C-kit Mutation Research Articles

OrienX010 oncolytic viral therapy in phase Ic trial of intralesional injection in liver metastases among patients with stage IV melanoma after standard treatment.

e21013 Background: The liver is one of the most common metastases in melanoma pts and associated with extremely poor prognosis. However, effective treatment has not been found. The safety and efficacy of OrienX010, a herpes simplex virus type 1-derived oncolytic immunotherapy with expression of gene encoding human GM-CSF, was tested in phaseⅠc trial of intralesional injection in liver metastases among patients with stage IV melanoma after standard treatment. Methods: We studied safety and efficacy of OrienX010 (8×107pfu/ml, 10ml per injection) q2w by ultrasound guided puncture. Efficacy was evaluated q8w by CT scan. The primary endpoint was toxicity. Secondary endpoints included Objective Response Rate (ORR), Disease Control Rate (DCR) as well as PFS time. Treatment continued until intolerance, or disease progression per Immune Related Response Criteria. Results: From May 2016, 15 pts received OrienX010 intralesional therapy. The median age was 47 y (32, 61), and 53.3% were female. The primary anatomic sites were: 40.0% (6 pts) from ocular, 33.3% (5 pts) from acral, 26.7% (4 pts) from mucosal. Only 1 patient with c-Kit mutation, 14 pts were wide-type in genotype. All pts had received at least one prior therapy, 53.3% (8 pts) had received transcatheter hepatic arterial chemoembolization. 73.3% (11 pts) had multiple liver metastases, 60.0% (9 pts) had extrahepatic metastases such as lung, bone, abdominal, distant lymph node. 60.0% (9 pts) had an elevation of serum LDH. Mean injection times were 6 (4-18). Mean size of all injectable lesions was 30.4mm (10.0-59.0). AEs were all grade 1/2, pyrexia 80.0%, fatigue 33.3%, injection site pain 26.7%, nausea/vomiting 20.0%, hepatotoxicity 20.0%, leucopenia 6.7%. 12 pts were eligible for evaluation till Jan 2017, and the median follow-up time was 6.0 months. ORR was 8.3% (1 PR), DCR was 41.7% (1 PR, 4 SD). Time to response was 8-16 wks, median PFS was 13.3 wks (95%CI 8.3-18.4), OS was not reached. Conclusions: This is the first trial for evaluating OrienX010 of intralesional injection in liver metastases among melanomas. It appears to be tolerable and with a potentially beneficial effect. Phase 2 and combination trial are pending.

The HMGCR inhibitor Fluvastatin induces apoptosis and autophagy in primary and neoplastic mast cells

Abstract Mast cells are best known for their role in inflammatory and allergic disease. Mast cell leukemia is a rare subtype of acute myelogenous leukemia, typically expressing the c-Kit mutation, D816V, that yields factor-independent growth. Understanding and treating mast cell hyperplasia and leukemia could be beneficial to both inflammatory and malignant diseases. Recently the family of statin drugs, widely employed as HMG CoA Reductase (HMGCR) inhibitors to lower serum cholesterol, have also been found to be immunosuppressive. However, statin effects on mast cell survival are not known. We report that Fluvastatin induces apoptosis in mouse bone marrow-derived mast cells (BMMC) and P815 mastocytoma cells in a dose- and time-dependent manner. Apoptosis was evidenced by DNA fragmentation and caspase activation. Importantly, the mastocytoma line P815, which expresses mutant c-Kit, was readily killed by Fluvastatin, with an IC50 of 5–10μM. P815 cell death was marked by c-Kit upregulation preceding mitochondrial membrane instability and Caspase-9 activation. Chemical blockade of autophagy reduced Fluvastatin-mediated apoptosis in primary mast cells, but augmented apoptosis in transformed mast cells. These data suggest that the HMGCR pathway may be targeted to suppress mast cell survival, and that its combination with autophagy inhibitors may be useful for treating mast cell neoplasias.

Differences in the Importance of Mast Cells, Basophils, IgE, and IgG versus That of CD4+ T Cells and ILC2 Cells in Primary and Secondary Immunity to Strongyloides venezuelensis.

There is evidence that mast cells, basophils, and IgE can contribute to immune responses to parasites; however, the relative levels of importance of these effector elements in parasite immunity are not fully understood. Previous work in Il3-deficient and c-kit mutant KitW/W-v mice indicated that interleukin-3 and c-Kit contribute to expulsion of the intestinal nematode Strongyloides venezuelensis during primary infection. Our findings in mast cell-deficient KitW-sh/W-sh mice and two types of mast cell-deficient mice that have normal c-kit ("Hello Kitty" and MasTRECK mice) confirmed prior work in KitW/W-v mice that suggested that mast cells play an important role in S. venezuelensis egg clearance in primary infections. We also assessed a possible contribution of basophils in immune responses to S. venezuelensis By immunohistochemistry, we found that numbers of basophils and mast cells were markedly increased in the jejunal mucosa during primary infections with S. venezuelensis Studies in basophil-deficient Mcpt8DTR mice revealed a small but significant contribution of basophils to S. venezuelensis egg clearance in primary infections. Studies in mice deficient in various components of immune responses showed that CD4+ T cells and ILC2 cells, IgG, FcRγ, and, to a lesser extent, IgE and FcεRI contribute to effective immunity in primary S. venezuelensis infections. These findings support the conclusion that the hierarchy of importance of immune effector mechanisms in primary S. venezuelensis infection is as follows: CD4+ T cells/ILC2 cells, IgG, and FcRγ>mast cells>IgE and FcεRI>basophils. In contrast, in secondary S. venezuelensis infection, our evidence indicates that the presence of CD4+ T cells is of critical importance but mast cells, antibodies, and basophils have few or no nonredundant roles.

