Search

Introduction The diagnosis and treatment of Gastro-intestinal stromal tumours (GISTs) has been revolutionized by molecular pathology and targeted therapy. Description This patient was diagnosed with locally advanced gastric GIST in 2009. He was initially treated neoadjuvantly with imatinib from 2009- 2010. He underwent laparoscopic resection in 2010. Pathology showed almost complete response with only 1.5mm focus of viable tumour. He did not receive adjuvant imatinib as this was not established practice in 2010. Recurrent disease was resected in 2011. Mitotic count was 200/50hpf. Adjuvant imatinib was given for 5 years then discontinued in 2016. Imaging showed no recurrence over this time period. Molecular testing showed Kit Exon 11 mutation- this is common in GISTs and associated with response to imatinib. Recurrent disease was diagnosed 2018 with a 10x9cm mass between residual stomach and liver– he recommenced imatinib with partial response (maximal response was reached in 2020, but a new 3cm lesion was noted) He underwent further resection of the residual stomach and liver segmentectomy in 2020. Histology showed acellular areas of myxoid degeneration, indicating treatment response however viable tumour remained. Sequencing was performed. This showed the expected mutation in exon 11 but also a mutation in exon 13 of KIT- this has been shown recently to confer resistance to imatinib. Discussion Over 90% of GISTs harbour mutations in c-KIT. Recent work has demonstrated that some tumours acquire secondary mutations conferring resistance, following prolonged TKI therapy. Radiological and histopathological features correlate with such events and assist in deciding surgical management.

Purpose Multiple gastrointestinal stromal tumors (GISTs) are rare. The aim of this study was to investigate the clinicopathologic characteristics and prognosis of multiple GISTs. Patients and methods Between May 2003 and June 2018, patients who underwent surgery for multiple GISTs were retrospectively analyzed. Exons 9, 11, 13, and 17 of the KIT gene, and exons 12, and 18 of the PDGFRA gene were examined in 34 tumors from 20 patients. Results A total of 20 patients with multiple GISTs were enrolled. There were 11 females and nine males with a median age of 59 years (range: 37-80 years). Of these cases, 16 were sporadic cases and four were associated with GIST syndromes (two cases of Carney triad and two cases of neurofibromatosis type 1 [NF1]). The most common presentation was gastrointestinal bleeding. Carney triad GISTs did not exhibit KIT/PDGFRA mutations. One of the NF1 patients was a KIT/PDGFRA wild-type, and the other patient had a PDGFRA mutation. Of the sporadic cases, one shared the same KIT gene mutation within each GIST and one had two lesions that were both wild-type for KIT and PDGFRA. Different KIT mutations among individual tumors were detected in seven patients. During the median follow-up period of 66 months (range: 3-183 months), four patients developed liver or abdominal metastases, three of whom expired due to the disease. The rates of recurrence-free survival and overall surviva at 5 years were 65.8% and 76.7%, respectively. Conclusion Multiple GISTs may occur as sporadic tumors or as an additional component of specific syndromes (eg, Carney triad and NF1) that display different clinicopathologic characteristics based on their particular underlying mechanisms. The overall prognosis of patients with multiple GISTs is comparable to that of patients with only a single GIST.

Gastrointestinal stromal tumor (GIST) is a neoplasm derived from the interstitial cells of Cajal. As a group, the tumors show variable clinical presentations, characteristic morphologic features, and distinct molecular signatures. This chapter reviews the most important issues regarding diagnosis, differential diagnosis, tumor classification and risk stratification, and therapeutic implication. It also discusses the most frequently encountered diagnostic challenges and pitfalls in clinical practice.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract; most of them have gain-of-function mutations of the KIT gene. There have been rare cases of families with multiple GISTs, that had autosomal dominant germline KIT mutations. Here, we present a case of multiple GISTs caused by a novel germline KIT mutation. Intraoperatively, the main tumor was present in the body of the stomach, and multiple small nodules were detected mainly in the upper and middle part of the gastric wall; several nodules were also present in the small bowel wall. The main tumor and surrounding nodules were resected. DNA sequencing of the tumor tissue, adjacent normal mucosal tissue, and peripheral blood leukocytes revealed that the patient had germline Asp820Gly mutation in exon 17 of the KIT gene. This is the first case with germline Asp820Gly mutation in exon 17 of the KIT gene.

We established an induced pluripotent stem cell (iPSC) line from peripheral blood mononuclear cells of a Chinese neonate with mastocytosis carrying heterozygous mutation (c.2447A > T (p.D816V)) in KIT gene by episomal vector (EV) reprogramming system. This iPSC line carrying KIT gene mutation, was free of exogenous gene, showed a normal karyotype, expressed pluripotency markers and exhibited differentiation potential.

The discovery of KIT gene mutation in gastrointestinal stromal tumor (GIST) has provided a paradigm shift in the classification, diagnosis and molecular-targeted therapy of gastrointestinal mesenchymal tumors. There is growing evidence of phenotype-genotype (KIT, platelet-derived growth factor receptor-alpha, succinate dehydrogenase or other driver gene mutation) and genotype-therapeutic (sensitivity to imatinib) correlations in GIST. Risk stratification based on mitotic counts, tumor size and rupture is useful for the prognostication and management of patients with GIST. Blood vessel invasion is a strong indicator of liver metastasis in GIST. In addition, novel biomarkers such as cell-cycle regulators, microRNAs and their targets have been discovered by using high throughput molecular analyses. In contrast, leiomyosarcoma of the gastrointestinal tract has become a very rare entity in the 'KIT' era, and its molecular pathogenetic mechanism is unclear. Recent studies have revealed a wide spectrum of cytological atypia, mitotic counts and biological behavior of gastrointestinal smooth muscle tumors, suggesting the necessity of establishing the criteria for malignancy. Collectively, both classical histopathological procedures and modern molecular investigations are indispensable for the evolution of diagnosis and treatment of GIST and mimics.

