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também desempenhar um papel relevante na progressão da doença298. É possível que mecanismos farmacocinéticos descritos para o imatinib estejam envolvidos noutros inibidores dos receptores tirosina-cinásicos, mas é necessária investigação adicional para conclusões definitivas.
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Estudos recentes descreveram que os doentes com GISTs que progrediram após diferentes opções terapêuticas (estabelecidas e/ou experimentais) podem obter benefício clínico quando é reintroduzido o mesmo agente terapêutico a que tinham previamente respondido (imatinib;
sunitinib)307, 308. Quando não existe outra opção, a manutenção do tratamento com o mesmo agente pode retardar a progressão da doença.
Na ausência de ensaios clínicos para tratamento alternativo, a continuação ou a reintrodução do inibidor tirosina-cinásico a que o doente já foi exposto pode ser uma opção adequada24, 138.
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71 OBJECTIVOS
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73 OBJECTIVOS
Objectivo geral
O objectivo geral da presente dissertação consistiu na caracterização clínico-patológica, imuno-histoquímica e molecular de uma série de doentes consecutivos com GIST, diagnosticados e tratados numa unidade de cuidados terciários de saúde (Hospital de São João) em Portugal.
Pretendia-se caracterizar os fundamentos do diagnóstico e clarificar os princípios do tratamento destes tumores, numa perspectiva multidisciplinar, com especial ênfase na terapêutica cirúrgica.
Adicionalmente, pela análise da sobrevida dos doentes, pretendia-se clarificar o possível impacto de parâmetros de prognóstico e preditivos da resposta terapêutica em doentes com GIST.
Objectivos específicos
1. A ressecção cirúrgica é o tratamento de eleição para os GISTs primários e pode ser curativa. No entanto, o impacto da extensão da ressecção sobre o prognóstico dos doentes continua a ser objecto de discussão.
Objectivo: Analisar o valor prognóstico da qualidade das margens cirúrgicas no tratamento dos doentes com GIST primário.
2. Os GISTs em localização extragástrica ou extra-intestinal são menos frequentes e têm sido pouco analisados. Nestes casos, o diagnóstico diferencial com outros tumores mesenquimatosos é particularmente importante, não só pelo risco de comportamento agressivo, mas também pelas implicações terapêuticas específicas, que incluem drogas dirigidas a alvos moleculares.
Objectivo: Avaliar os procedimentos de diagnóstico de GIST no esófago, que é uma localização rara deste tipo de tumores, e rever as opções terapêuticas mais adequadas nesta localização.
3. Há evidências que indicam que os GISTs incluem tumores geneticamente muito heterogéneos, com prognósticos distintos e respostas variáveis ao tratamento com os inibidores tirosina-cinásicos aprovados para o tratamento não-cirúrgico (ex: imatinib e sunitinib) dos doentes.
Objectivo: Avaliar o estado mutacional dos genes KIT e PDGFRA numa série de doentes, para correlacionar as características clínico-patológicas e moleculares identificadas e o seu impacto no prognóstico dos doentes com GIST.
4. Os GISTs resultam geralmente de mutações oncogénicas nos genes KIT ou PDGFRA, e numa percentagem variável (10-40%) de casos não se identificam alterações genéticas nestes genes (GISTs wild-type).
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Objectivo: Esclarecer as alterações moleculares alternativas em GISTs wild-type, procurando identificar alterações potencialmente úteis para o diagnóstico e eventualmente preditivas de resposta a terapêuticas dirigidas a novos alvos moleculares em doentes com GIST.
5. Os parâmetros com impacto prognóstico importante nos GISTs primários incluem: dimensão maior, localização no tubo digestivo/extragastrointestinal, ruptura tumoral, índice mitótico, tipo de mutações do KIT / PDGFRA e qualidade da ressecção cirúrgica. Contudo, alguns casos continuam a revelar comportamento biológico imprevisível, levando à necessidade da pesquisa de novos marcadores moleculares com valor prognóstico e/ou preditivo para os doentes.
Alguns marcadores (ex: proteína RKIP - Raf kinase inhibitory protein) são considerados supressores da metastização e têm sido reconhecidos como parâmetros de prognóstico em diferentes cancros (ex:
carcinomas da próstata, colo-rectais e gástricos).
Objectivo: Avaliar a expressão de RKIP, a sua associação com diferentes parâmetros clínico-patológicos, e esclarecer o papel deste marcador em doentes com GISTs primários.
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MATERIAL E MÉTODOS
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MATERIAL E MÉTODOS
Uma vez que cada subcapítulo da secção de resultados corresponde a um trabalho publicado com material e métodos, decidiu-se simplificar o mais possível a estrutura deste capítulo nesta dissertação.
Foram avaliados os registos clínicos e patológicos e o material relacionado com todos os casos consecutivos de tumores estromais gastrointestinais (GISTs) diagnosticados e tratados no período decorrido entre 1989 e 2006 (18 anos) nos serviços de Cirurgia Geral e Anatomia Patológica do Hospital de S. João do Porto, Portugal.
De entre todos os doentes admitidos com o diagnóstico de “tumor mesenquimatoso” do tubo digestivo neste período, foram identificados 114 casos em que se confirmou o diagnóstico de GIST.
