CT-based assessment of sarcopenia for differentiating wild-type from mutant-type gastrointestinal stromal tumor.

Non-invasive prediction for KIT/PDGFRA status in GIST is a challenging problem. This study aims to evaluate whether CT based sarcopenia could differentiate KIT/PDGFRA wild-type gastrointestinal stromal tumor (wt-GIST) from the mutant-type GIST (mu-GIST), and to evaluate genetic features of GIST. A total of 174 patients with GIST (wt-GIST = 52) were retrospectively identified between January 2011 to October 2019. A sarcopenia nomogram was constructed by multivariate logistic regression. The performance of the nomogram was evaluated by discrimination, calibration curve, and decision curve. Genomic data was obtained from our own specimens and also from the open databases cBioPortal. Data was analyzed by R version 3.6.1 and clusterProfiler ( http://cbioportal.org/msk-impact ). There were significantly higher incidence (75.0% vs. 48.4%) and more severe sarcopenia in patients with wt-GIST than in patients with mu-GIST. Multivariate logistic regression analysis showed that sarcopenia score (fitted based on age, gender and skeletal muscle index), and muscle fat index were independent predictors for higher risk of wt-GIST (P < 0.05 for both the training and validation cohorts). Our sarcopenia nomogram achieved a promising efficiency with an AUC of 0.879 for the training cohort, and 0.9099 for the validation cohort with a satisfying consistency in the calibration curve. Favorable clinical usefulness was observed using decision curve analysis. The additional gene sequencing analysis based on both our data and the external data demonstrated aberrant signal pathways being closely associated with sarcopenia in the wt-GIST. Our study supported the use of CT-based assessment of sarcopenia in differentiating the wt-GIST from the mu-GIST preoperatively.

Pre-and Postoperative Circulating Tumour DNA in Patients With Gastrointestinal Stromal Tumour - A Methodological Assessment Study.

BACKGROUND/AIM: Gastrointestinal stromal tumours (GISTs) harbour genetic aberrations in receptor tyrosine kinase KIT (KIT) or platelet-derived growth factor receptor A (PDGFRA) in 85-90% of the patients. Circulating tumour DNA (ctDNA) is a potential biomarker in patients with GIST. Previous studies investigating ctDNA around surgery in patients with GIST presented divergent results regarding the proportion of patients with detectable ctDNA. This study aimed to 1) investigate the feasibility of detecting and monitoring ctDNA pre-and postoperative, 2) compare two different circulating free DNA (cfDNA) extraction methods, and validate results obtained by next-generation sequencing (NGS) using Real-Time PCR technology. PATIENTS AND METHODS: Eight patients planned for immediate surgery or surgery after neoadjuvant oncological treatment were included in the study, from whom blood collection was performed pre- and postoperatively for ctDNA analysis. Furthermore, blood samples from six patients with GIST harbouring a point mutation in KIT or PDGFRA in tissues from primary tumours were used for comparison and validation sub-study. RESULTS: In this explorative study, none of the patients with very low to intermediate risk GIST harboured KIT, or PDGFRA mutated ctDNA in pre-or postoperative blood samples. The methods used for cfDNA extraction gave similar output, and the two methods for ctDNA analysis gave identical results. CONCLUSION: There is no benefit in analysing ctDNA around surgery in very low to intermediate-risk GIST patients. Larger studies investigating ctDNA in patients with high-risk GIST around surgery are warranted.

Human erythroblasts with c-Kit activating mutations have reduced cell culture costs and remain capable of terminal maturation.

A major barrier to the in vitro production of red blood cells for transfusion therapy is the cost of culture components, with cytokines making up greater than half of the culture costs. Cell culture cytokines also represent a major expense for in vitro studies of human erythropoiesis. HUDEP-2 cells are an E6/E7 immortalized erythroblast line used for the in vitro study of human erythropoiesis. In contrast to other cell lines used to study human erythropoiesis, such as K562 cells, HUDEP-2 cells are capable of terminal maturation, including hemoglobin accumulation and chromatin condensation. As such, HUDEP-2 cells represent a valuable resource for studies not amenable to primary cell cultures; however, reliance on the cytokines stem cell factor (SCF) and erythropoietin (EPO) make HUDEP-2 cultures very expensive to maintain. To decrease culture costs, we used CRISPR/Cas9 genome editing to introduce a constitutively activating mutation into the SCF receptor gene KIT, with the goal of generating human erythroblasts capable of SCF-independent expansion. Three independent HUDEP-2 lines with unique KIT receptor genotypes were generated and characterized. All three lines were capable of robust expansion in the absence of SCF, decreasing culture costs by approximately half. Importantly, these lines remained capable of terminal maturation. Together, these data suggest that introduction of c-Kit activating mutations into human erythroblasts may help reduce the cost of erythroblast culture, making the in vitro study of erythropoiesis, and the eventual in vitro production of red blood cells, more economically feasible.

