Sentinel node biopsy in breast cancer: results in a large series

Sentinel lymph node biopsy (SLNB) is an appropriate method for the evaluation of axillary status in cases of early breast cancer. We report our experience in treating cases evaluated using SLNB. We analyzed a total of 1192 cases assessed by means of SLNB from July 1999 to December 2007. SLNB processing was successfully completed in 1154 cases with the use of blue dye or radiolabeled 99mTc-Dextran-500, or both. Of these 1154 patients, 857 were N0(i-) (no regional lymph node metastasis, negative immunohistochemistry, IHC), 96 were N0(i+) (no regional lymph node metastasis histologically, positive IHC, no IHC cluster greater than 0.2 mm) and 201 were N1mi (greater than 0.2 mm, none greater than 2.0 mm). Most of the tumors (70%) were invasive ductal carcinomas and tumors were staged as T1 in 770 patients (65%). A total of 274 patients underwent SLNB and axillary dissections up to April 2003. The inclusion criteria were tumor size equal to or less than 3 cm in diameter, no clinically palpable axillary lymph nodes, no neoadjuvant therapy. In 19 cases, the SLN could not be identified intraoperatively. A false-negative rate of 11% and a negative predictive value of 88.2% were obtained for the 255 assessable patients. The overall concordance between SLNB and axillary lymph node status was 92%. SLNB sensitivity for nodes was 81% and specificity was 100%. The higher sensitivity, specificity, accuracy, and lower false-negative rates of SLNB suggest that this method may be an appropriate alternative to total axillary dissection in early breast cancer patients.

Détection moléculaire des métastases des ganglions lymphatique dans le cancer du col de l'utérus

Le Cancer du Col Utérin (CCU) chez la femme est provoqué par le virus oncogénique VPH. La métastase lymphatique ganglionnaire est un facteur pronostique majeur pour l’évolution de ce cancer et sa présence influence la décision thérapeutique. En général, l’envahissement ganglionnaire est diagnostiqué par histologie, mais cette méthode est laborieuse et parfois prise en défaut pour détecter les micrométastases et les cellules cancéreuses isolées et pour donner des résultats rapides en per opératoire. L’outil moléculaire que nous désirons développer pour combler cette lacune est basé sur une analyse d’ARN des gènes du VPH exprimés par les cellules du CCU. Ceci sera fait par transcription réverse de l’ARN cellulaire couplé à une réaction quantitative en chaine par polymérase en temps réel (RT-qPCR). Cette technique devrait nous permettre une détection et une évaluation rapide des micrométastases pour aider à déterminer immédiatement un pronostic fiable et la thérapie associée. C’est un test précis, sensible et rapide pour détecter un envahissement ganglionnaire dans le CCU visant à améliorer la gestion thérapeutique. Le projet est basé sur trois objectifs. En premier lieu, valider les marqueurs moléculaires E6 et E7 de VPH16 et 18 à partir des échantillons frais et des échantillons fixés dans des blocs de paraffine. En deuxième lieu, déterminer la fiabilité et la sensibilité des marqueurs pour la détection des macrométastases, des micrométastases et les cellules tumorales isolées en utilisant la technique de RT-qPCR. En troisième lieu et parallèlement au travail présenté dans ce mémoire, il est nécessaire de constituer une base de données des patientes qui ont le virus VPH16 et 18 intégré dans leur génome, qui ont été traitées et dont nous connaissons déjà le diagnostic final afin de valider la méthode (biobanque). Nous avons réussi à extraire de l’ARNm de haute qualité à partir d’échantillons complexes, à détecter les gènes E6 et E7 de VPH16 et 18 en RT-qPCR, et à déterminer précisément la limite de détection de E6 et E7 dans les échantillons frais qui est une proportion de 0,008% de cellules cancéreuses. Dans les échantillons fixés dans la paraffine, cette limite est de 0,02% et 0,05% pour E6-E7-VPH16 et E6-E7-VPH18 respectivement. Ceci comparativement à une limite de détection histologique de 1% qui est déterminée par immunohistochimie de CK19. Enfin, notre protocole est validé pour VPH18 dans les ganglions lymphatiques du CCU.