1 - Parâmetros clínicos e cirúrgicos
Foram estudados os seguintes parâmetros clínicos dos doentes: género, idade, localização do tumor primário, sintomas clínicos e sinais de apresentação. O tipo de ressecção cirúrgica (enucleação, ressecção atípica/em cunha, ressecção segmentar, ressecção total/subtotal de órgão e ressecção
“em bloco”) do tumor foi avaliado em cada um dos 96 casos com ressecção macroscópica completa. Os procedimentos cirúrgicos foram agrupados da seguinte forma: enucleação, ressecção em cunha/segmentar, ressecção total/subtotal de órgão e ressecção “em bloco”.
2 - Parâmetros anátomo-patológicos e imuno-histoquímicos
Todo o material anátomo-patológico disponível foi avaliado em cortes de 4-µm corados por hematoxilina-eosina (HE) [número médio de fragmentos estudados por caso: 6,1 (intervalo: 1-26)], e o diagnóstico de GIST foi estabelecido de acordo com a classificação da Organização Mundial de Saúde (OMS) 309. Os parâmetros analisados em cada tumor foram: o maior diâmetro (cm), estado das margens de ressecção [negativas (R0) e positivas (microscópicas/R1 e macroscópicas/R2)], tipo celular (fusiforme, epitelióide e misto), presença ou ausência de necrose, índice mitótico (número de mitoses por 50 CGA) e classificação de risco do GIST ─ muito baixo (MB), baixo (B), intermédio (I) e alto (A) risco 182.
Os estudos imuno-histoquímicos foram realizados em cortes de 3-µm representativos de cada tumor usando o princípio do complexo estreptavidina-biotina-peroxidase. Foram utilizados os
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seguintes anticorpos policlonais anti-humanos de coelho: CD117, actina, desmina, proteína S100 e CD34. Foram também utilizados anticorpos primários dirigidos contra o KIT, SCF, fosfo-ERK e RKIP. Os procedimentos imuno-histoquímicos foram realizados segundo metodologia já descrita pelo nosso grupo e por outros autores310-313, com algumas modificações. Muito resumidamente, as lâminas desparafinadas e rehidratadas foram incubadas durante 10 minutos em 3% de peróxido de hidrogénio em metanol, para inibir a peroxídase endógena. Após incubação com anticorpo primário, foi aplicado o anticorpo secundário biotinilado anti-polivalente de cabra durante 10 minutos, seguido de incubação com os complexos estreptavidina-peroxídase. A reacção imunológica foi visualizada utilizando DAB (diaminobenzidina) como cromogéneo. Qualquer imuno-reactividade (forte/fraca, focal, moderada ou difusa) membranar (CD117) e/ou citoplasmática (CD117, actina, desmina e CD34), e nuclear (proteína S100) das células foi considerada como positiva. No estudo imuno-histoquímico da expressão de KIT, SCF, fosfo-ERK e RKIP avaliou-se a extensão e a intensidade da reactividade. A extensão da imuno-reactividade foi avaliada numa escala de 0 a 3 (0, ausência de células positivas; 1, <25% de células positivas; 2, 26-50% de células positivas; e 3,>50% de células positivas) e a intensidade também numa escala de 0 a 3 (0, negativa; 1, fraca; 2, moderada; 3, forte). A pontuação final usada resultou da soma das pontuações atribuídas à extensão e à intensidade (negativa: 0 a 2; moderadamente positiva: 3 e 4; e fortemente positiva: 5 e 6). Foram incluídos controlos positivos e negativos apropriados para cada procedimento. Para os controlos negativos foram omitidos anticorpos primários. Todos os cortes foram contrastados com hematoxilina.
3 - Extracção de ADN
As amostras foram obtidas por dissecção de áreas seleccionadas, com pelo menos 85% de tecido tumoral, para um tubo de micro-centrifugação, utilizando agulha esterilizada (Neolus 25G - 0,5 mm).
A extracção de ADN foi realizada como previamente descrito314. Resumidamente, as amostras foram desparafinadas mediante extracção seriada com xilol e etanol em concentrações decrescentes (100%-70%-50%), e secagem ao ar. O ADN foi extraído utilizando o QIAamp® DNA Micro Kit da Qiagen.
4 - Análise de mutações nos genes KIT e PDGFRA
A análise mutacional do gene KIT foi realizada seguindo metodologia descrita previamente36, 311, 314. Nos GISTs wild-type para o gene KIT foram ainda investigadas mutações hotspot no gene PDGFRA.
O ADN foi submetido a amplificações por reacção de polimerização em cadeia (PCR; polymerase chain reaction) seguida de sequenciação directa, para os exões 11 do KIT e 12, 14 e 18 do PDGFRA, e de uma pré-selecção pela técnica de polimorfismo conformacional de cadeia simples (SSCP; single strand conformational polymorphism), seguida de sequenciação directa dos casos positivos, para os exões 9, 13, 14 e 17 do KIT. Resumidamente, a reacção PCR foi realizada com um volume final de 25µL,
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nas seguintes condições: 1x Buffer (Bioron, Germany), 1,5 mM de MgCl2, 200 µM de dNTPs (desoxirribonucleotídeos-trifosfato), 0,5 µM de iniciadores e 1 U de SuperHot Taq Polymerase. A análise SSCP dos exões 9, 13, 14 e 17 foi realizada num gel 1x MDE (mutation detection enhancement), com adição de percentagens diferentes de glicerol na análise de diferentes exões. Vinte microlitros de produto da PCR foram incubados a 95ºC durante 10 minutos com um volume igual do tampão de corrida de formamida [98% de formamida, 10 mM de ácido etilenodiamino tetra-acético (EDTA) e 1 mg/mL de azul de bromofenol e xileno cianol]. Os géis SSCP foram processados a 20ºC, e as amostras com padrão SSCP diferente do normal foram sequenciadas directamente.