An optimized protocol for the generation and functional analysis of human mast cells from CD34+ enriched cell populations.

The culture of mast cells from human tissues such a cord blood, peripheral blood or bone marrow aspirates has advanced our understanding of human mast cells (huMC) degranulation, mediator production and response to pharmacologic agents. However, existing methods for huMC culture tend to be laborious and expensive. Combining technical approaches from several of these protocols, we designed a simplified and more cost effective approach to the culture of mast cells from human cell populations including peripheral blood and cryopreserved cells from lymphocytapheresis. On average, we reduced by 30-50 fold the amount of culture media compared to our previously reported method, while the total MC number generated by this method (2.46±0.63×106 vs. 2.4±0.28×106, respectively, from 1.0×108 lymphocytapheresis or peripheral blood mononuclear blood cells [PBMCs]) was similar to our previous method (2.36±0.70×106), resulting in significant budgetary savings. In addition, we compared the yield of huMCs with or without IL-3 added to early cultures in the presence of stem cell factor (SCF) and interlukin-6 (IL-6) and found that the total MC number generated, while higher with IL-3 in the culture, did not reach statistical significance, suggesting that IL-3, often recommended in the culture of huMCs, is not absolutely required. We then performed a functional analysis by flow cytometry using standard methods and which maximized the data we could obtain from cultured cells. We believe these approaches will allow more laboratories to culture and examine huMC behavior going forward.

Avaliação da via do c-kit no adenoma pleomórfico e carcinoma adenoide cístico das glândulas salivares: estudo da expressão das proteínas da via, das mutações e da regulação pós-transcricional do gene kit / c-Kit signaling cascade in pleomorphic adenoma and adenoid cystic carcinoma of salivary glands: evaluation of cascade proteins, mutations and post-transcriptional regulation of gene Kit