The natural history and patterns of metastases from mucosal melanoma: an analysis of 706 prospectively-followed patients

We examined whether mucosal melanomas are different in their clinical course and patterns of metastases when arising from different anatomic sites. Our hypothesis was that metastatic behavior would differ from primary mucosal melanomas at different anatomical sites. Clinical and pathological data from 706 patients were compared for their stage distribution, patterns of metastases, CKIT/BRAF mutation status, and overall survival for different anatomical sites. The anatomic sites of the primary mucosal melanomas were from the lower GI tract (26.5%), nasal cavity and paranasal sinuses (23%), gynecological sites (22.5%), oral cavity (15%), urological sites (5%), upper GI tract (5%), and other sites (3.0%). At initial diagnosis, 14.5% were stage I disease, 41% Stage II, 21.5% Stage III, and 23.0% stage IV. Predominant metastatic sites were regional lymph nodes (21.5%), lung (21%), liver (18.5%), and distant nodes (9%). Oral cavity mucosal melanoma had a higher incidence of regional nodal metastases (31.7% versus 19.8%, P = 0.009), and a higher incidence of lung metastases (32.5% versus 18.5%, P = 0.007) compared to other primary mucosal melanomas. There was a 10% incidence of CKIT mutation and 12% BRAF mutation. Mucosal melanomas from nasal pharyngeal and oral, gastrointestinal, gynecological, and urological had a similar survival with a 1-year survival rate (88%, 83%, 86%), 2-year survival rate (66%, 57%, 61%), 5-year survival rate (27%, 16%, 20%), respectively. The largest sample size allows, for the first time, a comparison of primary melanoma stage and patterns of metastases across anatomical sites. With few exceptions, the presenting stages, incidence of nodal and distant metastases, the site of predilection of distant metastases, or overall survival were similar despite different primary anatomic sites. These findings suggest that clinical trials involving mucosal melanomas and the administration of systemic therapy can be applied equally to mucosal melanomas regardless of their primary anatomic site.

The Prognostic Value of BRAF, C-KIT, and NRAS Mutations in Melanoma Patients With Brain Metastases

Brain metastases are a common problem in patients with melanoma, but little is known about the effect of gene mutations on survival in these patients. We created a retrospective multi-institutional database of 823 patients with melanoma and brain metastases diagnosed between 2006 and 2015. Clinical parameters, gene mutation status (BRAF, C-KIT, NRAS), and treatment were correlated with survival. Treatment patterns and outcomes were compared with a prior era (1985-2005). BRAF status was known in 584 of 823 patients (71%). BRAF, NRAS, and C-KIT mutations were present in 51%, 22%, and 11% of tested patients, respectively. The median time from primary diagnosis to brain metastasis was 32months, and overall median survival (MS) from the time of initial treatment of brain metastases was 10months. MS for BRAF-positive and BRAF-negative patients was 13months and 9months, respectively (P=.02). There was no significant difference in MS in patients with or without NRAS or C-KIT mutations. The time from primary diagnosis to brain metastasis did not vary by mutation and was not associated with survival after the diagnosis of brain metastases. MS for the 1985 to 2005 and 2006 to 2015 cohorts was 6.7months and 10.0months, respectively (P<.01). Reflecting treatment-trend changes, use of whole-brain radiation therapy decreased from 48% to 26% during this period. Among BRAF-positive patients, 71% received targeted BRAF and/or MEK inhibitors and 57% received some combination of targeted therapy, chemotherapy, and/or immunotherapy. For melanoma patients with brain metastases, BRAF-positive patients survive longer than BRAF-negative patients and overall survival has improved from 1985-2005 to 2006-2015.

Update from the 4th Edition of the World Health Organization Classification of Head and Neck Tumours: Mucosal Melanomas.

The updated edition of The World Health Organization Classification of Tumours of the Head and Neck includes discussions on mucosal melanoma of both the sinonasal and oral cavity. Since the prior edition, sinonasal origin is now recognized as the most common site of occurrence of mucosal melanoma in the head and neck (66%) with oral cavity representing 25% of cases. Histologic features of mucosal melanomas vary widely from spindled, epithelioid, and pleomorphic to rhabdoid, plasmacytoid and undifferentiated. Additionally, mucosal melanomas are commonly amelanotic (or minimal pigmentation) (~50%) leading to overlapping features and diagnostic challenges in differentiating mucosal melanomas from other small cell/undifferentiated sinonasal tumors. Since the last edition, formal staging of head and neck mucosal melanomas was added to the American Joint Committee on Cancer entities, though the traditional histologic features that have prognostic significance in cutaneous melanomas fail to stratify mucosal melanomas (i.e. tumor thickness, ulceration). Interestingly, while melanomas of all sites are a malignancy derived from melanocytes, mucosal melanomas are now recognized to have distinct molecular alterations compared to cutaneous or uveal melanomas. BRAF V600E mutations are rare (<6%) in mucosally derived melanomas compared to a rate of 50% in cutaneous melanomas. CD117 (C-Kit) mutations are the most common alteration encountered (~25%) in mucosal sites with potential therapeutic targetability. The recognition of the distinct genetic changes in this subgroup of melanomas means that therapy advances in cutaneous melanomas may not translate to head and neck mucosal melanomas and clinical trials specific to this subgroup of patients are needed.