Todos os casos foram confirmados duas vezes, com nova amplificação por PCR e técnicas de SSCP e de sequenciação directa.
5 - Análise de mutações na região 3’ do exão 11 do KIT e nos genes H-RAS,K-RAS,N-RAS e BRAF
A pré-selecção das regiões hotspot do KIT (região 3’ do exão 11), H-RAS, K-RAS, N-RAS [exões 1 (codões 12-13) e 2 (codão 61)] e BRAF (exões 11 e 15) foi efectuada por PCR – SSCP, seguida pela realização de sequenciação directa do ADN nas amostras que evidenciaram uma alteração da mobilidade. Resumidamente, a reacção PCR foi realizada com um volume total de 25µL, consistindo em 1 µL de solução de ADN, 0,3 µM de iniciadores paralelos e anti-paralelos, 200 µM de dNTPs, 1,5 mM de MgCl2, 1x Taq Buffer incompleto e 1 U de Taq Superhot DNA Polymerase. A reacção consistiu numa desnaturação inicial a 96ºC durante 10 minutos, seguida por 40 ciclos de desnaturação a 96ºC durante 45 segundos, hibridação a 55-60ºC durante 45 segundos e extensão a 72ºC durante 45 segundos, seguida de uma extensão final de 10 minutos a 72ºC, num Thermocycler.
As sequências iniciadoras para todos os genes foram como anteriormente descrito315-317. Os produtos da PCR foram misturados com quantidade equivalente do tampão desnaturante de formamida [98% de formamida, 10 mM de EDTA, 1 mg ⁄ml de azul de bromofenol e xileno cianol]. Após desnaturação a 98ºC durante 10 minutos e arrefecimento em gelo, 20 µL da mistura foram adicionados a um gel 1x MDE e percentagens variáveis de glicerol para os genes KIT, K-RAS, H-RAS e N-RAS, e a um gel 0,8x MDE sem glicerol para o BRAF. As amostras que, na análise SSCP, apresentaram um padrão diferente do habitual foram sequenciadas directamente.
Todos os casos foram confirmados duas vezes, com nova amplificação por PCR independente seguida de sequenciação directa.
6 - Análise da metilação no promotor do gene RKIP
Foi determinado o padrão de metilação do ADN na região promotora do gene RKIP através da PCR específica para metilação (MSP; methylation-specific PCR), segundo técnica anteriormente descrita318, com algumas modificações. Foi previamente efectuado tratamento de 200 ng de ADN com bissulfito, usando o EZ DNA Methylation Golf Kit. Foram usados iniciadores específicos para
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distinguir o ADN metilado (produto de PCR com 204 pb) do ADN não metilado (produto de PCR com 205 pb), tal como os controlos de ADN metilado e ADN não metilado adequados.
7 - Follow-up e análise estatística
Os dados relativos ao follow-up dos doentes foram obtidos através de entrevistas directas com os doentes ou com familiares, bem como pela análise dos registos hospitalares. A análise da sobrevida dos doentes referenciados e seguidos em regime ambulatório baseou-se na actualização sistemática dos dados da avaliação clínica e imagiológica de cada doente. Foi possível obter informação para todos os casos seleccionados nas diferentes avaliações efectuadas. Os parâmetros clínicos e patológicos estudados foram analisados como possíveis factores de prognóstico para recidiva local ou à distância (metastização) e sobrevida especifica (SE), relacionada com a doença. A sobrevida livre de doença (SLR) foi calculada a partir da data da cirurgia até à primeira evidência de recidiva, local ou à distância (ou ambas), nos tumores submetidos a ressecção macroscópica completa; foram excluídos desta análise os tumores com ressecção R2. A sobrevida foi calculada a partir da data de diagnóstico até à morte relacionada ou não com a doença, ou censurada na data da última observação.
A análise da sobrevida global (SG), da SE e da SLR dos casos submetidos a ressecção foram determinadas pelo método de Kaplan-Meier e pelo teste Log rank, com o programa SPSS, versões 14.0 e 15.0 (SPSS Inc., Chicago, IL, USA). A associação entre variáveis clínico-patológicas ou moleculares e a recidiva do tumor foi analisada com o teste Qui-quadrado ou com o teste exacto de Fisher, consoante os casos. O hazard ratio (HR) foi calculado (na análise univariada) com o modelo de regressão de Cox separadamente para cada variável, e foi considerado estatisticamente significativo o valor de probabilidade inferior a 0,05 (p≤0,05). A análise multivariada foi efectuada com o modelo de riscos proporcionais de Cox e apenas foram incluídas as variáveis consideradas estatisticamente significativas na análise univariada.
81 RESULTADOS
Os cinco trabalhos que a seguir se apresentam reproduzem os estudos efectuados para o esclarecimento das questões levantadas no capítulo de objectivosdesta dissertação.
