No abstract

Our aim in this study was to describe the clinical, morphological, and molecular profile of gastrointestinal stromal tumor (GIST) metastatic to bone. We analyzed the morphological, phenotypic, and molecular characteristics of seven cases, and in addition reviewed 17 cases from literature. Sequence analysis of KIT and PDGFRA genes was possible for six cases. For the GIST cases with bone metastasis, the most common primaries were small intestine (29%), stomach (25%), and rectum (21%). Sites of bone metastases were vertebrae (11), pelvis (8), femur (8), ribs (6), humerus (5), skull (3), scapula (1), and mandible (1). The size ranged from 1.5 to 13 cm (median, 3.8 cm). Bone metastases without involvement of any other organ were seen in 17% of the cases and were solitary in 14 (58%). Adjacent soft tissue involvement was present in nearly half of the patients. Bone metastasis was either manifest at the time of diagnosis (28%) or occurred after a mean period of 4.7 years (3 months-20 years). Morphologically, neoplastic cells were spindle in 67%, epithelioid in 13%, and mixed epithelioid and spindle in 20%. CD117, DOG1, and CD34 were positive in 88, 86, and 85% of the cases, respectively. KIT Exon 11 mutations were the most frequent gene alteration (78%), followed by KIT Exon 13 mutations. Of 17 of the cases with available follow-up information, 7 (41%) patients developed bone metastasis under imatinib therapy. Five patients (29%) died of disease within a mean of 17 months. Bone metastases from GIST are usually found in patients with advanced disease and typically present as lytic masses with occasional soft tissue involvement. We could not identify any KIT or PDGFRA alterations predisposing to bone metastasis.

Abstract. Hepatocellular carcinoma (HCC) is the fifth most common male-predominant type of cancer worldwide. There is no effective treatment regimen available for advanced-stage disease and chemotherapy is generally ineffective in these patients. The number of studies on the prevalence of K-Ras mutations in HCC patients is currently limited. A total of 58 patients from 6 comprehensive cancer centers in 4 metropolitan cities of Turkey were enrolled in this study. Each center committed to enroll approximately 10 random patients whose formalin-fixed paraffin-embedded tumor tissues were available for K-Ras, exon 2 genotyping. Two methods were applied based on the availability of adequate amounts of tumor DNA. In the first method, the samples were processed using TheraScreen. The genomic DNA was further used to detect the 7 most frequent somatic mutations (35G>A; 35G>C; 35G>T; 34G>A; 34G>C; 34G>T and 38G>A) in codons 12 and 13 in exon 2 of the K-Ras oncogene by quantitative polymerase chain reaction (PCR). In the second method, the genomic DNA was amplified by PCR using primers specific for K-Ras exon 2 with the GML SeqFinder Sequencing System's KRAS kit. The identified DNA sequence alterations were confirmed by sequencing both DNA strands in two independent experiments with forward and reverse primers. A total of 40 samples had adequate tumor tissue for the mutation analysis. A total of 33 (82.5%) of the investigated samples harbored no mutations in exon 2. All the mutations were identified via a direct sequencing technique, whereas none were identified by TheraScreen. In conclusion, in our patients, HCC exhibited a remarkably low (<20%) K-Ras mutation rate. Patients harboring K-Ras wild-type tumors may be good candidates for treatment with epidermal growth factor inhibitors, such as cetuximab.

Formalin-fixed paraffin-embedded (FFPE) tissue is a widely available clinical specimen for retrospective studies. The possibility of long-term clinical follow-up of FFPE samples makes them a valuable source to evaluate links between molecular and clinical information. Working with FFPE samples in the molecular research area, especially using high-throughput molecular techniques such as microarray gene expression profiling, has come into prominence. Because of the harmful effects of formalin fixation process such as degradation of nucleic acids, cross-linking with proteins, and chemical modifications on DNA and RNA, there are some limitations in gene expression profiling studies using FFPE samples. To date many studies have been conducted to evaluate gene expression profiling using microarrays (Thomas et al., Thomas et al. (2013) [1]; Scicchitano et al., Scicchitano et al. (2006) [2]; Frank et al., Frank et al. (2007) [3]; Fedorowicz et al., Fedorowicz et al. (2009) [4]). However, there is still no generally accepted, efficient and standardized procedure for microarray analysis of FFPE samples. This paper describes the microarray data presented in our recently accepted to be published article showing a standard protocol from deparaffinization of FFPE tissue sections and RNA extraction to microarray gene expression analysis. Here we represent our data in detail, deposited in the gene expression omnibus (GEO) database with the accession number GSE73883. Four combinations of two different cRNA/cDNA preparation and labeling protocols with two different array platforms (Affymetrix Human Genome U133 Plus 2.0 and U133_X3P) were evaluated to determine which combination gives the best percentage of present call. The study presents a dataset for comparative analysis which has a potential in terms of providing a robust protocol for gene expression profiling with FFPE tissue samples.