Validation of an NGS mutation detection panel for melanoma

BackgroundKnowledge of the genotype of melanoma is important to guide patient management. Identification of mutations in BRAF and c-KIT lead directly to targeted treatment, but it is also helpful to know if there are driver oncogene mutations in NRAS, GNAQ or GNA11 as these patients may benefit from alternative strategies such as immunotherapy.MethodsWhile polymerase chain reaction (PCR) methods are often used to detect BRAF mutations, next generation sequencing (NGS) is able to determine all of the necessary information on several genes at once, with potential advantages in turnaround time. We describe here an Ampliseq hotspot panel for melanoma for use with the IonTorrent Personal Genome Machine (PGM) which covers the mutations currently of most clinical interest.ResultsWe have validated this in 151 cases of skin and uveal melanoma from our files, and correlated the data with PCR based assessment of BRAF status. There was excellent agreement, with few discrepancies, though NGS does have greater coverage and picks up some mutations that would be missed by PCR. However, these are often rare and of unknown significance for treatment.ConclusionsPCR methods are rapid, less time-consuming and less expensive than NGS, and could be used as triage for patients requiring more extensive diagnostic workup. The NGS panel described here is suitable for clinical use with formalin-fixed paraffin-embedded (FFPE) samples.

Cancer biology of small gastrointestinal stromal tumors (

IntroductionThe identification of small (<2 cm) gastrointestinal stromal tumors (GISTs) is increasingly recognized. The malignancy potential is not absent. However, data on risk is scarce. The aim was to review the existing data on perceived risk of small GISTs in population-based studies. MethodsA combined review of small GISTs (<2 cm) in a population-based dataset compared with a systematic review of available population-based studies. ResultsAbout one in every four (27%) patient has a small GIST, of which 79% were incidental in presentation, and all had a low-risk mitoses index (<5/hpf). The small GISTs had C-KIT mutations in exon 11 or 9 (66%), none had mutation in PDGFRA, and 33% were non-KIT/PDGFRA. The rate of small GISTs increased for each advancing age group. None of the small GISTs had a recurrence nor were there any GIST-specific deaths during follow-up. A “small GIST” rate at one-fourth of all GISTs reported was corroborated in other population-based studies from Norway, Iceland and Korea. Common to studies reporting mitosis index were an almost universal finding of very low-risk in small GISTs, with very few recurrences and an excellent long-term survival reported to 95–100% in several series. DiscussionOne in every four GIST diagnosed is a small GIST (<2 cm). Small GISTs are increasingly found with advanced age and have an overall excellent prognosis. Defining true risk-features for proper surveillance and treatment strategy is needed in order to avoid over- and under-treatment.

Comparative proteomic analysis of kit-positive gastrointestinal stromal tumors based on differential risk classification

Objective To screen new molecular markers related to invasion and metastasis of gastrointestinal stromal tumors (GISTs) by analyzing differential protein expression profiles between high-risk and low-risk GISTs with c-kit mutation. Methods Two dimensional polyacrylamide gel electrophoresis (2D-PAGE) was used to detect the differentially expressed proteins in 5 cases of high-risk and 5 cases of low-risk GIST tissues with c-kit mutation respectively, and the differentially expressed protein markers were analyzed and identified by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). Results By comparative analysis of protein expression profiles we found 41 differentially expressed protein spots. Using MALDI-TOF-MS technique for mass spectrometry analysis of differentially expressed protein spots we successfully identified 27 different protein molecules. Of these, 14 proteins including aldehyde dehydrogenase 1 family, member A2 (ALDH1A2), ubiquinol cytochrome c reductase core protein 1 (UQCRC1), chaperonin containing TCP1 subunit 2 (CCT-2), pyruvate dehydrogenase beta (PDHB), peroxiredoxin 3 (PRDX3), protease activator 28 beta (Pa28β), cytochrome c oxidase subunit 5A (COX5A), caseinolytic mitochondrial matrix peptidase proteolytic subunit (CLPP), Tu translation elongation factor, mitochondrial (TuFM), glutathione S-transferase omega 1 (GSTO1), hypocretin receptor 1 (HCRTR1), NM23-H1 and an unknown protein were upregulated in high-risk GISTs. 13 proteins included apolipoprotein A1 (APOA1), heme binding protein 1 (HEBP1), chromosome 1 open reading frame 123 (C1orf123), heat shock protein beta-6 (HSPB6), SH3 domain-binding glutamic acid-rich-like protein (SH3BGRL), glutathione S-transferase Mu 2 (GSTM2), tropomyosin alpha-1 chain (TPM1), protein disulfide-isomerase A3 (PDIA3), dimethylarginine dimethylaminohydrolase 1 (DDAH1), tetratricopeptide repeat domain 1 (TTC1), heterogeneous nuclear ribonucleoprotein C (HNRNPC) and DJ-1 were downregulated in high-risk GISTs. Conclusion The 27 differentially expressed proteins had the potential to be the specific tumor biomarkers for predicting the prognosis and warning of the metastasis of GISTs. Key words: Gastrointestinal stromal tumors; Proteomics; Molecular markers; Neoplasm metastasis

Expression of Phosphorylated KIT in Canine Mast Cell Tumor.