Introdução: As neoplasias das glândulas salivares têm amplo espectro histológico resultante da múltipla diferenciação celular tumoral. O adenoma pleomórfico (AP) e o carcinoma adenoide cístico (CAC) são as mais comuns neoplasias benignas e malignas provenientes do ducto intercalado, respectivamente, além de serem compostas por estruturas luminais e células mioepiteliais. Em estudo realizado previamente pelo nosso grupo, detectamos que a proteína c-kit está envolvida nos processos da morfogênese das glândulas salivares e no adenoma pleomórfico. A proteína c-Kit tem papel importante no desenvolvimento de muitos processos embrionários, incluindo a gametogênese, melanogênese e hematopoiese, e também na biologia de tumores. Sua ativação induz diversas respostas intracelulares através de cascatas de sinalização de vias como PI3K/AKT e MAPK. Em tumores da glândula salivar ainda há poucos estudos sobre as alterações do gene KIT e das proteínas relacionadas a sua via de sinalização, assim como sua regulação pós-transcricional, realizada principalmente por meio dos microRNAs. O presente estudo avaliou, em APs e CACs (a) a localização das proteínas das vias PI3K/AKT/mTOR e MAPK por meio da técnica de imunoistoquímica; (b) a expressão dos microRNAs 221 e 222, relacionados ao gene KIT (c) a associação dos achados laboratoriais com variáveis clínicas, patológicas e sobrevida. Resultados: Nos casos de AP a proteína c-Kit foi identificada em formações luminais e em raras células isoladas no parênquima tumoral. Já nos CAC, observou-se positividade na membrana das células ductais. Para a via de PI3K/AKT/mTOR, no AP, a proteína PI3K beta mostrou-se parcialmente positiva no citoplasma das células próximas à capsula tumoral, e as proteínas AKT e mTOR fosforiladas, foram expressas especialmente nas células epiteliais e em poucas células mioepiteliais. Já no CAC, a proteína PI3K beta e AKT fosforilada mostraram-se negativas na maioria dos casos, e a proteína mTOR fosforilada foi expressa no citoplasma das células epiteliais e em algumas células mioepiteliais. Para a via MAPK, as proteínas RAS, MEK-1 fosforilada e ERK 1/2 foram negativas na maioria dos AP e CAC; B-Raf e MEK-2 fosforilada foram observadas nas células luminais dos AP. Nos CAC, estruturas luminais neoplásicas foram positivas para a proteína MEK-2 fosforilada; B-Raf foi positivo nas células luminais e mioepiteliais. Além disso, os pacientes que expressaram as proteínas mTOR e MEK-2 fosforilada apresentaram sobrevida câncer-específica significativamente aumentada (p=0,040 e p=0,005, respectivamente). Na análise do microRNAs, a expressão do miR-221 foi variável nas 13 amostras analisadas, tendo baixa expressão em 30,77% dos casos, expressão normal em 38,46 e expressão aumentada em 30,77% dos casos. Já nos APs o miR-221 foi detectado em 19 amostras, sendo 36,84% com baixa expressão, 52,63% com expressão normal e expressão aumentada foi vista em 10,53% dos casos. A expressão do miR-222 foi detectada em 14 CACs, sendo que a maioria dos casos (8 casos ­ 57,1%) a expressão do miR-222 foi semelhante ao observado nas amostras não neoplásicas. Nos APs, o miR-222 foi detectado em 22 amostras, sendo 31,8% com baixa expressão, 31,8% com expressão normal e 36,4% com expressão aumentada. Conclusão: Apesar de a proteína c-Kit ser expressa em ambas as neoplasias ­ AP e CAC, sua influência sobre as vias de sinalização MAPK e PI3K/AKT/mTOR ainda permanece por ser estabelecida. Ainda, os microRNAs 221 e 222 não mostram correlação consistente com a expressão de c-Kit nos tipos tumorais estudados.

Introduction: Salivary gland tumors present broad histological spectrum resulting from multiple tumor cell differentiation. Pleomorphic adenoma (PA) and adenoid cystic carcinoma (ACC) are the commonest benign and malignant salivary gland neoplasms originated from the intercalated duct region, respectively, and are composed by luminal structures and myoepithelial cells. In a previous study we detected that protein c-kit is involved in the process of salivary gland morphogenesis and PA. c-Kit protein is important during embryogenesis, including gametogenesis, melanogeneis and hematopoiesis as well as in tumorigenesis. Its activation induces various intracellular responses through pathways such as MAPK and PI3K/AKT/mTOR signaling cascades. In salivary gland neoplasms, only a few reports have shown that alterations in KIT gene are present and proteins related to its signaling pathway as well as its post-transcriptional regulation. This study has aimed at evaluating in PA and ACC: (a) the proteins location of PI3K/AKT/mTOR and MAPK pathways using immunohistochemistry (IHC); (b) expression of miR-221 and miR-222, related to KIT gene; and (c) the association of these findings with clinical, pathological and survival data of patients. Results: In PA c-kit was positive in isolated luminal cells; in ACC, neoplastic luminal structures were positive for c-Kit. In PA, PI3K beta protein was shown to be partially positive in the cytoplasm of cells near the tumor capsule and phosphor AKT and phospho mTOR, are specifically expressed in epithelial cells and in a few myoepithelial. In ACC, PI3K and phosphor AKT protein showed to be negative in most of cases. Phospho mTOR protein was expressed in the cytoplasm of epithelial cells and some myoepithelial cells. In MAPK pathway, Ras, ERK1/2 and phosphor MEK-1 proteins were negative in most PAs and CACs; B-Raf and phospho MEK-2 were detected in luminal cells of PA. In ACC neoplastic luminal structures were positive for phospho MEK-2; B-Raf was also positive in myoepithelial and epithelial cells. In addition, cases with expressed phospho-mTOR and phosphor MEK-2 proteins were significantly associated with higher cancer-specific survival (p = 0.040 and p = 0.005, respectively). Moreover, expression of miR-221 was detected in 13 CAC samples and 19 PA samples. In CAC, expression of miR-221 was downregulated in 30,77% of the samples, upregulated in 30,77% samples, and normal in 38,46% samples. In PA, miR-221 expression was downregulated in 36,84% samples, upregulated in 10,53% samples, and normal in 52,63% samples. Expression of miR-222 was detected in 14 CAC samples and 22 PA samples. In the majority of CAC samples, the expression of miR-222 was similar to that observed in non-neoplastic samples. In PA samples, expression of miR-222 was downregulated in 31,8% samples, upregulated in 36,4% samples, and normal in 31,8% samples. Conclusion: Although c-Kit expression is detected in PA and ACC, its influence on the MAPK e PI3K/AKT/mTOR signaling cascades remains to be established. miR-221 e -222 did not show a robust correlation with c-Kit expression in the tumors studied.