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83 Trabalho I
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Surgical Margin Status and Prognosis of Gastrointestinal Stromal Tumor
Anto´nio M. GouveiaÆAmadeu P. Pimenta ÆAna F. CapelinhaÆ Dionı´sio de la CruzÆPaula Silva ÆJose´ M. Lopes
Published online: 7 August 2008
ÓSocie´te´ Internationale de Chirurgie 2008
Abstract
Background Surgery is the best treatment for primary GIST and may be curative, but resection extension/com-pleteness impact on the prognosis remains controversial.
The authors aim was to evaluate the clinicopathological (CP) parameters and surgical margins status influence on GIST patients’ outcome.
Materials and methods The study evaluated 113 con-secutive patients with sporadic GIST; the influence of CP parameters on recurrence-free survival (RFS) and disease-specific survival (DSS) was determined by univariate analysis (UA) and multivariate analysis (MA).
Results Of 104 cases, macroscopically complete resec-tion was achieved in 96: R0 surgical margin status in 78 and R1 in 18. Recurrence rates (12.5%) were significantly lower in R0 (9.0%) than in R1 (27.8%). Tumor[10 cm, mitotic count[5/50 high power field (HPF), and high-risk GIST predicted poor RFS and DSS (UA). Disease-specific survival was significantly shorter after macroscopic incomplete (R2) resection, for mixed cellular morphology, and in tumors with necrosis (UA). High-risk GIST (p = 0.016) and R2 resection (p =0.013) predicted poor DSS of patients (MA).
Conclusions High risk and positive macroscopic surgical margin status are parameters associated with poor disease-specific survival in GIST patients.
Introduction
Gastrointestinal stromal tumor (GIST) is the most common of the mesenchymal tumors, accounting for up to 3% of malignant tumors of the gastrointestinal tract (GI). Lack of reproducible diagnostic criteria led to previous classifica-tion of such tumors as leiomyomas, leiomyoblastomas, and leiomyosarcomas, avoiding accurate actual use based on older published work of GI sarcoma surgical management.
Recent reports define GIST as a c-KIT (CD117) positive mesenchymal tumor [1].
Gastrointestinal stromal tumors vary in their malignant potential, with a spectrum ranging from virtually benign tumors, to tumors with uncertain malignant potential, to highly malignant tumors. The proportion of overtly malignant or high-risk GISTs accounts for 20–45% of diagnosed GISTs [2,3].
Surgery is the best treatment for primary GISTs and may be curative [4–11]. After successful complete or en bloc removal of the tumor and surrounding tissue, surgery for GIST should establish a complete margin of normal tissue around the primary tumor site [4–7, 12–17]. Incomplete resection is considered a major factor predictive of poor prognosis in GIST patients. However, the effect of com-pleteness and extension of GIST resection on the prognosis of patients remains controversial [4,7,12,13,16,17].
Because controversy remains regarding the prognostic value of surgical margins in the management of patients with GIST, we aimed to develop a study in which to A. M. GouveiaA. P. Pimenta
Department of Surgery, Hospital de S. Joa˜o/Porto Medical School, Al. Prof. Hernani Monteiro, 4202-451 Porto, Portugal A. M. GouveiaA. P. PimentaP. SilvaJ. M. Lopes (&) Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), R. Dr. Roberto Frias, 4200-465 Porto, Portugal
e-mail: jmlopes@ipatimup.pt
A. F. CapelinhaD. de la CruzP. SilvaJ. M. Lopes Department of Pathology, Hospital de S. Joa˜o/Porto Medical School, Al. Prof. Hernani Monteiro, 4202-451 Porto, Portugal
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World J Surg (2008) 32:2375–2382 DOI 10.1007/s00268-008-9704-8
evaluate prognostic clinicopathological parameters and surgical margins status on the outcome in a series of 113 consecutive patients diagnosed with primary GIST at the University Hospital of S. Joa˜o, Porto, Portugal.
Materials and methods
Clinical and pathological files and material related to all consecutively primary gastrointestinal stromal tumors (GISTs) diagnosed in the 18 years between 1989 and 2006 were retrieved from the Surgery and Pathology Depart-ments of Hospital S. Joa˜o, Porto, Portugal.
Clinical and surgical parameters
Clinical parameters of the patients included gender, age, primary tumor site, clinical symptoms, and signs at pre-sentation. The type of surgical resection (enucleation, wedge resection, segmental resection, total/subtotal organ resection, en bloc resection) of the tumor was evaluated in each case. Surgical procedures were grouped as follows:
wedge/segmental, enucleation, total/subtotal organ resec-tion, and en bloc resection.
Pathological parameters
All available pathological material was evaluated on 4-lm hematoxylin & eosin–stained sections [mean number of fragments studied per case: 6.1 (range: 1–26)], and the diagnosis of GIST was established according to the WHO classification [1].
Analyzed parameters for each tumor included largest size (cm), negative (R0) and positive (microscopic-R1, and macroscopic-R2) resection margin status [18], cell type (spindle, epithelioid, and mixed), presence or absence of necrosis, mitotic activity [number of mitoses per 50 high power field (HPF), 9400], and risk group of the GIST—
very low (VL), low (L), intermediate (I), and high (H) [19].