Canine cutaneous mast cell tumor (MCT) is the most common canine skin tumor and exhibits variable biologic behavior. Signaling through the KIT receptor tyrosine kinase promotes cellular proliferation and survival and has been shown to play a role in MCT progression. Despite investigations into numerous biomarkers and the proposal of several grading schemas, no single marker or grading system can accurately predict outcome in canine MCT. The first aim of this study was to develop an immunohistochemical assay to measure phosphorylated KIT (pKIT) to investigate its association with 2 commonly used grading systems and other established prognostic markers for canine MCT. Thirty-four archived MCTs were evaluated for expression of pKIT and Ki-67, KIT localization, mitotic count, mutations in exons 8 and 11 in c-kit, and grading by the Patnaik and 2-tier systems. Expression of pKIT was significantly ( P < .05) correlated with the 2-tier grading scheme and c-kit mutation. Correlation approached significance ( P = .06) with Mitotic Index (MI) and Ki-67. An additional aim was to determine whether pKIT labeling provides a pharmacodynamic marker for predicting response to the receptor tyrosine kinase inhibitor toceranib (TOC). MCTs from 4 of 7 patients demonstrated a partial response to TOC. pKIT expression was assessed by immunohistochemistry in biopsies obtained before and 6 hours after the patients were treated with TOC. Reduced pKIT expression after TOC treatment was demonstrated in 3 of the 4 patients with a partial response compared to 1 of the 3 nonresponders. Collectively, these results demonstrate that immunohistochemical detection of pKIT may be a clinically relevant assay to evaluate the activation status of the major oncogenic pathway in canine MCT.

Clinical Outcome of Autologous Hematopoietic Cell Transplantation in Adult Patients with Acute Myeloid Leukemia: Who May Benefit from Autologous Hematopoietic Cell Transplantation?

The role of autologous hematopoietic cell transplantation (auto-HCT) for postremission therapy of acute myeloid leukemia is yet to be elucidated. We retrospectively analyzed 240 patients treated with auto-HCT in first remission. All patients were treated with standard induction chemotherapy, and CD34+ stem cells were collected at each cycle of consolidation. Stem cells were infused after total body irradiation (1200 cGy), cytarabine (9 g/m2), and melphalan (100 mg/m2). Estimated 5-year overall survival, disease-free survival (DFS), cumulative incidence of relapse (CIR), and nonrelapse mortality were 58.4%, 55.3%, 38.8%, and 5.9%, respectively. We identified that poor-risk karyotype showed very poor outcome after auto-HCT, and then analyzed 85 patients with good to intermediate-risk molecular cytogenetics with available molecular study results and markers for minimal residual disease (MRD) such as WT1 and core-binding factor (CBF) associated MRD (ie, AML1/ETO and CBFβ/MYH11). Our data identified that old age, pre-HCT markers for MRD, and high post-HCT WT1, high dose of CD34+ stem cell (≥4.5 × 106/kg) infusion, and c-kit or FLT3-ITD mutations were associated with higher relapse rate and poor DFS. Using pre-HCT parameters, except for post-HCT WT1, multivariate analysis revealed that patients with young age (<40 years old), no adverse mutations, and limited dose of CD34+ stem cells might be good candidate for auto-HCT (3-year DFS and CIR were 83.4% and 16.6%, respectively). Young patients with good- to intermediate-risk molecular cytogenetics may benefit from auto-HCT if stem cell dose is limited.

An update on the current management of head and neck mucosal melanoma.

Primary mucosal melanomas of the head and neck are rare and aggressive tumours that arise in the nasal cavity, paranasal sinuses and more rarely in the oral cavity. The current treatment options include radical surgical resection with adjuvant external beam radiotherapy being offered in high-risk patients. Although the latter can improve regional control, it does not reduce overall survival. Elective neck dissection is recommended for nodular oral mucosal melanoma, but its role in the clinically node negative neck is controversial. Systemic therapies including the use of tyrosine kinase inhibitors for tumours with c-KIT mutations are suitable for patients with advanced loco-regional and/or metastatic disease, but current results are variable. Patients with head and neck mucosal melanoma have a poor prognosis due to the high incidence of metastatic disease. This review assesses the latest evidence in the diagnosis and management of primary oral and head and neck mucosal melanoma including details of systemic therapies.

Unknown Primary Melanoma: Worldwide Survey on Clinical Management

Background: How to deal with melanoma of unknown primary (MUP) origin is a debated topic in the literature. Objective: We performed a worldwide survey to inquire what clinical and investigational workup is performed as well as the physicians' perception of this disease. Methods: A questionnaire was sent via mail to clinicians involved in melanoma care from December 2015 to April 2016 using the International Dermoscopy Society website. Results: 119 physicians from 47 different countries answered the questionnaire. The most reported examination was skin examination followed by CT and/or PET scans. All the participants declared asking about previous excisions of skin lesions with 81% of them asking for a histopathological slide review of previous biopsies. Half of the participants checked for a possible vitiligo phenomenon that may explain regression of the primary lesion. BRAF, cKIT, and GNAQ mutations were screened by 32% of participants. The majority of participants (76%) applied the same treatment protocols for MUP as patients with known primary melanomas of the same AJCC stage. Conclusion: Strong heterogeneity was found between physicians dealing with MUP. Thus, a consensus document should be strongly encouraged.