Introduction C-kit overexpression has previously been described in chromophobe renal cell carcinoma (cpRCC) and renal oncocytoma (RO). However, so far no KIT mutations have been found. The objective of our study was to analyse c-kit in a large cohort of renal tumors and to perform KIT mutation analysis in a subset cpRCC and RO cases with overexpression of c-kit. Materials and methods We studied the immunohistochemical expression of c-kit on tissue microarrays containing formalin-fixed, paraffin-embedded samples of 948 patients with renal tumors. CpRCC and RO cases with c-kit overexpression ( n = 23) were analyzed for KIT mutations in exons 9, 11, 13, 14, 15, and 17. Results Expression of c-kit was found in 6/642 (0.9%) clear cell RCC, 3/154 (1.9%) papillary RCC, 54/69 (78.3%) cpRCC, 37/45 (82.2%) RO and 2/30 (6.7%) of other unclassified tumor types. In none of the RO and cpRCC cases analyzed, a KIT gene mutation was found. Conclusion C-kit expression is found in the majority of cpRCC and RO, but these tumors do not harbor the usual c-kit activating mutations. This may have implications for the use of tyrosine kinase inhibitors in patients with advanced cpRCC and c-kit expression.

In the present study, the features of gastrointestinal stromal tumours (GIST) in patients with neurofibromatosis type 1 (NF1) are analysed, and the fundamental differences between NF1-associated GIST and the common types of GIST are illustrated. We report a case of NF1-associated GIST, and discuss the clinical and pathological data of 71 reported cases from the PubMed/MEDLINE database over the past two decades. The clinical characteristics, histopathology, biological behaviour, immunohistochemistry, phenotype and gene mutation status of GIST in NF1 are carefully reviewed. A total of 72 cases (35 females and 37 males; median age: 53 years) was included in this study. More than 80 per cent of lesions were located in the small intestine, most of which were multicentric (63.89 per cent), and the median diameter of the tumours was 4 cm (range: 0.3–20 cm). The positive expression rates for transmembrane type III receptor tyrosine kinase (KIT), CD34, S-100, smooth muscle actin, and desmin were 100 per cent, 94.34 per cent, 37.5 per cent, 35.9 per cent and 7.41 per cent, respectively. There were just six cases found with the KIT gene mutation; no cases were found with mutations in the platelet-derived growth factor receptor-α (PDGFRA) gene. Patients with NF1 have a high risk of developing GIST. NF1-associated GIST are significantly different from common GIST in terms of clinical characteristics, histopathology, biological behaviour, immunohistochemistry and the mutation status of the KIT and PDGFRA genes. In addition, in this research, we indicate that different pathogenetic mechanisms are involved in their evolution, and that imatinib has limited effects on GIST in NF1 patients.

Purpose: To investigate the presence of KIT gene mutations and immunoreactivity in 85 conjunctival melanocytic tumours and to clarify the role of KIT as a potential therapeutic target in this group of patients. Methods: Eighty-five conjunctival pigmented tumours [27 melanomas, 12 primary acquired melanosis (PAMs) and 46 nevi] were immunostained for KIT. Intensity and pattern of expression were evaluated. Molecular analysis to identify KIT mutations was performed in 15 selected cases (tumour-rich areas >50%). KIT immunostaining score and pattern were statistically related to patients’ age, sex, diagnostic category, presence of relapse, disease-free survival, presence of metastases, metastasis-free survival, limbal versus nonlimbal tumour location and thickness of melanomas. Results: KIT stains were documented in 48% of melanomas, 50% of PAMs and 24% of nevi. The mean score of KIT staining in the melanomas/PAMs group was significantly different from nevi (p = 0.0076). No statistically significant differences were detected between either c-kit immunostaining score or pattern and each of the other clinico-pathologic parameters considered. No KIT gene mutations were detected in melanomas and nevi. A silent mutation/polymorphism in KIT exon 13 was found in one PAM. Conclusions: Despite the high level of KIT immunostains in PAMs and melanomas, this parameter seems not to be a good predictor of the presence of molecular mutations. KIT-activating mutations should be considered an uncommon event in this tumour.

Multiple gastrointestinal stromal tumors (GISTs) caused by germline KIT gene mutations are an extremely rare autosomal dominant disorder. We report a case of a 21-year-old woman who presented to the emergency department with a 2-week history of asthenia, palpitations and upper gastrointestinal bleeding. After further clinical evaluation one gastric and two small bowel GISTs were diagnosed, which were surgically resected after neoadjuvant therapy with Imatinib. Diffuse hyperplasia of the interstitial cells of Cajal was also seen in the background gastric and small intestinal walls. Somatic mutational analysis of the KIT gene revealed a substitution at codon 576 in exon 11 (p.Leu576Pro) in all tumors and normal ileal mucosa. The germline nature of this mutation was confirmed by mutation analysis in peripheral blood leukocytes. However, she had no familial history of GISTs and her parents did not carry the respective germline mutation.