An in vitro model for the assessment of stem cell fate following implantation within the infarct microenvironment identifies ISL-1 expression as the strongest predictor of c-Kit(+) cardiac progenitor cells' therapeutic potential.

Cell therapy has the potential to drastically improve clinical outcomes for the 1.45 million patients suffering from a myocardial infarction (MI) each year in the U.S. However, the limitations associated with this treatment - including poor engraftment, significant cell death and poor differentiation potential - have prevented its widespread application clinically. To optimize functional improvements provided by transplanted cells, there is a need to develop methods that increase cellular retention and viability, while supporting differentiation and promoting paracrine signaling. Current in vivo models are expensive, difficult to access and manipulate and are time consuming. We have developed an in vitro model of MI which allows for a straightforward, consistent and relatively accurate prediction of cell fate following injection in vivo. The model demonstrated how the infarct environment impairs cellular engraftment and differentiation, but identified an implantation strategy which enhanced cell fate in vitro. Multivariate linear regression identified variables within the model that regulated vascular differentiation potential including oxygen tension, stiffness and cytokine presence, while cardiac differentiation was more accurately predicted by Isl-1 expression in the original cell isolate than any other variable present within the model system. The model highlighted how the cells' sensitivity to the infarct variables varied from line to line, which emphasizes the importance of the model system for the prediction of cell fate on a patient specific basis. Further development of this model system could help predict the clinical efficacy of cardiac progenitor cell therapy at the patient level as well as identify the optimal strategy for cell delivery.

The stem cell factor/Kit signalling pathway regulates mitochondrial function and energy expenditure.

Cell growth is tightly coupled with mitochondrial biogenesis in order to maintain energy and organelle homeostasis. Receptor tyrosine kinase Kit and its ligand, stem cell factor (SCF), play a critical role in the growth and survival of multiple cell lineages. Here we report that the expression of SCF and Kit in adipose tissues is responsive to food availability and environmental temperature, and is altered in obese mice and human patients. Mice carrying a loss-of-function mutation in Kit develop obesity as a result of decreased energy expenditure. These phenotypes are associated with reduced PGC-1α expression and mitochondrial dysfunction in brown adipose tissue and skeletal muscle. We further demonstrate that SCF/Kit directly promotes Ppargc1a transcription and mitochondrial biogenesis. Blocking Kit signalling in mice decreases PGC-1α expression and thermogenesis, while overexpressing SCF systemically or specifically in brown adipose tissue increases thermogenesis and reduces weight gain. Collectively, these data provide mechanistic insight into the regulation of mitochondrial function by SCF/Kit signalling and lay a foundation for exploring SCF/Kit signalling as a therapeutic target for metabolic diseases.

Extended adjuvant therapy with imatinib in patients with gastrointestinal stromal tumors : recommendations for patient selection, risk assessment, and molecular response monitoring.

On the basis of the recently published results of a clinical trial comparing 12 and 36 months of imatinib in adjuvant therapy for gastrointestinal stromal tumors (GISTs), which demonstrated clinical benefit of longer imatinib treatment in terms of delaying recurrences and improving overall survival, both the US Food and Drug Administration and the European Medicines Agency have updated their recommendations and approved 36 months of imatinib treatment in patients with v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT)-positive GISTs (also known as CD117-positive GISTs) at high risk of recurrence after surgical resection of a primary tumor. This article discusses patient selection criteria for extended adjuvant therapy with imatinib, different classifications of risk of recurrence, and assessment of the response to therapy.