Gastrointestinal Stromal Tumors: Whether Mutation Guided Diagnosis Matter

Introduction: Gastrointestinal Stromal Tumor (GIST), now the most common mesenchymal tumor of the Gastrointestinal Tract (GIT), spans a clinical spectrum from benign to malignant. It has been frequently studied, especially with regard to its successful targeted therapy using imatinib mesylate. Approximately 70-80% of GISTs have gain of function mutation of the KIT gene. The aim of the study is to describe spectrum of clinical presentation and histomorphologic characteristics in classic GIST with CD117 immunostaining in a tertiary care hospital from north east India. Design: This is a descriptive cross sectional study of cases encountered over one-year period. Cases included after histomorphology and immunohistochemistry staining with CD117. Results: A total of 10 cases of GIST like histomorphology were studied and 6 were confirmed as GIST and included in the study. Four were males and two females with age ranged from 32 to 55 years. The cases had variable clinical presentations with abdominal pain and lump, melena and bleeding per rectum. Two of the cases were located in the stomach, one in the duodenum involving the periampullary area, one each in the ileocaecal region, transverse colon and rectum. Most of the cases grossly presented as a polypoidal growth. Four of the cases showed typical spindle cell morphology composed of interlacing bundles of uniform spindle shaped cells. One had a mixed morphology with features of both spindle cell and epithelioid pattern and one case mostly epithelioid type. Two cases showed intense immunoreactivity for CD117 in 90% of the tumour cells, three cases showed immunoreactivity for CD117 in 70-80% and in one case 50%. The management by surgical resections followed by 6 months imatinib mesylate treatment of 5 cases in the study had been showing good response for last two years. Conclusion: The molecular phenotype is an important consideration in the treatment of patients from prognostic point of view. Those with mutations in KIT or PDGFRA often respond to the tyrosine kinase inhibitor imatinib in contrast, tumors without these mutations are generally resistant. So a uniform category of cases with cKIT mutation may be helpful to study treatment outcome besides standard predictor of biologic behavior. It would be interesting to see whether mutation guided diagnosis has any bigger role in prognosis of classic GIST in future studies involving larger sample size.

Stomach (Gastrointestinal Stromal Tumor)GIST in a Patient of Situs Inversus Totalis: A Rare Association and Brief Case Report

Situs inversus is a rare congenital condition in which the major visceral organs are reversed from their normal positions. Usually, it involves complete transposition (right to left reversal) of all of the abdominal organs and only a small percentage is associated with GIST which is the rare mesenchymal tumors of the alimentary tract. Nowadays GISTs represents 0.1% to 3% of all gastrointestinal malignancies, making it a diagnostic challenge. Lesions are frequently located in stomach and proximal small intestine but rarely elsewhere in the abdomen. They are believed to result from mutations of proto-oncogenes c-Kit or platelet-derived growth factor receptor alpha polypeptide, this increase tyrosine kinase receptor activity, leading to uncontrolled proliferation of stem cells that differentiate into cells of Cajal. They can occur at any age but predominantly in middle-aged people and in elderly. We report a very rare case of stomach GIST in Situs inversus totalis. A 56-yearold male presented to our hospital complaining of pain in the epigastrium with melena, findings suggested a gastric GIST. Cytologic and immunohistochemistry analysis confirm diagnosis of GISTs. The reverse location of the internal organs caused no significant difficulties for surgical treatment.

Analysis of a panel of druggable gene mutations and of ALK and PD-L1 expression in a series of thymic epithelial tumors (TETs)

IntroductionThymic epithelial tumors (TETs) are rare neoplasms with different prognosis lacking consistent molecular alterations possibly leading to targeted therapy. We collected a consecutive series of TETs aimed at investigating the mutational status of druggable genes (EGFR, c-KIT, KRAS, BRAF, PDGFR-alpha and −beta, HER2 and c-MET) and the expression of ALK and PD-L1. Patients and methodsOne hundred twelve consecutive cases of TETs and relative clinico-pathologic features were collected. Immunohistochemical expression of ALK (clone D5F3) and PD-L1 (clone E1L3N), molecular analysis of EGFR (exons 18–21), c-KIT (exons 9,11,13,14,17), KRAS (exon 2), BRAF (exon 15), PDGFR-alpha (exon 12) and -beta (exons 12, 14, 18), HER-2 (exons 19 and 20) and c-MET (exons 14, 17, 18, 19) mutations were performed. Immuno-molecular results were then statistically matched with clinico-pathologic characteristics. ResultsPatients were male in 54% of cases, with a median age of 61 years (range 19–83) and affected mainly by thymoma (78%) in stage II (45%). At molecular analysis, there were 4 c-KIT mutations (occurring in exon 11 V559A, L576P, Y553N and exon 17 D820E) in thymic carcinomas (typeC), but not in other tumor types (p=0.003). No mutations were detected in other genes and none case was ALK positive. Twenty-nine (26%) cases were PD-L1 positive (65% of thymic carcinomas and 18% of thymomas). High PD-L1 expression was statistically associated with WHO classification stage type C (p<0.001) and Masaoka stage III–IV disease (p=0.007). In univariate analysis, WHO classification type C, advanced Masaoka stage and absence of myasthenia, but not PD-L1 expressions were correlated with worse survival; at multivariate analysis, only WHO type C confirmed its negative prognostic role. ConclusionA subset of TETs as thymic carcinomas can harbor c-KIT mutations and elevated PD-L1 expression that could represent targets of potential therapeutic use.