Identificação do linfonodo-sentinela em pacientes com carcinoma de colo uterino invasor estádio I-B 1

Objetivo: determinar a viabilidade da identificação do linfonodo-sentinela em pacientes com câncer invasor de colo uterino estádio Ib1. Material e Métodos: 16 pacientes consecutivas com câncer de colo uterino agendadas para histerectomia radical com linfadenectomia pélvica bilateral realizaram estudo para detecção de linfonodo-sentinela. Onze pacientes injetaram 1 mCi de tecnécio 99 (99Tc) em quatro pontos do estroma superficial do colo uterino ao redor do tumor, às 12, 3, 6 e 9 h ( 16 horas antes da cirurgia ). No dia da cirurgia, as pacientes foram submetidas ao mapeamento linfático com gamma-probe e azul patente injetado nos mesmos pontos que o 99Tc.Cinco pacientes realizaram a detecção apenas com azul patente. Resultados: foi detectado pelo menos um (1 a 3 por paciente) linfonodo-sentinela em cada uma das 15 pacientes (93,7 %) que realizaram a técnica combinada.Foi detectado pelo menos 1 linfonodo-sentinela em 4 pacientes ( 80% )com azul patente apenas. A maioria dos linfonodos-sentinela foi localizada nas regiões: obturadora (37 %), ilíaca externa (22,2 %) e inter-ilíacas (18,5 %). Seis pacientes (40 %) tiveram linfonodos-sentinela bilaterais. Dos 27 linfonodos-sentinela detectados, 11 (40,7 %) foram detectados pelo corante, 9 (33,3%) pela radioatividade e 7 (26 %) pela radioatividade e corante. O índice de detecção intra-operatória com o gamma-probe foi de 90,9 % ( 11 pacientes ). Destes, 7 linfonodos foram azul e quente (31,8 %), 6 linfonodos foram apenas azuis (27,2 %) e 9 linfonodos foram apenas quentes (40,9 %). A sensibilidade, especificidade e valor preditivo negativo para a detecção do linfonodo-sentinela foram 100%, 85,7 % e 100 % respectivamente. Conclusão: A combinação do radiofármaco 99Tc e azul patente é efetiva na detecção do linfonodosentinela em câncer de colo uterino inicial.

Immune response pattern of the popliteal lymph nodes of dogs with visceral leishmaniasis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Polymorphisms of Lewis and Secretor genes are related to breast cancer and metastasis in axillary lymph nodes

ABH and Lewis antigen expression has been associated with cancer development and prognosis, tumor differentiation, and metastasis. Considering that invasive ductal breast carcinoma (IDC) presents multiple molecular alterations, the aim of the present study was to determine whether the polymorphism of ABO, Lewis, and Secretor genes, as well as ABO phenotyping, could be associated with tumor differentiation and lymph nodes metastasis. Seventy-six women with IDC and 78 healthy female blood donors were submitted to ABO phenotyping/genotyping and Lewis and Secretor genotyping. Phenotyping was performed by hemagglutination and genotyping by the polymerase chain reaction with sequence-specific primers. ABO, Lewis, and Secretor genes were classified by individual single nucleotide polymorphism at sites 59, 1067, 202, and 314 of the Lewis gene, 428 of the Secretor gene, and 261 (O1 allele), 526 (O2 and B allele), and 703 (B allele). No association was found between breast cancer and ABO antigen expression (P = 0.9323) or genotype (P = 0.9356). Lewis-negative genotype was associated with IDC (P = 0.0126) but not with anatomoclinical parameters. Nonsecretor genotype was associated with axillary lymph node metastasis (P = 0.0149). In conclusion, Lewis and Secretor genotyping could be useful to predict respectively breast cancer susceptibility and axillary lymph nodes metastasis.