Allogeneic stem cell transplantation applications have improved tremendously over the past quarter of a century. The use of new immunosuppressive protocols and elimination of T cells by CD34+ cell enrichment or T cell depletion on apheresis products increases the chance of using partially matched or haploidentical grafts. This is without increasing the risk of graft-versus-host disease, which is observed as a major complication of hematopoietic stem cell transplantation. The aim of this protocol is to evaluate the results obtained from 6 different process cycles performed on 6 different days. We used the CliniMACS Plus system located in our Cell and Tissue Manufacturing Center Quality Control Unit which is already calibrated as a class D room and includes a class A microbiological safety cabinet inside. The average purity of the end products was 95.66%, excluding only one end product which was 70%; this was higher than the values in current studies in the field. Superior to the reported studies, the CD3 quantity in each end product was below the dedicated thresholds. BactecTM FX40 blood culture system test results were detected as negative for each end product. Endotoxin testing suggested the absence of endotoxin within the products. The consistent outcomes obtained from these 6 different process cycles confirmed that the CliniMACS® Plus process cycles performed in accordance with our well-defined quality management system procedure is sufficient for the routine application of high-quality and safe CD34+ enrichment processes within our clean room area.

Bu çalışmada küçük hücreli dışı akciğer kanserli Türk topolumunda epidermal büyüme faktör reseptör mutasyonlarının sıklığı, dağılımı ve morfolojik/immünhistokimyasal özellikleri araştırıldı ve materyal tipi ve mutasyon analizinin teknik başarısı arasındaki muhtemel ilişki incelendi. Ça lış ma pla nı:Eylül 2012 -Aralık 2015 tarihleri arasında Sanger sekanslama yöntemi ile epidermal büyüme faktör reseptör mutasyon testi yapılan, daha önce tedavi edilmemiş, primer veya metastatik küçük hücreli dışı akciğer kanserli toplam 499 ardışık hasta (437 erkek, 163 kadın; ort. yaş 61 yıl; dağılım, 30-84 yıl) retrospektif olarak incelendi. Arşiv kayıtları ve hematoksilen-eozin ve immünohistokimya boyalı kesitler yeniden değerlendirildi. Doku array bloklarında tiroid transkripsiyon faktör-1 ve napsin A immünohistokimyasal boyamaları yapıldı.Bul gu lar: Yetmiş hastada (%14) 75 mutasyon saptandı. Sitolojik materyallerde analizin başarı oranı ve intakt deoksiribonükleik asit parça uzunluğu, doku materyallerine kıyasla, anlamlı olarak daha yüksekti (p<0.001). Kadınlarda mutasyon oranı %33.9 ve erkeklerde %9.4 idi. En sık görülen mutasyon ekzon 19'da L746-E750del (%29.3) olup, bunu ekzon 21'de L858R (%28) mutasyonu izledi. Mutasyon oranı en yüksek mikropapillerde (%40) ve en düşük solid adenokarsinomalarda (%5.4) izlendi. Biri hariç, epidermal büyüme faktör reseptör mutasyonlarının tümünde tiroid transkripsiyon faktör-1 pozitifliği mevcut idi. Hastaların %79.6'sında ekzon 20'de tekli nükleotid polimorfizmi (Q787Q) izlendi.So nuç: Küçük hücreli dışı akciğer kanserli Türk hastalarda epidermal büyüme faktör reseptör mutasyonlarının görülme sıklığı ve dağılımı Avrupa toplumları ile benzerdir. Ayrıca bu sonuçlar, epidermal büyüme faktör reseptör mutasyon analizi için sitolojik materyallerin son derece güvenilir olduğunu göstermekle birlikte, tiroid transkripsiyon faktör-1 negatif olgularda mutasyona rastlama ihtimali çok düşüktür.Anah tarsöz cük ler: Epidermal büyüme faktör reseptörü, napsin A, küçük hücreli dışı akciğer kanseri, tiroid transkripsiyon faktör-1, Türk toplumu.

To compare our parameters as regards: i) cell count via two different automated cell count techniques, and ii) viability via automated trypan blue exclusion and 7-aminoactinomycin D (7-AAD) staining. Method: We used the trypan blue exclusion technique and an automated cell counter and for viability testing, and the trypan blue exclusion technique and the 7-AAD evaluation by flow cytometry. The trypan blue exclusion and the radio frequency techniques were used for automated cell counting. Flow cytometric analysis was performed by evaluating the yielded cellular products for 7-AAD uptake during the cell count of CD34+ cells. Results: The mean values for cell count were estimated as 3.44±1.22x10 6 /ml (range, 2.48-5.71x10 6 /ml) and 4.14±1.94x10 6 /ml (range, 1.77-7.43x106/ml) for the trypan blue exclusion and radio frequency techniques, respectively. Additionally, the mean values for viability analyses via the automated trypan blue exclusion and 7-AAD were 93.38±6.09% (range, 79.00-98.00%) and 99.49±0.60% (range, 98.40-100.00%), respectively. Conclusions: Our study has responded to two fundamental questions: whether the results of both of the automated techniques for cell count correspond with each other, and whether the results of the automated viability assessment conform those of the 7-AAD technique during the manufacturing processes of cellular therapy products intended for clinical use. Even though we have the opportunity to use the hemocytometer in our laboratory setting, the automated trypan blue exclusion technique gives cell count results in concordance within the range of the expectations of our Quality Management System (QMS).