The immunohistochemistry analysis was performed on representative 3-lm sections of each tumor using the streptavidin-biotin-peroxidase complex principle. Rabbit polyclonal anti-human antibodies used were raised against CD117 (dilution 1:500; clone A 4502, DAKO A/S, Den-mark), actin (dilution 1:100; clone HHF35, DAKO, A/S, Denmark), desmin (dilution 1:50; Zymed Laboratories, South San Francisco, CA), S100 protein (dilution 1:1000;
DAKO A/S, Denmark), and CD34 (dilution 1:40; clone QBEnd/10, Novocastra Laboratories Ltd, Newcastle-upon-Tyne, UK).
Briefly, deparaffinized and rehydrated slides were sub-jected to 10 min incubation in 3% hydrogen peroxide in methanol, in order to inhibit endogenous peroxidase. No
antigen retrieval was used. After incubation with primary antibody at room temperature for 30 min, the secondary biotinylated goat anti-polyvalent antibody was applied for 10 min, followed by incubation with the streptavidin-per-oxidase complex. The immune reaction was visualized by DAB as a chromogen (Ultravision Detection System Anti-polyvalent, HRP/DAB; Lab Vision, Fremont, CA). Any (strong/weak, focal, moderate, or diffuse) membrane (CD117) and/or cytoplasm (CD117, actin, desmin, and CD34), and nuclear (S100 protein) immunoreactivity of the cells was considered as positive staining. Appropriated positive and negative controls were included in each run:
interstitial cells of Cajal in a section of normal intestine were used as positive control for CD117; smooth layers, for actin and desmin; small nerves, for S100 protein; and vessels, for CD34. For negative controls, primary anti-bodies were omitted. Mast cells, smooth layers, small nerves, and vessels were used as internal positive controls in the cases tested. All sections were counterstained with hematoxylin.
Follow-up and statistical analysis
Follow-up information was collected through direct inter-view with patients or their relatives, and by reinter-view of in-hospital patient files. Outcome data were available for all patients as of December 2007. Studied clinical and patho-logical parameters were analyzed as possible prognostic factors for local or distant (metastasis) recurrence and dis-ease-specific survival (DSS). Recurrence-free survival (RFS) was calculated from the time of surgery until the first evidence of recurrence, either distant or local, or both, in tumors with macroscopic complete resection; tumors with R2 resection were excluded for this calculation. Survival was calculated from the time of diagnosis until death related or not with the disease, or censored at the time of latest follow-up. Eight GIST patients with surgically resected tumors were treated with imatinib, as follows: because of tumor recurrence in five patients; as neodjuvant treatment in one case; as adjuvant treatment in one en-bloc-resected high-risk tumor; and because of R2 status margin in one patient. Recurrence-free survival, overall survival (OS), and DSS of all resected tumors were determined by Kaplan-Meier analysis [20], with SPSS software for Windows (version 14.0). Association between clinicopathological variables (e.g., risk group and margin status) and tumor recurrence was evaluated with the chi-square test or Fish-er’s exact test, as appropriate. Crude hazard ratio (HR) was computed (univariate analysis) with separate Cox regres-sions for each variable, and a probability value of less than 0.05 (pvalue\0.05) was considered statistically significant.
Multivariate analysis was performed with the Cox propor-tional hazards model, and only variables that were deemed
2376 World J Surg (2008) 32:2375–2382
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statistically significant by univariate analysis were consid-ered for the final Cox model.
Results
Clinical data
The patients entered into this study were 113 Caucasian patients with 114 sporadic tumors; one patient had metachronous gastric and colon GISTs. The series included 59 (52.2%) women and 54 (47.8%) men, with a median age of 66 years (range: 20–88 years). At presentation, major symptoms and signs included gastrointestinal bleeding (37.0%), abdominal pain (12.9%), abdominal mass (7.4%), tumor rupture (6.5%), and bowel obstruction (3.7%). Some 24.1% of patients were asymptomatic and their tumors were incidental findings on radiological examination and after surgery for other conditions.
By site, the tumors were located as follows: stomach (58.6%), small bowel (30.6%), colon/rectum (4.5%), mes-entery (3.6%), esophagus (1.8%), and omentum (0.9%); in three cases accurate localization was not possible because of the large dimension of the tumors.
One patient, diagnosed by endoscopic biopsy, was not submitted to surgery because of extensive local disease and poor general condition. Surgical resection was macro-scopically complete in 96 (84.9%) and incomplete in 8 tumors. In the remaining 9 (8%) cases, tumor resection was not performed because of the presence of liver metastases and diffuse peritoneal disease (3 cases), and extensive local invasion (6 cases).
The median follow-up time of patients was 42.6 months (range: 1–206 months), and the 5-year cumulative overall survival was 63.8%.
Surgical treatment
Only patients with surgically resected tumors (n = 104) were considered for further analysis. The mean and median ages of those men and women were 63.4 years and 66 years, respectively (range: 20–88 years), and the male/
female ratio was 0.8/1. Tumor frequency by primary site was as follows: 62 (59.6%) stomach, 33 (31.7%) small bowel, 4 (3.8%) colon and rectum, 3 (2.9%) mesentery, and 1 each in the esophagus and omentum.
The surgical procedures by primary site of tumor were as follows: stomach: wedge resection (29 [46.8%]), enu-cleation (12), distal/subtotal gastrectomy (12), total gastrectomy (7), and extended en bloc resection (2); small bowel: segmental resection (24 [72.7%]), enucleation (7), pancreaticoduodenectomy (1), and extended resection (1);
colon: segmental resection (2), total colectomy (1), and en
bloc resection (1); for esophagus: Ivor-Lewis resection (1);
for mesentery: enucleation (2) and segmental resection (1);
for omentum: enucleation (1).