Background Nilotinib inhibits the tyrosine kinase activities of ABL1/BCR-ABL1, KIT, and platelet-derived growth factor receptors (PDGFRs). The results of a phase III clinical trial indicated that nilotinib could not be recommended for broad use as first-line therapy for gastrointestinal stromal tumor (GIST). However, some clinical studies have reported the effectiveness of nilotinib. We report here the cases of two patients who underwent surgical resections of nilotinib-resistant lesions after long-term nilotinib administration. Case presentation Two Japanese female patients, aged 66 and 70 years, experienced peritoneal recurrence of intestinal GIST several years after surgery. Both were registered in the ENESTg1 trial and received nilotinib therapy. Although they continued nilotinib administration with a partial response according to the protocol, nilotinib-resistant lesions, which were diagnosed as focally progressive disease, developed and complete surgical resection was performed. Pathological examination revealed that the tumors were composed of viable KIT-positive spindle cells, and the recurrent tumors were diagnosed as nilotinib-resistant GIST. In gene mutation analysis, a secondary KIT gene mutation was detected in one case. Both patients have survived more than 5 years after the first surgery. Conclusions Of patients who were registered in this trial, we have encountered two patients with long-term effects after nilotinib administration. Moreover, secondary mutations in the KIT gene, similar to those involved in resistance to imatinib, might be involved in resistance to nilotinib.

We screened for natural mutations in Crl:CF1 closed colony mice using an ordinary backcrossing system. Five of 30 CF1 males carried novel genes that caused white spots on colored coats. Their backcross progenies showed a white spot phenotype. The white spot gene was mapped to approximately 39 cM on chromosome 5, where the Kit gene is known to reside. Allelism testing between this spot gene and the Kit gene was performed using two already known Kit alleles, Kit(W), and Kit(W-v). We demonstrated that the spot mutation was semidominant and a novel allele of the Kit gene, which was tentatively named Kit(W-Ham). No infertility or anemia was observed in Kit(W-Ham) homozygotes. However, a reduced number of germ cells and mast cells was observed in Kit(W-Ham)/Kit(W) and Kit(W-Ham)/Kit(W-v) transheterozygotes. Sequencing of the 21 exons of the Kit gene in the Kit(W-Ham) mutants revealed that a unique guanine-to-adenine (G-A) transition at nucleotide position 545 (c.545G>A) of exon 3 changes arginine (R) to glutamine (Q) at position 182 in the extracellular domain of the KIT protein (p.R182Q). This extracellular KIT domain is a binding site for stem cell factors (SCF). It was concluded that the Kit(W-Ham) mutant may serve as a new model of human piebaldism.

J Oral Pathol Med (2012) 41: 415–423 Background: While overexpression of KIT protein has been well documented in adenoid cystic carcinomas (ACCs), mutation of KIT gene has been a controversial issue. We wanted to evaluate clinical value of the KIT mutation and protein expression in ACC. Methods: We analyzed 33 cases of ACC. Gene mutations in KIT exons 9, 11, 13, and 17 were analyzed using paraffin-embedded tissue, and two different sets of primers with direct sequencing after polymerase chain reaction (PCR) for exon 9, 11, 13, and 17, and cloning of PCR products for exon 11. KIT protein expression was assessed by immunohistochemistry. The correlation between clinicopathological findings and these biomarkers was analyzed. Results: No KIT mutation was observed in all of the 33 cases. With one primer set, KIT mutation was found in nine of 33 cases (27.3%). However, these mutations were not reproducible in the experiment using another primer set. KIT protein overexpression was detected in 22 of 33 patients (66.7%). KIT protein expression was not statistically correlated with either clinicopathological factors or survival. Patients with metastasis showed a tendency of longer progression-free survival (P = 0.052) and overall survival (P = 0.080) when the tumor overexpressed KIT protein. Conclusion: This study supports that mutational study using paraffin-embedded tissue should be interpreted with great caution. KIT gene mutation is very rare in ACC, and gene mutation is not the cause of protein overexpression. KIT protein expression may have a potential value for better prognostic factor in patients with metastasis.

Background: Mastocytosis is a myeloproliferative neoplasm characterized by the excessive proliferation of mast cells. Mast cell leukaemia (MCL), the aggressive form of this disease, requires cytoreductive therapy, such as cladribine, interferon-alpha-2b and, most recently, tyrosine kinase inhibitors – dasatinib or imatinib. Patient and methods: We present a case of a 56-yr-old female patient with aleukaemic MCL in whom the typical KIT-D816V mutation was not detected. Sequencing of the entire coding sequence of KIT gene revealed a somatic mutation in exon 9 (p.A502_Y503dup). This mutation was previously reported in patients with gastrointestinal stromal tumours (GIST). Considering the good response to imatinib in such patients, therapy with imatinib was attempted in our patient. The treatment tolerance and outcomes were very good, with reduced mast cell infiltration of the bone marrow, normalization of the serum tryptase concentration and resolution of the clinical signs and symptoms. Conclusions: In the absence of the KIT-D816V in systemic forms of mast cell proliferation, a search for other mutations is indicated, preferably by sequencing the entire KIT gene, as this can influence the choice of treatment. The finding of the p.A502_Y503dup in exon 9, a mutation which has been observed in GIST but not previously reported in any form of aggressive mastocytosis, can be associated with a good response to imatinib in both diseases.