Colorectal poorly differentiated neuroendocrine carcinomas and mixed adenoneuroendocrine carcinomas: insights into the diagnostic immunophenotype, assessment of methylation profile, and search for prognostic markers.

Colorectal poorly differentiated neuroendocrine carcinomas (NECs) and mixed adenoneuroendocrine carcinomas (MANECs) are well-recognized entities generally known to be associated with biological aggressiveness and poor patient survival. However, a few published papers have highlighted the existence of a subgroup of tumors with a better survival than expected; however, to date, there are no established parameters that usefully identify this category. In the present study we have investigated the morphologic features, the CpG methylator phenotype (CIMP), microsatellite instability (MSI), and the immunohistochemical profile, including the expression of transcription factors (TTF1, ASH1, CDX2, and PAX5), stem cell markers (CD117 and CD34), and cytokeratins 7 and 20, in a series of 39 carcinomas (27 NECs and 12 MANECs) to better characterize such neoplasms and to search for prognostic indicators. No different patient survival was observed between NECs and MANECs. Neoplasms showed a heterogenous spectrum of morphologic and immunohistochemical features; however, only large-cell subtype, significant peritumoral lymphoid reaction, CD117 immunoreactivity, vascular invasion, and MSI/CIMP+ status were significantly correlated with prognosis on univariable analysis. Furthermore, vascular invasion and CD117 immunoreactivity were independent prognostic markers on multivariable analysis. In addition to these prognostic features, neoplasms showed different expression of transcription factors, stem cell markers, and cytokeratins that should be considered for diagnostic purposes and, especially, for discriminating among possible differential diagnoses.

Budgetary impact of treatment with adjuvant imatinib for 1 year following surgical resection of Kit-positive localized gastrointestinal stromal tumors.

BACKGROUND: Imatinib mesylate, an orally administered kinase inhibitor that targets the Kit (CD117) protein, currently has 10 approved indications including treatment of chronic myelogenous leukemia and metastatic gastrointestinal stromal tumors (GIST). Treatment with adjuvant imatinib following surgical resection of localized Kit-positive GIST, the most recent FDA-approved indication (December 2008), has been shown to significantly improve recurrence-free survival (RFS) compared with surgical resection alone. Although adjuvant imatinib has proven effective in clinical trials, it is important to consider the economic impact to health plans of introducing imatinib in accordance with its new labeled indication. OBJECTIVE: To evaluate the budgetary impact over a 3-year time horizon of treating patients with localized Kit-positive GIST with 1 year of adjuvant imatinib following surgical resection. METHODS: A Markov model was developed to predict patients' transitions across health states defined by initial treatment (surgical resection followed by adjuvant imatinib 400 milligrams [mg] daily versus surgical resection alone), recurrence, and progression. Treatments for a first recurrence were (a) imatinib 400 mg daily for recurrences following resection only or after completion of 1 year of treatment with imatinib 400 mg daily and (b) imatinib 800 mg daily for recurrence during active treatment with imatinib 400 mg daily. Treatments for further progression were imatinib 800 mg daily, sunitinib, or best supportive care (BSC) following imatinib 400 mg per day, and sunitinib or BSC following imatinib 800 mg daily. Recurrence rates were derived from the American College of Surgeons Oncology Group (ACOSOG) Z9001 clinical trial, which compared 1 year of adjuvant imatinib following surgical resection with surgical resection only. The total number of patients with localized and surgically resected GIST (incidence rate of 0.36 per 100,000) was estimated from epidemiologic studies of GIST. Uptake of treatment with imatinib was estimated from unpublished data from qualitative market research funded by the study sponsor. The uptake rate assumptions reflected both (a) the percentage of patients with Kitpositive disease and (b) the percentage of clinically eligible patients who would use imatinib. Costs were estimated by combining unit costs from published sources with expected resource utilization based on the clinical trial publication and National Comprehensive Cancer Network guidelines on the treatment of patients with GIST. To obtain estimates of the budgetary impact, we compared estimated health care costs with versus without adjuvant imatinib, where health care costs with imatinib reflected the costs of treatment minus cost offsets associated with delayed or avoided recurrence or progression. All "with" scenarios assumed no additional uses other than surgically resected localized Kit-positive GIST (i.e., no change in off-label use of imatinib). The budgetary impact was estimated for the first 3 years after the introduction of adjuvant imatinib in accordance with its new labeled indication in a hypothetical plan population of 10 million persons. Results were calculated both as total budgetary impact and as per member per month (PMPM) cost in 2009 dollars. Sensitivity analyses were performed to test the robustness of model results to changes in parameter estimates. RESULTS: The model predicted 36 incident resected GIST cases per year in a health plan of 10 million members. The estimated counts of cases treated with adjuvant imatinib were 10.8, 16.2, and 21.6 in the first, second, and third years after introduction, respectively, with the annual increases attributable to changes in the proportion of patients with resected GIST assumed to use imatinib (30% in year 1, rising to 45% in year 2 and 60% in year 3). The model predicted that treatment of these cases with imatinib will increase pharmacy costs by an additional $505,144 in the first year, $757,717 in the second year, and $1,010,289 in the third year. Increased resource use associated with monitoring patients during and after treatment with adjuvant imatinib would cost an additional $21,564, $38,145, and $56,605 in the first, second, and third years, respectively. Recurrence would be avoided or delayed in 7 patients over the 3-year period. Avoided or delayed recurrences would result in cost offsets of $61,583 in the first year, $156,702 in the second year, and $233,849 in the third year. The net budgetary impact was estimated to be $465,126 in the first year (less than $0.01 PMPM), $639,159 in the second year ($0.01 PMPM), and $833,044 in the third year ($0.01 PMPM). Results of sensitivity analyses indicated that the budgetary impact in the third year is most sensitive to changes in the price of adjuvant imatinib and recurrence rates. CONCLUSIONS: The model predicted that the introduction of adjuvant imatinib for treatment of surgically resected, localized, Kit-positive GIST will lead to a net budgetary impact of $0.01 PMPM in the third year after introduction assuming change in use only in accordance with the new labeled indication. Approximately 11.7%-21.9% of the cost of adjuvant imatinib is offset by the reduction in costs associated with GIST recurrence.