In 7 cases tumor rupture occurred in association with either peritonitis (4 cases) or hemoperitoneum (3 cases);
tumor resection was accomplished in 5 patients, and postoperative death occurred secondary to peritonitis and shock/multiorgan failure in two patients; despite tumor resection, peritoneal recurrence leading to death occurred in 2 patients.
Pathological parameters
The mean and median tumor sizes were 6.8 cm and 5.1 cm, respectively (range: 0.5–24 cm). The tumor dimension was B5 cm in 52 cases, from[5 toB10 cm in 30 cases, and [10 cm in 22 cases.
Tumor cell morphology of the GISTs was spindle in 66.3%, epithelioid in 12.5%, and mixed in 21.2%. Strong membrane and/or cytoplasm tumor cell immunoreactivity for CD117 was found in variable focal, moderate, or dif-fuse areas in 96 (92.3%) of the GISTs. In three cases, CD117 immunoreactivity was weak. Eight (7.7%) GISTs did not disclose CD117 immunoreactive tumor cells.
Interstitial cells of Cajal and mast cells, as internal positive controls, were variably observed in every case. The fre-quency and expression features of the other antibodies was variable from case to case, and within the same tumor, as follows: focal/rare tumor cells for actin (52.1%), desmin (6.4%), and S100 protein (17.9%); and moderate/diffuse for CD34 (72.7%)—data not shown. Six of eight CD117-negative GISTs expressed CD34 without any tumor cell expression for other markers tested.
Tumor necrosis was observed in 55 cases. The mean/
median mitotic rates were 4.6/3.0 mitoses/50 HPF (range:
0–27 mitoses). Mitotic count wasB5 mitoses/50 HPF in 80 tumors and[5 mitoses/50 HPF in 24 tumors. The GISTs were classified according to risk group as follows: 70 (67.3%) very low/low/intermediate risk (14 VL; 33 L; 23 I); and 34 (32.7%) high risk.
Margin status was classified as R0 in 78 tumors, R1 in 18 (9 enucleations; 9 wedge/segmental resections), and R2 in 8 (1 enucleation; 3 wedge/segmental resections; 2 total/
subtotal organ resections; 2 en bloc resections).
Follow-up and outcome
The 5-year recurrence-free survival (RFS) was 89.8% and 77.3% at 10 years, with a median follow-up time of 46 months (range: 1–206 months). The overall recurrence rate was 12.5% (12 cases) distributed as follows: local stomach serosa (1); diffuse peritoneal metastases (4); liver metastases (4); and synchronous peritoneal and liver
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metastases (3). The percentage of recurrence was signifi-cantly lower (p = 0.045) in R0 tumors (9.0%) than in R1 (27.8%) tumors. The recurrence rate in very low/low and intermediate risk tumors (4.4% and 4.5%, respectively) was significantly lower (p = 0.001) than in high-risk (34.6%) GISTs. Imatinib treatment (400 mg daily) was carried out in five recurrent GISTs with escalation to 600 mg daily whenever indicated. Surgical treatment was attempted in three cases: (a) R0 resection (local gastric recurrence); (b) R2 resection (extensive peritoneal metas-tases); and (c) surgical biopsy (synchronous peritoneal and liver metastases). These patients are alive without (a) and with (b, c) evidence of disease at last follow-up.
The 5-year/10-year cumulative disease-specific survival (DSS) was 87.7%/81.7%, with a median follow-up time of 45.9 months (range: 1–206 months). None of the 39 (37.9%) patients who died, 5 of them within the immediate postoperative (one month) period, were submitted to autopsy study. In 13 patients the cause of death was the tumor. In the surviving patients, 5 are with and 59 are without evidence of disease; 7 are under imatinib treat-ment, without evidence of disease (n = 2), with a partial response (n= 2), and with stable disease (n= 3).
Prognostic evaluation
Table1 summarizes clinicopathological parameters of the series. In the univariate analysis, tumor size[10 cm and number of mitoses per 50 HPF[5 were predictive of a poor RFS (HR = 5.61;p= 0.015 and HR = 4.22;p= 0.013, respectively) and DSS (HR = 8.83; p = 0.006 and HR = 5.24; p = 0.004, respectively). Patients with high-risk GISTs had a significantly shorter RFS and DSS than those with very low/low/intermediate risk GISTs (HR = 7.63;p= 0.002 and HR = 22.8; p\0.003, respectively). The DSS was shorter after macroscopical incomplete (R2) resection (Fig.1), and this difference was statistically significant (HR = 13.56;p\0.001); RFS showed a trend to be shorter after R1 resection (HR = 3.03;p= 0.059) Disease-specific survival was shorter for mixed cellular type and in tumors with necrosis (HR = 3.81; p = 0.027 and HR = 5.73;p = 0.023, respectively). Age, gender, and primary site of tumor were not significant prognostic parameters.
Prognostic value of risk group and margin status for DSS was significant in the multivariate study (Table2): high-risk tumors (p= 0.016) and macroscopic positive (R2) margin status (p= 0.013) were predictive of poor survival in GIST patients.