Introduction Gastrointestinal stromal tumours (GIST) are mesenchymal neoplasms that usually carry an activating mutation in KIT or platelet-derived growth factor receptor alpha ( PDGFRA ) genes with predictive and prognostic significance. We investigated the extended mutational status of GIST in a patient population of north-western Greece in order to look at geopraphic/genotypic distinctive traits. Patient and methods Clinicopathological and molecular data of 38 patients diagnosed from 1996 to 2016 with GIST in the region of Epirus in Greece were retrospectively assessed. Formalin-fixed paraffin-embedded tumours were successfully analysed for mutations in 54 genes with oncogenic potential. Next generation sequencing was conducted by using the Ion AmpliSeqCancer Hotspot Panel V.2 for DNA analysis (Thermofisher Scientific). Results Among 38 tumours, 24 (63.16%) and seven (18.42%) of the tumours harboured mutations in the KIT and PDGFRA genes, respectively, while seven (18.42%) tumours were negative for either KIT or PDGFRA mutation. No mutations were detected in five (13.16%) cases. Concomitant mutations of BRAF and fibroblast growth factor receptor 3 ( FGFR3 ) genes were observed in two patients with KIT gene mutation. Two patients with KIT / PDGFRA wild-type GIST had mutations in either KRAS or phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha ( PIK3CA ) genes. There was no significant survival difference regarding the exonic site of mutation in either KIT or PDGFRA gene. The presence of a mutation in pathway effectors downstream of KIT or PDGFRA , such as BRAF , KRAS or PIK3CA , was associated with poor prognosis. Adverse prognosticators were also high mitotic index and the advanced disease status at diagnosis. Conclusions We report comparable incidence of KIT and PDGFRA mutation in patients with GIST from north-western Greece as compared with cohorts from other regions. Interestingly, we identified rare mutations on RAS , BRAF and PIK3CA genes in patients with poor prognosis.

Journal of Thoracic Oncology • Volume 8, Number 10, October 2013 CASE REPORT A 46-year-old never-smoker man presented to the emergency room with a 4-day history of progressive cough and right-side chest wall pain in October 2011. A computed tomography scan of his chest showed large anterior mediastinal and right pleural-based masses associated with pleural effusion. The tumor was confirmed to be squamous cell carcinoma by percutaneous gun biopsy of the right pleural-based mass (Fig. 1A). The tumor cells expressed KIT (Fig. 1B) and p63 proteins, and the patient was diagnosed with thymic cancer. Even after two cycles of paclitaxel/cisplatin chemotherapy and a subsequent line of cyclophosphamide/doxorubicin/cisplatin chemotherapy, his pleural effusion increased and the primary anterior mediastinal mass progressed. He required frequent thoracentesis to relieve dyspnea. At that time, we evaluated whether the initially biopsied tumor specimen harbored KIT mutation, and identified a mutation in exon 11 (loss of aspartic acid at position 579: D579del). After administration of imatinib (400 mg daily) since December 2011, the tumorous lesion has decreased to the range of partial response by the Response Evaluation Criteria

Objectives and background: According to the World Health Organization (WHO) classification, the diagnosis of Systemic Mastocytosis (SM) relies on bone marrow (BM) examination and is based on a major and four minor criteria. The somatic ‘autoactivating’ point mutation D816V in the KIT receptor gene is one of the minor criteria, founded in the great majority of patients (90%) and it plays a central role in the pathogenesis of the disease. Indolent Systemic Mastocytosis (ISM) is the most common variant of SM, characterized by a very low MC burden and associated with very different clinical pictures. A highly sensitive diagnostic methods for D816V detection are required to assure an appropriate diagnosis and to reduce false-negative results. The recent development of “ultra-deep amplicon sequencing” (UDS) technologies has opened the way to a more accurate characterization of molecular aberrations with higher sensitivity of screening for known and unknown mutations. Our aims were: i) to set-up and optimize a UDS-based mutation screening strategy of the KIT gene on the Roche GS Junior Instrument; ii) to test the sensitivity of our UDS assay to detect the D816V mutation; iii) to investigate the presence of additional KIT mutations in SM. Methods: We decided to take advantage of a next generation sequencing approach to perform an UDS KIT gene mutation analysis on 20 bone marrow (BM) samples from patients whit ISM that were negative for the D816V mutation by Sanger Sequencing which has a sensitivity of 20%. Fusion primers were designed to generate ten partially overlapping amplicon covering the whole KIT transcript (exons 1-21) by RT-PCR. To determine the lower detection limit of our UDS-assay, serial dilutions of the HMC-1 cell line (harboring the D816V mutation) into an unmutated K562 cell line in ratios such as to simulate the following mutation loads were sequenced: 50%, 37.5%, 25%, 12.5%, 5%; 2.5%, 1.25%, 0.5%, 0.25%. Results and significance: UDS of cell line dilutions showed a high accuracy of D816V mutation detection and linearity of mutation calling over the entire range down to 0.25%. The UDS technology allowed to detected the D816V mutation, below the lower detection limit of Sanger Sequencing, with an abundance from 0.5% to 11%, in 12/20 ISM patients. Two additional sequence variations were detected in a large proportion of patients. These two variations included a 3bp in-frame deletion in exon 15 (GenBank X06182.1: c.2164_2166delAGC; p.S715del) found in 11/20 patients and a 12bp in frame-deletion in exon 9 in all patients, whit an abundance ranging from 83% to 97% (GenBank X06182.1: c.1550_1561delGTAACAACAAAG; p.G510_K513del). Previously published studies indicate that the KIT Gly-Asn-Asn-Lys510-513+/- alternatively spliced located immediately downstream to the extracellular KIT domain and KIT Ser715+/-, an interkinase KIT domain, are expressed in normal human hematopoietic cell, leukemic cell lines, acute myeloid leukemia blast and GISTs and represent rather a splice variant of KIT transcript. Interestingly our results showed the presence of the transmembrane domain M541L (GenBank X06182.1: c.1642A>C; p.Met541Leu) KIT-activating mutation in exon 10, with an abundance of 50%, in addition to D816V, in 2/20 ISM. This mutation is known to retain sensitivity to imatinib mesylate. Conclusions: Our preliminary results suggest that our-UDS based KIT gene mutation screening assay might be a reliable and sensitive alternative to conventional sequencing methods for the detection of the D816V. We are now planning to investigate whether the greater sensitivity of UDS allows to detect the D816V mutation in peripheral blood mononuclear cells from patients with a suspected clonal mast cell disorder. These results could represent a starting point to plan other extensive studies to better understand the exact role of KIT receptor alterations in SM. Supported by ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. Disclosures Cavo: Celgene: Consultancy, Honoraria, Speakers Bureau. Martinelli:Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy; ARIAD: Consultancy.