Assessment of "grading" with Ki-67 and c-kit immunohistochemical expressions may be a helpful tool in management of patients with flat epithelial atypia (FEA) and columnar cell lesions (CCLs) on core breast biopsy.

It is essential to reach a better understanding of "flat epithelial atypia/columnar cell lesions" (FEA/CCLs) in breast core biopsies. Our aim was to explore their biological nature, in order to predict the likelihood of an upgrade to carcinoma. "Cytological grading" has been specially focused, in view of its possible utility in the choice of management. One hundred thirty of a total of 900 cases core needle (CN)/vacuum-assisted biopsies (VABs), with diagnoses of "hyperplasia" and "atypia" were retrospectively re-evaluated. Pathological findings of further excision biopsies (FEBs) performed in 40/75 patients with follow-up were compared with the previous diagnoses. In all cases, both Ki-67 and c-kit immunoreactivities were explored and compared with both normal breast tissues and subsequently documented cancers, with special reference to the hyperplastic FEA/CCLs, with "mild" atypia (FEA/CCHAm). Sixteen cases were re-diagnosed as "usual ductal hyperplasia" (UDH), 60 as "columnar cell hyperplasia" (CCH), and 54 as FEA/CCHA, 30 of which FEA/CCHAm and 24 FEA/CCHAh (with high atypia). Significantly, the Ki-67 index proved to be on the increase and c-kit expression on the decrease in FEA/CCHA lesions, mainly in the FEA/CCHAh group and in the subsequently observed cancers, compared with either benign tissues or the FEA/CCH cases. It was also significant that most of the carcinomas were found in FEBs within the FEA/CCHAh group. In this study cytological grading, together with Ki-67 and c-kit indices, proved to be helpful in FEA/CCLs evaluation. With regard to FEA/CCHAm lesions, an adequate surveillance appears to be a more appropriate management tool than FEB, as a result of their biological nature and behavior.

Early response assessment in gastrointestinal stromal tumors with FDG PET scan 24 hours after a single dose of imatinib.