Study on the Pathogenesis and Clinical Significance of FLT3 and C-KIT Mutations in Core-Binding Factor AML

Objective1. To analyze the clinical and laboratorial characteristics of 176 patients with de novo CBF-AML who received cytogenetics and molecular genetics analysis in the First Affiliated Hospital of Soochow University.2. To clone two kinds of rare FLT3-TKD mutants in CBF-AML and explore their roles in leukemogenesis via in vitro experiments.3. To investigate the frequency of C-KIT mutation and prognosis in t(8;21) AML with trisomy 4.Methods1. Cytogenetic studies were performed on 176 patients' BM samples at their diagnosis. The karyotype obtained was described in accordance with the recommendations of the ISCN 2005.2. PCR was performed to acquire two rare FLT3-TKD mutations which was afterwards inserted to a transfer plasmid-LV5, respectively. The mutants were transfected into BaF3 cells by DNA-calcium phosphate, respectively.3. This retrospective study was conducted from February 2005 to January 2013. 145 AML patients with t(8;21) were included in the present study.Results1. The clinical and laboratorial characteristics of 176 newly diagnosed patients with CBF-AMLWe investigated the clinical and laboratorial characteristics of 176 CBF-AML patients including 139 patients with RUNX1-RUNX1T1 and 37 with CBFβ-MYH11 fusion transcripts. The patients consisted of 101 males and 75 females with a median age of 35 (range, 11-77).174 cases had cytogenetics analyzed successfully and 84 cases had additional chromosomal aberration. The most frequent additional chromosomal aberration was loss of X/Y identified in 55 (31.3%) patients, followed by del(9q) (n = 10, 5.7%), trisomy 22 (n = 10, 5.7%), trisomy 4 (n = 7, 4.0%) and trisomy 8 (n = 2, 1.1%).Overall, 73 patients (41.5%) had no detected mutations, a single gene was mutated in 97 (55.1%) patients, and more than one gene in six (3.4%) patients. The frequency of C-KIT mutation was 34.7% (n = 61) and most of the mutation occurred in exon 17 (90.2%, 55/61). FLT3 mutation was presented in 12.5% of the patients (22/176), among which FLT3-ITD was 40.9% (9/22) and FLT3-TKD was 59.1% (13/22), respectively. Mutation of FLT3-TKD was predominantly occurred in exon 20 (84.6%, 11/13), resulting in variable amino acid changes (D835Y, D835E or D835H). More t(8;21) AML patients suffered C-KIT mutation than patients with inv(16) (39.6% vs. 16.2%) (P<0.01). We also identified two rare FLT3-TKD mutants, namely FLT3 N676K and FLT3 H671Q, respectively.2. Molecular cloning and function analysis of the two rare FLT3-TKD in leukemogenesisThe mutated FLT3 was cloned and ligated to the LV5 vector plasmid. BaF3 cell lines stably expressing various FLT3 constructs were established. Through cell viability detection, we observed that the mutants could accelerate the proliferation of BaF3 cell and make the cells grow cytokine-independently to some extent.3. Frequency of C-KIT mutation and prognosis in t(8;21) AML with trisomy 4In 145 t(8;21) AML patients with trisomy 4, 91.7% (11/12) of patients were identified with C-KIT mutation, which was significantly higher than those without trisomy 4 (26.3% for the latter, P < 0.01). More importantly, the follow-up data showed that the patients with trisomy 4 correlated with a lower 3-year overall survival (OS) (15% vs. 56%, P < 0.01) and disease-free survival (DFS) (0% vs. 51%, P < 0.01) when compared with patients without trisomy 4. Furthermore, the subgroup of t(8;21)AML patients with both trisomy 4 and C-KIT mutation had a worse OS and DFS than the patients without trisomy 4 and C-KIT mutation (P < 0.05).Conclusion1. Loss of X/Y chromosome is the most frequent additional chromosomal aberration and C-KIT mutation is the most frequent gene mutation in CBF-AML, especially in t(8;21) AML. We also identified two rare FLT3-TKD mutants, namely FLT3 N676K and FLT3 H671Q.2. We successfully cloned the mutants and constructed the stable transfected cell lines. We observed that the mutants could accelerate the proliferation of BaF3 cell and make the cells grow cytokine-independently to some extent.3. We firstly identified high frequency of C-KIT mutation in t(8;21) AML patients with trisomy 4. And patients with trisomy 4 demonstrate an adverse implication in t(8;21) AML patients, which indicates it may define a distinctive subtype of t(8;21) AML. DisclosuresNo relevant conflicts of interest to declare.