Sir, mesenchymal tumors of the pancreas are exceedingly rare, accounting for less than 1% of all pancreatic tumors, and most of them have been reported in small series or as single case reports. This group of pancreatic tumors includes leiomyosarcoma, malignant peripheral nerve sheath tumor, malignant fibrous histiocytoma, liposarcoma, rhabdomyosarcoma,hemangiopericytoma,schwannomaandsolitary fibrous tumor. In the gut, mesenchymal tumors are more common, and the majority of these are formed by gastrointestinal stromal tumors (GISTs). GISTs are defined as KIT-positive mesenchymal spindle cell or epithelioid neoplasms showing differentiation toward the interstitial cell of Cajal. GISTs occur most commonly in the stomach (60–70%), small intestine (20–25%), colorectum (5%) and esophagus (

Summary Gain-of-function mutations involving c-kit protein, a cell-surface transmembrane receptor for stem cell factor, have been identified as a key oncogenic driver in a variety of solid tumours. Coupled with the development of tyrosine kinase inhibitors such as imatinib, c-kit has emerged as a viable drug target in what seems to be a validated therapeutic concept. This review will focus on gastrointestinal stromal tumours and melanomas, two types of solid tumours most closely associated with KIT gene mutations. The biology of KIT mutations in both conditions, as well as the value of KIT mutation testing in predicting disease and treatment outcomes are discussed. Since initial response to imatinib is largely influenced by mutation status, genotyping these tumours serves to facilitate personalised oncology. We also summarise our experience with diagnostic reporting of KIT mutation analysis over a period of 3 years, and briefly survey future developments in treatment, which indeed look very promising.

Background: Core binding factor acute myeloid leukemia (CBF-AML) is a form of AML associated with the chromosomal abnormalities t(8;21)(q22;q22) (t(8;21)AML) and inv(16)(p13.1q22)/t(16;16)(p13.1;q22) (inv16 AML). It accounts for approximately 8% of AML cases and is considered to be a karyotype with a favorable prognosis. Mutations of c-kit gene which constitute type III tyrosine kinase receptor were found in approximately 30% of patients with CBF-AML. In CBF-AML, KIT mutation is observed mainly in two domains: the extracellular domain (exon8), and the tyrosine kinase domain (exon17), and has frequently been reported to be an unfavorable prognostic factor for early relapse and for shorter survival. On the other hand, some reports conclude that KIT mutation has no prognostic impact in CBF-AML, and in 2016's guidelines of NCCN, CBF-AML with KIT gene mutation was not assigned to the intermediate prognosis group. One potential reason why the reported prognostic impact of KIT mutation in CBF-AML differs between studies is that CBF-AML cases with t(8;21) and those with inv(16) have differing clinical profiles. Another reason is that KIT mutation occurs at various locations on the gene. We have previously reported that KIT D816 in CBF-AML is associated with a higher relapse rate than KIT N822K and has unfavorable prognosis. The aim of the present study was to clarify that these two types of KIT mutations have differing prognostic impacts in CBF-AML. Methods: We analyzed 138 cases of CBF-AML who achieved complete remission (CR), retrospectively. Mutation analyses were performed by direct sequence analysis Mutation Biased PCR, direct sequence analysis, and the next-generation sequencer Ion PGMTM. Results: Average age was 45 years (15-80 years). The inv16 AMLand t(8;21) AMLwere28 cases and 110 cases, respectively. KIT mutations were found in 62 (45%) cases of patients with CBF-AML. Patients with KIT mutations were significantly more frequently associated with male gender (p=0.029) and higher white blood cell count (>1×104/μl) (p=0.002) compared to KIT wild type cases. No significant differences were found in other clinical characteristics between those with and without KIT mutations. Analyzing all CBF-AML patients and inv16 AML, rates of relapse free survival (RFS) and overall survival (OS) in those with KIT mutations were significantly lower than those in patients without c-kit mutations (All patients: RFS, 38% vs 58% at 3 years after CR1, p=0.040; OS, 61% vs 77%, p=0.033; inv16 AML: RFS, 46% vs 82%, p=0.044; OS, 75% vs 100%, p=0.045). However, there was no significant difference between t(8;21)AML with and without KIT mutations (RFS, 35% vs 51%, p=0.219, OS, 58% vs 70%, p=0.161). Next, we analyzed prognosis of CBF-AML according to the types of c-kit mutations. D816, N822K, co-expression of D816 and N822K, and other mutations of KIT gene were detected in 29 cases (21%, D816A:1 case, D816Y:2 cases, D816V:26 cases), 20 (14%), 7 (5%, D816H:1 case, D816V:6 cases), and 6 cases (4%), respectively. (RFS, 58.2% vs 21.6% vs 58.9% vs 14.3% vs 60.0%, p=0.005; OS,77.2% vs 40.0% vs 78.9% vs 60.0% vs 100.0%, p Conclusions: In ASH meeting of 2014, we showed that the D816V confers higher proliferation activity by JAK-STAT and Src family kinase compared to N822K by in vitro assay. In this study, we showed that patients with D816 had a significantly poorer prognosis than those with N822K or other mutations of KIT in CBF-AML. Going forward, a study is needed in which a trial of autologous stem cell transplantation in first CR or conventional chemotherapy with dasatinib or novel molecular target drug such as PKC 412 for CBF-AML with D816. Disclosures No relevant conflicts of interest to declare.