Discussion
Gastrointestinal stromal tumors occur predominantly in middle-aged and older patients (median age 63 years [21,
22]), without gender predominance. They are infrequent in patients younger than 40 years. They originate in the GI tract with a frequency as follows: stomach (40–70%), small intestine (20–40%), colon and rectum (5–15%), esophagus (\5%), and rarely in the mesentery, omentum, retroperi-toneum, and other intra-abdominal structures [4, 15, 22–
28]. Our series, like those in most reports, reveals no influence of either age or gender on the survival of GIST patients.
Small GISTs (B2 cm) are virtually benign, usually asymptomatic, and are detected during investigations or surgical procedures for unrelated disease. The symptoms of GIST are most commonly related to mass effect or bleed-ing. The most common symptom at presentation is GI bleeding [21] resulting from erosion of the tumor into the lumen of the GI tract. Tumor rupture into the abdominal cavity is also possible, causing life-threatening hemor-rhage. Patients with GIST may also exhibit various other symptoms that include early satiety, abdominal pain or discomfort, nausea, vomiting, obstruction, abdominal mass, dysphagia, jaundice, and anemia-related symptoms [21,29]. In our series, asymptomatic GIST comprised 1/4 (24.1%) of all cases, and this may account for the lower incidence of synchronous metastatic disease and more favorable prognosis compared with most of the reported data.
Gastrointestinal stromal tumors can be composed of spindle (70%), epithelioid (20%), or mixed cells [19]. Our histological findings are consistent with the results obtained by most groups. Immunohistochemistry, as an important tool in the differential diagnosis from other neoplasms, disclosed 7.7% CD117-negative tumors in our series. Molecular analysis of KIT and PDGFRA hot spot genes described in GISTs and already published by our group [30] fits with reported series and may help the final diagnosis of difficult cases.
Many clinical and pathological parameters may influ-ence survival of GIST patients [4,10,11,13,24,31–42]. In our study, parameters found in multivariate analysis to significantly influence DSS were incomplete macroscopic (R2) tumor resection and high risk group. Other parame-ters, such as tumor size[10 cm, mitotic count[5/50 HPF, cellular morphology, and tumor necrosis, showed statistical significance in the univariate analysis for DSS. Prognosis after primary surgical resection is dependant on GIST malignant potential [4,5], but prognostic grading systems to identify high risk or malignant GIST remain contro-versial and are still under investigation [10,19,36,43,44].
With various grading systems, the rate of recurrence after primary surgical treatment of high-grade GISTs reported in the literature is high [5, 10–13, 19, 37, 45] and their prognosis is poor [4,5,10,11,32,33,37,38,46,47]. With the consensus grading system [19], the RFS and OS after
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primary surgical treatment of high-risk GIST is short [11, 19,37,47]. The recurrence rate in our series was signifi-cantly associated with the risk grade of GIST, and high-risk tumors were associated with poor DSS in both the uni-variate and the multiuni-variate analyses.
Several reports showed higher risk for recurrent disease and shorter OS after primary resection of large tumors ([5 cm or[10 cm) [4,5,10,24,31,32,36–38,46–52]. We found tumor size larger than 10 cm to be a predictive factor for poor DSS in the univariate analysis. Primary tumor site Table 1 Clinicopathological parameters and recurrence-free survival/disease-specific survival of Gastrointestinal Stromal Tumor (GIST) patients
Parameters RFSa DSS
n(%) Events (n) HR 95% CIb pValue n(%) Events (n) HR 95% CIb pValue Agec(years)
B60 37 (39) 4 1 – 39 (38) 4 1 –
[60 59 (61) 8 1.63 0.49–5.44 ns 64 (62) 9 1.61 0.49–5.25 ns
Genderc
Female 54 (56) 5 1 – 57 (55) 4 1 –
Male 42 (44) 7 1.83 0.58–5.79 ns 46 (45) 9 2.92 0.89–9.51 0.075
Tumor site
Stomach 60 (63) 7 1 – 62 (59) 6 1 –
Small bowel 29 (30) 3 0.91 0.24–3.54 ns 33 (32) 5 1.56 0.47–5.10 ns
Other 7 (7) 2 2.43 0.50–11.78 ns 9 (9) 2 2.36 0.48–11.74 ns
Type of surgery
Wedge/segmental resection 53 (55) 6 NA NA 56 (54) 7 NA NA
Enucleation 21 (22) 2 NA NA 22 (21) 0 NA NA
Total/subtotal organ resection 20 (21) 4 NA NA 22 (21) 4 NA NA
En bloc resection 2 (2) 0 NA NA 4 (4) 2 NA NA
Tumor size (cm)
B5 51 (53) 3 1 – 52 (50) 2 1 –
[5 toB10 29 (30) 3 1.44 0.29–7.18 ns 30 (29) 3 1.56 0.22–11.11 ns
[10 16 (17) 6 5.61 1.40–22.50 0.015 22 (21) 8 8.83 1.87–41.77 0.006
Cellular morphology
Spindle 67 (70) 5 1 – 69 (66) 5 1 –
Epithelioid 12 (12) 3 3.13 0.75–13.15 ns 13 (13) 2 2.06 0.39–10.63 ns
Mixed 17 (18) 4 3.13 0.84–11.66 ns 22 (21) 6 3.81 1.16–12.50 0.027
Necrosis
Absent 49 (51) 4 1 – 49 (47) 2 1 –
Present 47 (49) 8 2.43 0.73–8.08 ns 55 (53) 11 5.73 1.27–25.89 0.023
Mitotic count (/50 HPF)
B5 77 (80) 6 1 – 80 (77) 5 1 –
[5 19 (20) 6 4.22 1.36–13.11 0.013 24 (23) 8 5.24 1.71–16.06 0.004
Risk group
Very low/low/Intermediate 69 (72) 3 1 – 70 (67) 2 1 –
High 27 (28) 9 7.63 2.06–28.19 0.002 34 (33) 11 22.8 2.93–176.29 0.003
Margin status
R0 78 (81) 7 1 – 78 (75) 5 1 –
R1 18 (19) 5 3.03 0.96–9.56 0.059 18 (17) 3 2.44 0.58–10.21 ns
R2 NA – – – – 8 (8) 5 13.56 3.89–47.26 \0.001
a Excluded R2 cases
b 95% confidence interval for HR
c One patient with two metachronous tumors
RFS,recurrence-free survival;DSS, disease-specific survival;HR, hazard ratio;HPF, high power field (9400);NA, not applicable;ns, not significant
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(stomach or small bowel) was not correlated with RFS or DSS in our patients. A number of previous reports showed better OS for stomach primary GISTs [24,53,54], but the frequency of lower versus higher risk GISTs varies among the different sites. Lower risk tumors usually outnumber malignant GISTs in the stomach, probably because GIST is often an incidental finding in this location [36].