MRD Is an Independent Prognostic Factor in Acute Myeloid Leukemia Carrying t(8;21) Chromosomal Abnormalities

Background:Minimal residual disease (MRD) based on quantitativeRT-PCR has become an important prognostic factor for leukemia. There are few cohort based studies to investigate prognostic factors for t(8;21) AML, especially after MRD is integrated.Objectives:To retrospectively investigatethe prognostic factors in t(8;21) AML when MRD is integrated.Patients and Methods:From August 2010 to January 2016, newly diagnosed adultpatients (15 to 55 years old) with AML were consecutively enrolled in a prospective trial(ChiCTR-TRC-10001202). Patients were assigned standard dose HAD (homoharringtonine,cytosine arabinosine(Ara-c), and daunorubicin) induction therapy or HAD containing intermediate dose Ara-c. All patients who achieved CR received either high dose or intermediate dose Ara-c. Allogeneic SCT was based on c-Kit mutation and patients' intention. Patients received 6 courses of consolidation therapy if they did not receive transplantation. MRD based on quantitative RT-PCR was performed after induction therapy and each consolidation therapy.Results:113 enrolled patients were t(8;21) AML (median age 33 years old).108 patients (95.6%) achieved CR. Six of them receivedallogeneicstem-cell transplantation in CR1.The median follow-up is 14.6(0.6-59.2) months. Three years relapse-free survival (RFS) and overall survival (OS) is 57.2% and 71.8%, respectively.Analyses of prognostic factors were performed. In univariate analysis, high WBC count (cut-off value at 25X109/L) (P=0.007), c-Kit mutation (P=0.017), and MRD positive during therapy (P=0.001)were adverse prognostic factors for RFS. But Age (cut-off value at 45 years old) (P=0.957), gender (P=0.418), induction regimen (P=0.055), and consolidation regimen (P=0.311) were not prognostic factors for RFS. MRDpositive during therapy (P=0.004) was the only adverse prognostic factors for OS. High WBCcount (P=0.824), c-Kit mutation (P=0.052), Age (P=0.596), gender (P=0.714), induction regimen (P=0.222), and consolidation regimen (P=0.299) were not prognostic factors for OS. In multivariate analysis, WBC count and MRD were independent prognostic factors for RFS. And MRD was the only prognostic factors for OS.MRD tests were performed in 83 patients after induction and during consolidation therapy. All of tests were positive in 37 (44.6%) patients. 46 (55.4%) patients achieved negative at least one of tests. The cumulative MRD negative was 3.6%, 15.7%, 34.9%, 39.8%, 47.0%, 50.6%, 55.4% after induction therapy, the first, the second, the third, the forth, the fifth, and the sixth consolidation therapy, respectively. Patients with negative MRD had better 3 years RFS (78.6% vs. 32.1%, P=0.001) and OS (85.3% vs. 55.2%, P=0.004) than patients with positive MRD.C-Kit mutation analysis was performed in 105 patients. 27(25.7%) patients had c-Kit mutation and 78(74.3%) patients had wild type c-Kit. 3 years RFS in patients with wild type c-Kit is superior to that in patients with c-Kit mutation (57.5% vs. 35.8%, P=0.017). There was no statistically difference between wild type and mutated c-Kit in 3 years OS (73.6% vs. 42.4%, P=0.052). MRD became negative during consolidation therapy in 66.1% (39/59) patients with wild type c-Kit, which is significantly higher than patients with mutated c-Kit (27.8%, 5/18, P=0.004). But there was no significant difference in RFS or OS between MRD negative and positive in c-Kit mutated patients (P=0.847 for RFS, P=0.11 for OS).To evaluate prognosis earlier, we analyzed MRD after induction therapy. MRD after induction therapy was performed in 76 patients. MRD decreased to ≤0.1%, >0.1% to≤1%, and >1% in 15 (19.7%), 45 (59.2%), and 31(40.8%) patients, respectively.Patients with MRD ≤1% after induction therapy had favorable 3 years RFS (75.4% vs. 29.7%, P=0.004) and OS (77.9% vs. 53.3%, P=0.013) compared with patients with MRD >1% after induction therapy.In multivariate analysis, in addition to c-Kit mutation and WBC count, MRD after induction therapy was still an independent prognostic factor for RFS. And MRD was the only prognostic factors for OS.Conclusion:MRD is an independentprognostic factor for OS and RFS for patients with t(8;21).Acknowledgments:This study was supported, in part, by State Key Program of National Natural Science of China (81430004), Foundation for Innovative Research Groups of the Natural Science Foundation of China (81421002) DisclosuresNo relevant conflicts of interest to declare.

Clinical and Biological Features of Pediatric Acute Myeloid Leukemia with Primary Induction Failure in the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-05 Study