Gastrointestinal stromal tumors (GIST), rare mesenchymal tumors of the gastrointestinal tract, are gaining the interest of researchers because of the impressive metabolic response to the targeted molecular therapeutic drug imatinib mesylate. FDG PET is now routinely used to assess treatment response in cases of GIST because this has proven to give metabolic information, which demonstrates response earlier than anatomic imaging modalities. A 50-year-old man presented with abdominal pain and the CT scan showed a large lobulated heterogeneously enhancing mass in the abdomen. Fine needle aspiration cytology (FNAC) confirmed GIST with strong immunoreactivity to C-Kit protein. A baseline FDG PET done before initiation of therapy showed intense nonhomogenous FDG uptake in the mass (standard uptake value maximum, SUVmax of 13.45). A whole body FDG PET, repeated 24 hours after a single dose of imatinib mesylate 400 mg, showed a significant reduction in FDG uptake with a SUVmax of 4.26.

A study of histopathological assessment criteria for assessing malignancy of gastrointestinal stromal tumor, from a clinical standpoint.

BACKGROUND: As no established histopathological criteria exist for assessing the malignant potential of gastrointestinal stromal tumor (GIST), recurrence or metastasis is occasionally observed in lesions diagnosed histopathologically as benign. The present study aimed to clarify the histopathological criteria for assessing the malignancy of GIST, from a clinical standpoint. METHODS: The subjects were 22 patients with GIST expressing CD117 (c-kit) and/or CD34, who were followed up for more than 2 years. Clinically, GIST malignancy was diagnosed if any of the following criteria were met: peripheral invasive growth, lymph node metastasis, metastasis to another organ, peritoneal dissemination, recurrence, or death. GIST was also categorized as either benign or malignant by a new histological malignancy classification system, based on the determination of significant factors indicating malignancy in the clinical classification system above. RESULTS: Significant factors for malignancy identified in the clinical malignancy classification were: tumor hemorrhage/necrosis (present vs absent; P = 0.0053), tumor size (<5 cm vs > or =5 cm; P = 0.0022), and Ki-67 labeling index (<3% vs > or =3%; P = 0.0002). A new histological malignancy classification, based on a combination of these three factors, was developed. A significant correlation existed between the clinical system and the new histological malignancy classification system (P = 0.0008). The recurrence-free survival rate was 100% in the histologically benign cases and 37.5% in the histologically malignant cases (P = 0.0012). CONCLUSIONS: The new histological malignancy classification for GIST was demonstrated to be useful from a clinical standpoint.

Essential role of extracellular matrix (ECM) overlay in establishing the functional integrity of primary neonatal rat Sertoli cell/gonocyte co-cultures: an improved in vitro model for assessment of male reproductive toxicity.

The development of in vitro models for testicular toxicity may provide important tools for investigating specific mechanisms of toxicity in the testis. Although various systems have been reported, their application in toxicological studies has been limited by the poor ability to replicate the complex biochemical, molecular, and functional interactions observed in the testis. In the present study, we evaluated a significantly improved Sertoli cell/gonocyte co-culture (SGC) system that employs a 3-dimensional extracellular matrix Matrigel (ECM) applied as an overlay instead of a substratum. We explored the dose- and time-dependent effects of the addition of such an ECM overlay on cytoskeletal and morphological changes in the SGC system, and the resulting effects on cellular integrity. Furthermore, we correlated the latter effects with the ECM-dependent modulation of stress and survival signaling pathways and, most critically, the expression levels of the spermatogonia-specific protein, c-Kit. Finally, we applied this co-culture system to investigate the dose- and time-dependent effects on the morphology and induction of apoptosis of cadmium. We observed that the dose-dependent addition of an ECM overlay led to an enhanced attachment of Sertoli cells and facilitated the establishment of SGC communication and cytoskeletal structure, with a dramatic improvement in cell viability. The latter was consistent with the observed dose- and time-dependent modulation of both stress signaling pathways (SAPK/JNK) and survival signaling pathways (ERK and AKT) in the presence of the ECM overlay. Furthermore, the dose-dependent stabilization of c-Kit protein expression confirmed the functional integrity of this co-culture system. We conclude that this modified SGC system will provide investigators with a simple, efficient, and highly reproducible alternative in the screen for testicular cell-specific cytotoxicity and the assessment of molecular mechanisms associated with both normal development and reproductive toxicity induced by environmental toxicants.

