutura na pele, sendo que o Grupo 2 (controle) submeteu-se apenas a operacao, enquanto o Grupo 3 recebeu, ainda, solucao salina a 0,9% e os Grupos 4 e 5 receberam 10mg/kg/dia de hidrocortisona local e sistemica, respectivamente. Avaliaram-se a resistencia cicatricial e a variacao ponderal nos setimo e 21o dias pos-operatorios. RESULTADOS: Os camundongos que receberam corticoide, Gupos 4 e 5, apresentaram decrescimo ponderal significativo (p < 0,02). Quanto a resistencia cicatricial da pele, os Gupos 3, 4 e 5 apresentaram valor inferior ao Grupo 2 no setimo dia pos-operatorio (p < 0,02). No 21o dia, a queda foi observada apenas no grupo submetido a solucao salina (p < 0,05). CONCLUSAO: Os resultados indicam uma diminuicao da resistencia cicatricial apenas nos camundongos tratados com corticoide em intervalos menores de tratamento.

Concordância interobservador na interpretação imuno-histoquímica da superexpressão do Her2 detectada por cinco diferentes anticorpos em array de carcinomas mamários

UNLABELLED Whole slide imaging (WSI) technology has been used for training, teaching, researching, and remote consultation. Few studies compared HER2 expression using optical microscopy (OM) and WSI evaluations in breast carcinomas. However, no consensus has been achieved comparing both assessments. MATERIAL AND METHODS Sections from tissue microarray containing 200 preselected invasive breast carcinomas were submitted to immunohistochemistry applying three anti-HER2 antibodies (HercepTest™, CB11, SP3) and in situ hybridization (DDISH). Slides were evaluated using OM and WSI (Pannoramic MIDI and Viewer, 3DHISTECH). Sensitivity and specificity were calculated comparing the anti-HER2 antibodies and DDISH. RESULTS WSI and OM HER2 evaluations agreement was considered good (SP3, k=0.80) to very good (CB11 and HercepTest™, k=0.81). WSI evaluation led to higher sensitivity (ranging from 100 of SP3 and HercepTest™ to 97 of CB11) and lower specificity (ranging from 86.4 of SP3 to 89.4 of HercepTest™) compared to OM evaluation (sensitivity ranged from 92.1 of CB11 to 98 of SP3 and specificity ranged from 95.2 of SP3 and HercepTest™ to 97.1 of CB11 and SP3). CONCLUSION High agreement was achieved between WSI and OM evaluations. All three antibodies were highly sensitive and specific using both evaluations. WSI can be considered a useful tool for HER2 immunohistochemical assessment.

Rabbit antibodies for hormone receptors and her2 evaluation in breast cancer

A superexpressao de receptores hormonais e Her2 avaliada pela imuno-histoquimica (IHQ) e amplamente validada como fator preditivo em câncer de mama. A qualidade da reacao imuno-histoquimica e influenciada pela fixacao do tecido e seu processamento. A fixacao insuficiente ou demasiada afeta profundamente os resultados da IHQ. A reativacao antigenica pode melhorar os resultados da IHQ, porem nao recupera tecidos com autolise ou com excessiva fixacao. A escolha do anticorpo primario para a IHQ, considerando sua sensibilidade e sua especificidade de acordo com a resposta terapeutica, representa uma importante etapa. Alem de anticorpos monoclonais de camundongo, novos anticorpos monoclonais de coelho sao comercialmente disponiveis, tais como clones SP1 e B644 anti-RE, SP2 e B645 anti-RP, e SP3 e 4B5 anti-Her2. Eles representam uma alternativa para avaliacao de receptores hormonais e Her2 atraves da IHQ. Novos sistemas de deteccao polimericos nao-biotinilados tambem sao disponiveis e permitem marcacao exata e forte sem marcacao estromal ou citoplasmatica inespecifica devido a biotina endogena. O cut off mais recomendado para receptor de estrogenio (RE) e receptor de progesterona (RP) e acima de 1% de celulas positivas com marcacao moderada ou forte (sistema de escore de Allred). Novas recomendacoes para avaliacao de Her2 atraves da IHQ apontam um cut off de mais de 30% de celulas positivas com marcacao forte (3+), que melhor se relaciona com amplificacao genica. Os casos 2+ sao agora considerados indeterminados e devem ser confirmados por hibridacao in situ por fluorescencia (FISH) ou hibridizacao in situ colorimetrica (CISH). Um controle de qualidade de fases pre-analitica, analitica e pos-analitica da IHQ e recomendado para a otimizacao dos resultados.

Efeito da suplementação nutricional com glicina e glutamina, por via oral, na cicatrização colônica em coelhos

AIM: To examine interobserver agreement in immunohistochemical evaluation of Her2 overexpression using five different antibodies on breast cancer array. MATERIAL AND METHOD: Material and method: One array was built with two cores (2 mm diameter each) from 25 breast carcinomas. Serial sections from the array were submitted to immunohistochemistry using five anti-Her2 antibodies: SP3 (NeoMarkers), HercepTest and A0485 (Dako), CB11 (Novocastra), and 4D5 (Genentech). One slide immunostained for each antibody (total = five slides) were independently scored by five observers following HercepTestTM scoring system. Interobserver agreement was evaluated in three different analysis: I (0; 1+; 2+; 3+); II (0 and 1+; 2+ and 3+) and III (0 and 1+; 2+; 3+), and the kappa statistics was applied. RESULTS: There was a good rate of interobserver agreement when the four scores were considered (0; 1+; 2+; 3+). When the scores were considered in two categories (0 and 1+; 2+ and 3+) the interobserver agreement rate was considered substantial for cases stained for SP3 and CB11, and almost perfect for cases stained for A0485, HercepTest and 4D5. For analysis III (0 and 1+; 2+; 3+), a moderate rate of interobserver agreement was considered for cases stained for CB11, and a substantial rate for other antibodies. CONCLUSION: The overall interobserver agreement was considered moderate to substantial in the evaluation of cases stained for the five antibodies. The lowest rate of agreement was obtained in the evaluation of the cases scored as weak (1+) and moderate (2+). The observers experience altered the concordance rates.