Background:Imatinib therapy has been successful in gastrointestinal stromal tumours containing mutation of the KIT gene. However, there are few reported cases of successful imatinib therapy in patients with melanoma containing KIT gene mutation or c-kit protein expression.Methods and results:A 52-year-old man developed metastatic melanoma from a primary melanoma in the left side of the nasopharynx. The tumour was positive for c-kit protein, and there was a KIT mutation in exon 11. He was treated with imatinib. A follow-up scan one year later showed a complete response. Treatment targeting the biological characteristics of melanoma proved successful in this patient.

Core binding factor (CBF)-positive acute myeloid leukemia (AML) presents a favorable prognosis, except for patients with KIT mutation, especially D816 mutation. The current retrospective study attempted to validate a prognostic role of KIT mutation in 121 Korean patients with CBF AML. The study patients consisted of 121 patients with CBF AML (82 patients with RUNX1/RUNX1T1 [67.8 %] and 39 patients with CBFB/MYH11 [32.2 %]) recruited from eight institutions in Korea. All patients received idarubicin plus cytarabine or behenoyl cytosine arabinoside 3 + 7 induction chemotherapy. The KIT gene mutation status was determined by direct sequencing analyses. A KIT mutation was detected in 32 cases (26.4 %) in our series of patients. The KIT mutation was most frequent in exon 17 (n = 18, 14.9 %; n = 16 with D816 mutation), followed by exon 8 (n = 10, 8.3 %). The presence of KIT D816 mutation was associated with adverse outcomes for the event-free survival (p = 0.03) and for the overall survival (p = 0.02). The unfavorable impact of D816 mutation was more prominent when the analysis was confined to the RUNX1/RUNX1T1 subtype. The KIT mutation was detected in 26.4 % of Korean patients with CBF AML. The KIT D816 mutation demonstrated an unfavorable prognostic implication, particularly in the RUNX1/RUNX1T1 subtype.

Submucosal tumors (SMTs) of the gastrointestinal (GI) tract can be potentially difficulty to diagnose pathologically. We report a case of a gastric SMT that was resected by laparoscopic partial gastrectomy. Although the initial histological and immunohistochemical examinations considered the tumor as a schwannoma, mRNA-based KIT genotyping indicated that the tumor included cells with KIT gene expression, and that a small number of cells carried a deletion mutation in exon 11. Additional histopathological investigations revealed small aggregates of enlarged spindle to epithelioid cells, which were positive for KIT, CD34 and DOG1, and negative for S-100, scattered among the S-100-positive schwannoma cells. We consider that the cells carrying the KIT gene mutation are microscopic buds of a gastrointestinal stroma tumor (GIST), and to the best of our knowledge, this is the first report of probable GIST tissues identified in a schwannoma. Our observations raised the significance of genotyping for diagnosis of GI tract SMTs.

BACKGROUND: Dysgerminoma, the ovarian counterpart of seminoma, is the most common type of malignant ovarian germ cell tumor. The role of KIT mutation and amplification in the development of dysgerminoma is not currently established. The purpose of this study was to analyze alterations of the KIT gene in a large series of dysgerminomas and correlate the findings with clinicopathological parameters. METHODS: Dysgerminoma cells from 22 patients were analyzed for KIT mutations at exon 17 codon 816. KIT amplification and chromosome 12p anomalies were investigated by way of dual color fluorescence in situ hybridization. KIT protein expression was also examined by way of immunohistochemistry. RESULTS: KIT exon 17 codon 816 mutations and KIT amplification were each detected in 6 cases of dysgerminoma (27%); however, there was no correlation between these 2 factors. KIT expression was detected in 87% of dysgerminomas. The KIT mutation was associated with advanced pathological stage (P < .05), and KIT amplification was associated with elevated KIT protein expression (P < .05). Chromosome 12p anomalies were found in 82% of the dysgerminomas and did not correlate with KIT abnormalities. CONCLUSIONS: KIT mutations occur in approximately one-third of cases of dysgerminomas and are associated with advanced stage at presentation. KIT is a potential therapeutic target for those dysgerminomas that have the mutation. Cancer 2011. © 2010 American Cancer Society.

Piebaldism is an autosomal dominant genetic disorder characterized by cogenital patches of skin and hair from which melanocytes are completely absent. A similar disorder of mouse, dominant white spotting (W), results from mutations of the c-Kit protooncogene, which encodes and receptor for mast/stem cell growth factor. We identified a KIT gene mutation in a proband with classic autosomal dominant piebaldism. This mutation results in a Gly----Arg substitution at codon 664, within the tyrosine kinase domain. This substitution was not seen in any normal individuals and was completely linked to the piebald phenotype in the proband's family. Piebaldism in this family thus appears to be the human homologue to dominant white spotting (W) of the mouse.