Conventional chemotherapy and external beam radio-therapy are well-known unsuccessful treatments for advanced GISTs. Imatinib has been effective in patients with metastatic and recurrent GIST [55–59]. Furthermore, it may be used as neoadjuvant treatment by experienced multidisciplinary teams, particularly when function-sparing surgery is the goal (e.g., rectal or esophageal tumors). I-matinib treatment is acceptable for patients with high-grade GISTs, after incomplete resection, or tumor rupture, to control the risk of recurrence [15, 44, 58, 60, 61]. The results achieved in the few cases of our series treated with imatinib fit with those reported in literature.
Five-year survival rates after GIST resection is reported to range from 32% to 76% [4,7,9,10,12,13,24,31–33, 36,37,39,52,62], probably as a result of different clini-copathological features and management procedures of the studied cohorts. Series concerning primary GISTs report higher resectability rates and longer overall survival. In addition, old published series of GI sarcomas may be contaminated with other (non-GIST) histology tumor types [4, 24,52, 63–65]. Only a few series have evaluated the DSS of GIST patients, showing higher 5-year DSS than 5-year OS [4, 10–12, 33, 35, 37–39, 66]. In our series, surgical resection of primary GISTs was associated with 87.7% 5-year DSS, which compares favorably with reported studies. In our patients, 5-year RFS after macro-scopic tumor resection was 89.8%. At variance with the findings reported by Ng et al. [33], our results show that only 5/12 recurrences occurred during the first 2 years of follow-up, and 4/12 occurred after 5 years of follow-up. As expected, most recurrences were abdominal metastasis (liver metastasis, peritoneal sarcomatosis, or both), a result reported by others [4, 13,35,67]. In one case with local recurrence complete tumor resection was achieved.
Because GISTs tend to protrude from the primary site and displace the surrounding organs/structures, wedge or segmental resection is usually an adequate surgical option.
The surgeon should make every effort to achieve complete resection of the tumor, which may require a subtotal or total organ resection or the removal of adjacent organs. In our study, resection of the tumor was achieved in 85%, which compares favorably with other reports (range: 48–
90%) [4, 7, 9, 44,52]. Considering extension of the sur-gical procedure to achieve complete resection, the role of lymphatic dissection may be an issue [4,5,7,12,13,33, 65,68] in the treatment of GISTs. Because of the low rate of reported lymph node involvement (4%) and/or sub-sequent lymph node metastases [5], there seems to be no added survival benefit from radical resection or extensive lymphadenectomy in the surgical treatment of GISTs. Even in high-grade GISTs, lymphadenectomy is indicated only when there is evidence of lymph node tumor involvement.
The postoperative course of patients with preoperative tumor rupture or tumor rupture during resection surgery was similar to that of patients with R2 resection, as in our series, with shorter overall survival (median survival of 17 months) in a reported series of GISTs [33]. Thus, as others do, we believe that laparotomy is in general more appropriate than laparoscopy in the treatment of tumors with features predictive of friability and of large GISTs.
Review of major published series shows several authors who emphasize complete macroscopic resection [4,5,15, 33, 35, 41, 47], whereas others highlight R0 resections [8–10,12,13,17,32,42,69] as good standard of care in the surgical treatment of GISTs. Some report that Fig. 1 Disease-specific cumulative survival according to margin
status of Gastrointestinal Stromal Tumors (GISTs,n=104). Cumu-lative survival is significantly lower in cases with R2 tumor resection (p\0.001)
Table 2 Multivariate analysis of prognostic factors of disease-specific survival of GIST patients
Parameter HR 95% CIa p
Risk group
Very low/low/intermediate 1.00
High 13.87 1.65–116.86 0.016
Margin status
R0 1.00
R1 1.54 0.34–7.08 0.57
R2 5.72 1.44–22.71 0.013
a 95% confidence interval for HR
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