INTRODUCTIONTreatment outcome of pediatric acute myeloid leukemia (AML) patients has improved over the past few decades due to intensive chemotherapy. Primary induction failure (PIF), defined as failure to achieve complete remission after the second induction chemotherapy, occurs in around 10% of children with newly diagnosed AML. Effective treatment strategy for these patients is not established and their poor prognoses have been reported elsewhere. Here we investigated the clinical and biological features of pediatric AML patients with PIF who were registered in the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-05 Study (AML-05). METHODSWe conducted a retrospective survey of patients who were registered in the AML-05. We analyzed probabilities of overall survival (OS) according to gender, age at diagnosis, white blood cell count (WBC) at diagnosis, FAB classification, etc. Genetic analysis of primary leukemic blasts was performed. We screened fusion genes and gene mutations as follows: RUNX1-RUNX1T1, CBFB-MYH11, NUP98-NSD1, DEK-NUP214, MLL-PTD, WT1, NPM1, and MLL-rearrangements, gene mutations of KIT and FLT3-ITD. Furthermore, we evaluated the clinical and biological background of these PIF patients to define their prognostic factors statistically. RESULTS: Among the 443 patients registered in the AML-05 between November 2006 and December 2010, 43 patients (9.7%) had PIF. The observation period from the date of PIF was 5-78 months (median, 20 months). The age at diagnosis was 0.1-17 years (median, 6.1 years). There was no significant difference in gender (28 males vs15 females). The 3-year OS rate was 19.0% (95% confidence interval 1.0%-38.0%). This was extremely poor compared to the patients who achieved complete remission (CR): 66.7% for high-, 73.2% for intermediate-, and 92.6% for low-risk groups, respectively. Disease recurrence is the major cause of death (n=19, 46.3%). The mean WBC at diagnosis of PIF patients was 96 x 109/L: this is significantly higher than that of non-PIF patients (51 x 109/L, P<0.01, Cox regression analysis). Morphologically, FAB classification M7 was more frequently detected in the PIF group compared with non- PIF group, although not statistically significant: 11 patients (25.6%) vs 43 patients (9.1%) (P=0.07). Unfavorable prognostic cytogenetics such as FLT3-ITD (n=16, 37.2%), NUP98-NSD1 (n=6, 15.4%), and complex karyotype (n=9, 20.9%) were detected more frequently in PIF patients, as well as MLL-PTD (n=7, 16.2%), c-KIT mutations (n=6, 14.0%), DEK-NUP214 (n=2, 4.7%), and monosomy 7 (n=6, 14%). There were no cases with WT1 or NPM1 mutations. Only 3 patients had core binding factor leukemia with RUNX1-RUNX1T1 or CBFB-MYH11 in the PIF patients. Thirty-seven PIF patients received stem cell transplantation (SCT). The patients without SCT did not achieve CR, and died of primary disease. The patients who had achieved CR before SCT showed significantly better OS (P=0.03). There was no significant difference in donor source and conditioning regimen. Younger age at diagnosis (<10 years of age, P=0.02) was associated with better outcome. High-dose cytarabine-containing re-induction chemotherapy seemed effective on univariate analysis, which indicates the type of re-induction chemotherapy to be prognostic. Finally, multivariate analysis showed only age at diagnosis (<10 years of age) to be a significant prognostic factor. CONCLUSIONSThe prognosis of pediatric AML patients with PIF was extremely poor. In our survey, unfavorable molecular biological background of leukemia was associated with PIF. It is necessary to explore more effective treatment strategies based on leukemia biology for these patients in the future. DisclosuresNo relevant conflicts of interest to declare.

C-KIT- Mutated t(8;21)AML Patients with >3log Reduction of MRD Conferred a Very High Relapse and Need HSCT to Improve Outcome

[Objective] Previous studies showed that c-KIT mutation and <3log reduction of minimal residual disease (MRD) were two poor prognostic markers for acute myeloid leukemia (AML) with t(8;21) . However, How to deal with c-KIT- mutated t(8;21)AML patients with >3log reduction of MRD remains undefined. To answer this question, we performed this study.[Methods] The current retrospective study consisted of 70 newly diagnosed patients with c-KIT- mutated t(8;21)AML during July 2005 and March 2016 in Peking University People's Hospital. Induction treatment included standard 3+7' regimen or HAA regimen. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) was recommended to all patients with an available donor after 2-3 cycles of consolidation treatment with high-dose cytarabine(HDAC). Patients without a donor or refusing HSCT received HDAC-based intensive treatment. MRD was determined by RUNX1/RUNX1T1 transcript levels detected by Q-PCR. The last follow-up time was July 2016.[Results] Sixty-nine out of 70 patients achieved complete remission(CR).Two patients relapsed after first cycle of consolidation treatment. There were 60 patients having the results of MRD after 2 cycles of consolidation treatment (Figure 1). Thirty-four patients achieved >3log reduction of RUNX1/RUNX1T1 transcript levels (defined as major molecular remission, MMR). There were 16 and 18 patients finally receiving chemotherapy and allo-HSCT. Twenty-six patients did not achieve MMR, and 12 and 14 patients finally receiving chemotherapy and allo-HSCH, respectively. For patients achieving MMR, the 4-year cumulative incidence of relapse (CIR) and disease-free survival (DFS) were 84.8% vs.6.7% (p<0.001) and 20.6% vs.87.5% (p<0.001) when receiving chemotherapy or allo-HSCT(Figure 2). For patients not achieving MMR, the 4-year CIR and DFS were 100% vs.30.8% (p<0.001) and 0% vs.60% (p<0.001) when receiving chemotherapy or allo-HSCT (Figure 2). Multivariate analysis revealed that MRD status (MMR or non-MMR and treatment choice (HSCT or chemotherapy) were independent prognostic factors for relapse, DFS.[Conclusion] We concluded that c-KIT- mutated t(8;21)AML patients with >3log reduction of MRD conferred a very high relapse and need allo-HSCT to improve outcome. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.