Effects of aspirin on mesenteric lymph nodes of rabbits as basis for its use on lymph nodes metastases

OBJETIVO: Avaliar os efeitos do ácido acetilsalicílico a 10% e 20% em linfonodos mesentéricos de coelhos para posterior embasamento e uso em metástases linfonodais. MÉTODOS: Um total de 20 linfonodos de 20 coelhos (divididos aleatoriamente em quatro grupos) foi avaliado. As soluções de aspirina a 10% (grupos A e C) e 20% (grupos B e D) foram injetadas em linfonodos mesentéricos de coelhos sadios e seus efeitos macroscópicos e histológicos foram avaliados em 24 horas (grupos A e B) e em sete dias (grupos C e D). RESULTADOS: Nos grupos avaliados em 24 horas (A e B) foi verificada intensa necrose e hemorragia, aumento importante de apoptose em todo o linfonodo, com alargamento dos seios medulares e aumento dos centros germinativos. Nos grupos avaliados em sete dias (C e D) também houve aumento da apoptose, com maior elevação de histiócitos e diminuição importante da necrose; a hemorragia foi ausente e aumento de células gigantes foi visualizado, conferindo processo inflamatório crônico do tipo corpo estranho. Não houve diferença entre as concentrações utilizadas (10 e 20%) em nenhuma das comparações. CONCLUSÕES: A injeção de aspirina em linfonodos causou necrose e um aumento de apoptose após 24 horas e após sete dias de tratamento, houve regeneração dos gânglios linfáticos, com diminuição intensa de necrose e grande aumento de apoptose. Uma vez que o aumento de apoptose é um dos pilares dos tratamentos antineoplásicos, estes resultados experimentais embasam eventual aplicação clínica da aspirina no tratamento de metástases linfonodais.

Isolation of coccidioides brasiliensis from armadillos (Dasypus noveminctus) captured in an endemic area of paracoccidioidomycosis

Paracoccidioides brasiliensis, the causative agent of paracoccidioidomycosis (PCM), was first isolated from armadillos from the Amazonian region where the mycosis is uncommon. In the present study, we report on the high incidence of PCM infection in armadillos from a hyperendemic region of the disease. Four nine-banded armadillos (Dasypus novemcinctus) were captured in the endemic area of Botucatu, São Paulo, Brazil, killed by manual cervical dislocation and autopsied under sterile conditions. Fragments of lung, spleen, liver, and mesenteric lymph nodes were processed for histology, cultured on Mycosel agar at 37 degrees C, and homogenized for inoculation into the testis and peritoneum of hamsters. The animals were killed from week 6 to week 20 postinoculation and fragments of liver, lung, spleen, testis, and lymph nodes were cultured on brain heart infusion agar at 37 degrees C. Paracoccidioides brasiliensis was isolated from three armadillos both by direct organ culture and from the liver, spleen, lung, and mesenteric lymph nodes of hamsters. In addition, one positive armadillo presented histologically proven PCM disease in a mesenteric lymph node. The three armadillos isolates (Pb-AL, Pb-A2, and Pb-A4) presented thermodependent dimorphism, urease activity, and casein assimilation, showed amplification of the gp43 gene, and were highly virulent in intratesticularly inoculated hamsters. The isolates expressed the gp43 glycoprotein, the immunodominant antigen of the fungus, and reacted with a pool of sera from PCM patients. Taken together, the present data confirm that armadillos an a natural reservoir of P. brasiliensis and demonstrate that the animal is a sylvan host to the fungus.

Direct Detection of Mycobacterium bovis in Bovine Lymph Nodes by PCR

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Leishmune (R) vaccine blocks the transmission of canine visceral leishmaniasis - Absence of Leishmania parasites in blood, skin and lymph nodes of vaccinated exposed dogs

Leishmune (R) vaccine is the first licensed vaccine against canine visceral leishmaniasis. It contains the Fucose-Mannose-ligand (FML) antigen of Leishmania donovani. The potential Leishmune (R) vaccine effect on the interruption of the transmission of the disease, was assayed by monitoring, in untreated (n = 40) and vaccinated dogs (n = 32) of a Brazilian epidemic area: the kala-azar clinical signs, the FML-seropositivity and the Leishmania parasite evidence by immunohistochemistry of skin and PCR for Leishmanial DNA of lymph node and blood samples. on month I I after vaccination, untreated controls showed: 25% of symptomatic cases, 50% of FML-seropositivity, 56.7% of lymph node PCR, 15.7% of blood PCR and 25% of immunohistochemical positive reactions. The Leishmune (R)-vaccinated dogs showed 100% of seropositivity to FML and a complete absence of clinical signs and of parasites (0%) in skin, lymph node and blood PCR samples (P < 0.01). The positivity in FML-ELISA in untreated dogs significantly correlates with the PCR in lymph node samples (p < 0.001) and with the increase in number of symptoms (p = 0.006) being strong markers of infectiousness. The absence of symptoms and of evidence of Leishmania DNA and parasites in Leishmune (R)-vaccinated animals indicates the non-infectious condition of the Leishmune (R)-vaccinated dogs. (c) 2005 Elsevier Ltd. All rights reserved.