Recent data suggested an increased frequency of KIT aberrations in mucosal melanomas, whereas c-KIT in most types of cutaneous melanomas does not appear to be of pathogenetic importance. However, studies investigating the status of the KIT gene in larger, well-characterised groups of patients with mucosal melanomas are lacking. We analysed 44 archival specimens of 39 well-characterised patients with mucosal melanomas of different locations. c-KIT protein expression was determined by immunhistochemistry, KIT gene mutations were analysed by PCR amplification and DNA sequencing of exons 9, 11, 13, 17 and 18. c-KIT protein expression could be shown in 40 out of 44 (91%) tumours in at least 10% of tumour cells. DNA sequence analysis of the KIT was successfully performed in 37 patients. In 6 out of 37 patients (16%) KIT mutations were found, five in exon 11 and one in exon 18. The presence of mutations in exon 11 correlated with a significant stronger immunohistochemical expression of c-KIT protein (P=0.015). Among the six patients with mutations, in two patients the primary tumour was located in the head/neck region, in three patients in the genitourinary tract and in one patient in the anal/rectal area. In conclusion, KIT mutations can be found in a subset of patients with mucosal melanomas irrespective of the location of the primary tumour. Our data encourage therapeutic attempts with tyrosine kinase inhibitors blocking c-KIT in these patients.

Aggressive systemic mastocytosis (ASM) is a very rare form of mast cell neoplasm that does not benefit from conventional chemotherapy. The majority of adult mast cell neoplasms and gastrointestinal stromal tumors (GISTs) have mutations in the proto-oncogene c-kit, which encodes the KIT receptor tyrosine kinase. The c-kit gene mutations are generally confined to the tyrosine kinase II domain in mast cell neoplasms, but are often observed at the juxtamembrane domain in GISTs. We found a case of ASM with a juxtamembrane-type mutation, Val559Ile, and in this report the mutation was characterized through transfection of the mutated c-kit cDNA into human embryonic kidney cells. Phosphorylation of KIT and its possible downstream signaling molecules were examined in the presence or absence of imatinib, a selective tyrosine kinase inhibitor. Ligand-independent autophosphorylation was observed in the mutant KIT with Val559Ile as well as that with Val559Asp, as found in GISTs. Imatinib, at a concentration of 10 microM, inhibited autophosphorylation of the mutant KIT with Val559Asp, but not that with the Val559Ile. Phosphorylation of MAPK and STAT5 was also inhibited by imatinib at the same concentration, in cells expressing Val559Asp but not in those expressing Val559Ile. These results suggest that different mutations, even at the same codon, in juxtamembrane domain of the c-kit gene show different inhibitory effects of imatinib, and that patients with GISTs or mast cell neoplasms possessing this Val559Ile mutation are resistant to imatinib therapy.

Background and Aims: The term Systemic Mastocytosis (SM) identifies a poorly understood group of rare and clinically heterogenous myeloproliferative neoplasms characterized by abnormal growth and activation of mast cells (MCs) and their precursors in the bone marrow and in various tissues and organs. Based on phenotype and extent of organ infiltration/dysfunction, a spectrum of disease variants can be recognized ranging from indolent SM (ISM) to aggressive SM (ASM) and mast cell leukemia (MCL). The fact that in all cases, including ISM who have a (near) normal life expectancy, neoplastic MCs display the same D816V KIT gene mutation points to additional mechanisms and molecular defects as responsible for ASM and MCL. So far, however, this issue has mainly been addressed with targeted resequencing studies of candidate gene panels. We thus decided to undertake an integrated molecular characterization study of ASM and MCL to identify novel, functionally relevant molecular lesions and/or clinically actionable signaling pathways. Methods: A discovery panel including 6 patients with ASM and 6 patients with MCL was studied using whole exome sequencing (WES) and copy number variation (CNV) analysis. WES (80x) was performed on a Hiseq 2500 (Illumina). CNV was done using Cytoscan HD Arrays (Affymetrix). Paired normal/MC DNA was analyzed in all but 2 archival MCL cases for whom germline DNA was not available. A validation panel of 30 ISM, 5 smoldering SM and 20 additional ASM was also included in this study. Results: In the discovery panel, WES identified a total of 1554 point mutations, small insertions and deletions. Seven hundred and eighty-five were non-silent mutations in 698 genes, with an average of 51 (range, 30-186) non-silent mutations per patient. Non-silent mutations included 354 missense mutations, 188 nonsense mutations, 145 frameshift insertions/deletions, 98 non-frameshift insertions/deletions. C to T transitions were by far the most frequent. Orthogonal validation estimated the accuracy of mutation calls at >95%. Interrogation of the COSMIC and OMIM databases revealed 42 known cancer genes. Among the missense mutations, 87 were predicted to have a high probability of being deleterious by Condel. MCL cases were found not to harbour a higher mutation load as compared to ASM cases. High resolution CN analysis showed that focal amplifications/deletions/loss-of-heterozygosity (LOH) were prevalent over arm-level alterations (found in 3 patients only). Genes were selected for further assessment when recurrently mutated in ≥2 patients or concurrently identified in WES and CNV analyses or previously associated with leukemogenesis or cancer pathogenesis. Among these, genes already reported to be affected by mutations in SM included TET2, NRAS, ASXL1, CBL, IDH1, SRSF2, SF3B1, RUNX1. We also identified genetic alterations in genes not previously implicated in SM pathogenesis including TP53BP1, RUNX3, NCOR2, CDC27, CCND3, EI24, MLL3, ARID1B, ARID3B, ARID4A, SETD1A, SETD1B, KDM1B, PRDM1, ATM, WRN. A long tail of infrequently mutated genes dominated, resulting in significant intertumoural heterogeneity. However, when genes were assigned to functional pathways to discern patterns of mutations across different patients, we found that PI3K/Akt and MAPK pathways, calcium pathway, chromatin modification, DNA methylation, and DNA damage repair were consistently affected (Figure 1). Further assessment of the mutation frequency of selected genes within each pathway and functional validation at the protein level are currently ongoing in the validation panel. Preliminary findings on a tumor suppressor selected among those identified by WES show transcript and/or protein downmodulation due to inactivating mutations, transcriptional silencing or enhanced degradation in 17/20 ASM. Detailed results will be presented at the meeting. Conclusions: WES and CNV analyses of ASM and MCL revealed a complex landscape, not unexpected when considering the clinical heterogeneity of these patients. Nonetheless, key pathways were found to be recurrently altered. Further investigation of selected candidate genes and pathways is warranted and will cast light on the cooperative genetic (and epigenetic?) events underlying the more aggressive forms of SM - paving the way to a better prognostic stratification and more effective treatment. <>This study was supported by ELN, AIL, AIRC, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. Disclosures Soverini: Ariad: Consultancy; Bristol-Myers Squibb: Consultancy; Novartis: Consultancy. Valent:Novartis: Consultancy, Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria; Pfizer: Honoraria; Celgene: Honoraria. Cavo:Janssen-Cilag, Celgene, Amgen, BMS: Honoraria. Martinelli:Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; ROCHE: Consultancy; Pfizer: Consultancy; Ariad: Consultancy; AMGEN: Consultancy; MSD: Consultancy.