Clinicopathologic assessment of postradiation sarcomas: KIT as a potential treatment target.

PURPOSE: Postradiation sarcoma, a sarcoma developing in a previously irradiated field, is a rare tumor. Surgery appears to be the only curative treatment option. In general the prognosis is poor, and new treatments options are needed. One study reported the expression of KIT receptor tyrosine kinase in two postradiation angiosarcomas. Success of inhibition of KIT in malignant gastrointestinal stromal tumors with imatinib mesylate seems mutation-dependent, with a favorable response in the presence of exon 11 mutations. EXPERIMENTAL DESIGN: We performed a clinical, immunohistochemical, and genetic assessment of postradiation sarcomas, including angiosarcomas. Archival tumor tissue was available from 16 patients diagnosed with a postradiation sarcoma between 1978 and 2001. Data on the first and secondary tumor, treatment, and follow-up was documented. KIT expression was assessed by immunohistochemistry. For comparison, 23 spontaneous soft tissue sarcomas of similar histological types were analyzed. Exon 11 of the c-kit gene was analyzed by direct DNA sequencing. RESULTS: Fifteen patients received initial irradiation for malignant disease and 1 patient for a benign condition. The median delivered dose was 50 Gy. The median latency period between irradiation and diagnosis of postradiation sarcomas was 222 months. Histological types included: angiosarcoma, fibrosarcoma, malignant fibrous histiocytoma, osteosarcoma, rhabdomyosarcoma, and unspecified sarcoma. In concordance with the literature, patients had a poor outcome. Only 3 of 16 patients were disease-free 43, 60, and 161 months after being diagnosed of postradiation sarcoma, all 3 having favorable tumor and treatment characteristics. Fourteen of 16 tumor samples were KIT-positive (88%). In 8 cases >80% of tumor cells stained positively. Five of 23 (22%) spontaneous soft tissue sarcomas of comparable histological types, including 2 angiosarcomas, were KIT-positive. Molecular genetic analysis of exon 11 of the c-kit gene was attainable for 13 of the 16 postradiation sarcomas. No mutations were found. CONCLUSIONS: Postradiation sarcomas are aggressive malignancies, seldom amenable to curative treatment. A majority of the analyzed tumors showed extensive expression of the KIT protein, but no mutations in exon 11 of the c-kit gene were found. Still, without the availability of effective therapies, treatment with the KIT inhibitor imatinib mesylate might be considered for patients with postradiation sarcomas.

Prognostic assessment of gastrointestinal stromal tumor.

The term "gastrointestinal stromal tumor" (GIST) has been applied to a collection of distinctive mesenchymal tumors occurring within the human gastrointestinal tract. As new drug therapy becomes available, data regarding the natural history of these unusual tumors are necessary to provide selection factors for treatment. Ninety-eight patients had light microscopy compatible with GIST at a single institution from 1989 to 2000. After immunostaining with c-kit and histopathologic review, 69 were judged to be GIST. All prognostic indicators were determined for gastric GIST, intestinal GIST, and all locations combined. The location of the GIST did not have a significant impact on survival. Clinically, tumor size, peritoneal cancer index, and completeness of cytoreduction had a significant impact on prognosis for GIST at all locations. Pathologically, cytologic atypia, necrosis, invasion and number of mitoses were significant prognostic indicators for GIST. Criteria to separate three pathologic groups of GIST according to the tumor size and the mitotic count were useful to evaluate the tumor behavior; in the borderline pathologic group invasion and cytologic atypia were statistically significant prognostic criteria. The cell phenotypes, as determined by immunostains, correlated with the prognosis of gastric GIST but not intestinal GIST. A correlation between the immunostain Ki-67 but not CD-34 or desmin and the prognosis was observed. It is possible to select clinical and pathologic parameters of GIST that impact on prognosis. Invasion and necrosis help to determine the prognosis with borderline tumors. The immunostain Ki-67 correlated with the prognosis and may be helpful to assess prognosis when dealing with small biopsy specimens.