Anatomopathological analysis of sentinel and nonsentinel lymph nodes in breast cancer: Hematoxylin-eosin versus immunohistochemistry

The authors compare the detection of metastases in sentinel lymph nodes (SLNs) and nonsentinel lymph nodes (NSLNs) using hematoxvlin-eosin (HE) staining versus immunohistochemistry (IHC). Thirty-six patients with breast carcinoma undergo exeresis of the primary tumor and of 50 SLNs and 491 NSLNs. Sentinel lymph nodes are sectioned into transverse slices of 2- to 3-mm thickness, and a cytologic smear and a frozen section were obtained from each slice. The slices are completely cut into serial sections at 100-mu m intervals. Two consecutive 4-mu m-thick sections are then obtained from each level and were prepared for HE staining and IHC. Nonsentinel lymph nodes are evaluated similarly to SLNs. The authors obtain 4076 SLN sections and 32 012 NSLN sections, fora total of 36 088 sections. A comparison of HE staining versus IHC based on the total number of sections shows a sensitivity of 93.8%, a negative predictive value of 98.9%, and an accuracy of 99.1 %. The values obtained by HE staining are similar to those obtained by IHC.

Missing association between telomerase activity and clinicopathological features in patients with early stage carcinoma of the cervix

Objectives: This study was undertaken to evaluate the association between the telomerase activity in the tumor and clinicopathological findings in patients with stage IB-IIA (FIGO) carcinoma of the cervix. Methods: Thirty-eight patients with carcinoma of the cervix submitted to radical hysterectomy were prospectively from January 1998 to November 2001. Samples from the tumor were taken and analyzed by the telomerase PCR-TRAP-ELISA kit. Clinicopathological characteristics such as age, stage, tumor size, grade of differentiation, lymphatic vascular space invasion (LVSI), parametrial involvement and status of pelvic lymph nodes were also recorded. Results: Patient's mean age was 49.3 ± 1.99 years (29-76 years). The clinical stage (FIGO) was IB in 35 patients (92.1%) and IIA in 3 patients (7.9%). The histological classification identified squamous cell carcinoma in 33 patients (86.8%) and adenocarcinoma in 5 patients (13.2%). There was no association between age, clinical stage, histological classification, tumor size, grade of differentiation and presence of LVSI with tumoral telomerase activity. The telomerase activity was not associated with the presence of vaginal involvement (P = 0.349), parametrium involvement (P = 0.916), pelvic lymph node metastasis (P = 0.988) or tumoral recurrence (P = 0.328) in patients with carcinoma of the cervix. Conclusions: Telomerase activity in the tumor is not associated with clinicopathological findings or tumor recurrence in patients with early stage cervical carcinoma. © 2006 Springer-Verlag.

Expression of the nonclassical HLA-G and HLA-E molecules in laryngeal lesions as biomarkers of tumor invasiveness

Introduction: HLA-G and HLA-E are two nonclassical class I molecules, which have been well recognized as modulators of innate and adaptive immune responses, and the expression of these molecules in virus infected cells has been associated with subversion of the immune response. Objective: In this study we performed a cross-sectional study, systematically comparing the expression of HLA-G and HLA-E in benign, premalignant and malignant laryngeal lesions, correlating with demographic and clinical variables and with the presence of high-risk and low-risk HPV types. Materials and methods: Laryngeal lesions were collected from 109 patients and stratified into 27 laryngeal papillomas, 17 dysplasias, 10 in situ laryngeal carcinomas, 27 laryngeal carcinomas without metastases, 28 laryngeal carcinomas with metastasis along with their respective draining cervical lymph nodes, and 10 normal larynx specimens. The expression of HLA-G and HLA-E molecules was determined by immunohistochemistry. HPV DNA detection and typing was performed using generic and specific primers. Results: HLA nonclassical molecules showed a distinct distribution pattern, according to the larynx lesion grade. HLA-G expression increased in benign and premalignant lesions, and gradually decreased in invasive carcinomas and in respective draining cervical lymph nodes. Conversely, HLA-E expression increased as far as lesion grade increased, including increased molecule expression in the draining lymph nodes of malignant lesions. Only 17 (15.6%) patients were HPV DNA positive. Conclusions: Overexpression of HLA-E and underexpression of HLAG appear to be good markers for malignant larynx lesion.