In a South African girl of Xhosa stock with severe piebaldism and profound congenital sensorineural deafness we identified a novel missense substitution at a highly conserved residue in the intracellular kinase domain of the KIT proto-oncogene, R796G. Though auditory anomalies have been observed in mice with dominant white spotting (W) due to KIT mutations, deafness is not typical in human piebaldism. Thus, the occurrence of sensorineural deafness in this patient extends considerably the phenotypic range of piebaldism due to KIT gene mutation in humans and tightens the clinical similarity between piebaldism and the various forms of Waardenburg syndrome.

Aims : Imatinib mesylate specifically inhibits KIT tyrosine kinase activity, and has been proven to be effective in the treatment of gastrointestinal stromal tumours. Because other KIT-expressing malignancies might benefit from Imatinib therapy, we evaluated the distribution and expression of KIT in 1166 cases of malignant lymphoma. Materials and results : Tissue microarrays (TMAs) containing 824 non-Hodgkin's lymphoma (NHL) and 342 Hodgkin's lymphoma (HL) cases were immunohistochemically analysed for the expression of the KIT protein. Two KIT-positive NHLs were sequenced using polymerase chain reaction analysis. One T-cell lymphoma and one follicular lymphoma of the 747 NHL cases (0.3%) were positive for KIT. All HLs were Kit-negative. None of the KIT-positive cases showed a kit gene mutation. Conclusions : KIT expression is a very rare event in NHL and virtually absent in HL. In the few positive cases, the aberrant expression is not caused by a mutation in the ‘hot-spots’ of the kit gene, indicating that treatment of these tumours with Imatinib may be ineffective.

Human piebaldism is a rare autosomal dominant disorder characterized by congenital depigmented patches of skin and hair. Piebaldism results from mutations of the kit proto-oncogene, which encodes a cell-surface receptor, tyrosine kinase, whose ligand is the stem/mast cell growth factor. We report four unrelated patients with piebaldism and consider the variations in phenotype in relation to the site of the kit gene mutation.

Abstract 1477 Introduction: When compared to older patients, infants with acute leukemia exhibit distinct cytogenetic features, such as higher prevalence of MLL gene rearrangement (MLL-R), and are known to have higher vulnerability to intensive cytotoxic therapy, such as hematopoietic stem cell transplantation. In contrast to acute lymphoblastic leukemia (ALL), there have been few reports on acute myeloid leukemia (AML) in infants. To develop more appropriate therapeutic strategies for infants with AML, it is necessary to elucidate the distinct clinical features of this subgroup. We therefore performed a retrospective analysis on infant AML in Japan. Patients: Infants with AML, aged less than 1 year at diagnosis, registered in any of the 6 Japanese AML clinical trials between 1991 and 2010 (TCCSG M91-13, TCCSG M96-14, AML99, CCLSG9805, CCLSG9805RE, and JPLSG AML-05) were included in this study. Patients with Down syndrome were excluded. Results: A total of 122 infant AML patients were included in the present analysis, which comprised approximately 10% of all pediatric AML patients. The most frequent FAB classification type was M5 (28.7%), followed by M7 (22.9%) and M4 (10.8%). About 30% of patients had 11q23 abnormalities/MLL -R, but there was no impact on prognosis. Several cases with normal karyotype were revealed to be MLL -R on FISH analysis or on MLL -fusion chimeric transcript analysis by RT-PCR. t(8;21), inv(16) and t(15;17) cases were very rare among the infant cohorts. Furthermore, 7.8% had t(1;22)(p13;q13), and 2.5% had t(7;12)(q36;p13). Genetic mutation results could be obtained in 11 cases in the AML99 study; only one case each was confirmed to have NRAS, KRAS or KIT gene mutation. No cases with FLT3-ITD were detected among the 11 cases in the AML99 or the 44 cases in the AML-05 study. Survival rate varied based on treatment received; 5-year OS rate was 58.3% to 71.4%, and 5-year EFS rate was 49.4% to 64.2%. Discussion: Survival rate in infant AML was identical to that in older pediatric AML. However, there was a possible underestimation of MLL -R patients based on sole chromosome analysis; the prevalence of MLL -R was less than 50% in infant AML patients, without any prognostic impact. Other well-known genetic alterations in pediatric AML also had no effect on outcome of infant AML. Infant AML is a heterogeneous subgroup of pediatric AML, and further studies, as well as novel biomarkers, will be necessary to fully understand its biology. Disclosures: No relevant conflicts of interest to declare.