Detecção do lindonodo sentinela com 99m Tc-Fitato em pacientes com carcinoma invasor do colo uterino

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Expressão das proteínas p53, Ki-67 e CD31 no tumor e nas margens vaginais após histerectomia radical em pacientes com carcinoma invasor do colo uterino

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Estudo clínico, morfológico e imuno-histoquímico de série de casos de tuberculose pleural e ganglionar

A dificuldade no diagnóstico definitivo da tuberculose extrapulmonar persiste principalmente devido a baixa resolutividade dos métodos convencionais disponíveis para a detecção do Mycobacterium tuberculosis. Esse estudo teve como objetivos avaliar a contribuição da técnica imuno-histoquímica (IHQ) para a detecção de Mycobacterium spp. em casos de tuberculose pleural e ganglionar com histoquímica negativa, assim como, investigar alguns aspectos clínicos, laboratoriais e morfológicos da doença. Para obtenção desta amostra fez-se a busca dos casos no Núcleo de Vigilância Epidemiológica (NVE) e Divisão de Arquivo Médico e Estatística (DAME) do Hospital Universitário João de Barros Barreto (HUJBB) e no Departamento de Anatomia Patológica da Universidade Federal do Pará (UFPA), selecionando-se aqueles que haviam realizado exame histopatológico para esclarecimento diagnóstico do caso. Foram incluídos 50 pacientes, sendo 25 com diagnóstico presuntivo de tuberculose pleural e 25 de tuberculose ganglionar. Para obtenção dos dados clínicos e laboratoriais os respectivos prontuários foram revisados, e para confirmação dos aspectos morfológicos foi realizada a revisão de todas as lâminas selecionadas. Posteriormente, cada amostra foi submetida à técnica IHQ com anticorpo polyclonal Mycobacterium bovis BCG. Encontrou-se no grupo investigado, maior frequência do sexo masculino, cuja média de idade foi de 33,8 anos (desvio padrão: 14,1) sendo a maioria procedente da cidade de Belém-Pará e com nível de escolaridade de sete ou menos anos de estudo. Os sintomas constitucionais mais frequentes em todo o grupo foram a febre e perda ponderal. Nos pacientes com tuberculose pleural, os sintomas específicos mais encontrados foram tosse, dor torácica e dispneia, e naqueles com a forma ganglionar da doença, o envolvimento da cadeia cervical isolada foi mais frequente. Infecção pelo vírus da imunodeficiência humana (HIV) ou Síndrome da Imunodeficiência Adquirida (Sida) e etilismo foram as condições de risco mais frequentemente associadas. Na tuberculose pleural, 20% dos casos cursaram com derrame pleural associado à lesão parenquimatosa, e em 60% o líquido pleural foi do tipo exsudativo. Enquanto, na forma ganglionar, em 50% dos casos evidenciou-se lesão parenquimatosa à radiografia do tórax. Neste estudo, foi inexpressiva a quantidade de participantes nos quais foi realizada a pesquisa direta e cultura para bacilo álcool - ácido resistente (BAAR) nos diversos espécimes clínicos analisados (líquido pleural, tecido pleural e ganglionar, escarro e lavado broncoalveolar) O padrão morfológico predominante em ambas as formas da doença foi o granuloma tipo tuberculoide com necrose caseosa, independente do status sorológico para o HIV. A técnica IHQ contribuiu para o diagnóstico de tuberculose pleural em 21% (4/19) das amostras de tecido pleural e em 37,5% (9/24) de tecido ganglionar. Um resultado imuno-histoquímico positivo define o diagnóstico de micobacteriose, e quando associado aos achados clínicos, laboratoriais e morfológicos torna-se uma ferramenta de grande utilidade para melhorar o diagnóstico da tuberculose extrapulmonar.