The optimal approach of EBUS-FNA rapid on-site evaluation (ROSE): a five-year experience from a large academic medical center.

INTRODUCTION: Rapid on-site evaluation (ROSE) performed during endobronchial ultrasound-guided fine needle aspiration (EBUS-FNA) has shown significant value. However, ROSE may not be available for some pulmonary centers. Performing ROSE can be challenging and stressful due to time constrains for preparing, staining and reviewing the cytology slides between passes. MATERIALS AND METHODS: A retrospective cytology report review of EBUS-FNA procedures performed between October 2014 and May 2019 revealed 516 cases that were included in the study. The number of passes for each procedure was documented. The adequacy rates were assessed at 4 different study points; ≤3 passes, ≤5 passes, at odd passes only, and the even passes only. The study groups results were compared to the overall ROSE and the final cytology adequacy. RESULTS: The overall ROSE interpretation was adequate in 370 (71.7%) and inadequate in 146 (28.3%). After reviewing the Papanicolaou stained slides and cell blocks, the final cytology results were adequate in 473 (91.7%) and inadequate in 43 (8.3%) of the cases. The number of passes per procedure ranged from 1 to 17. Our results showed that ROSE evaluation of the first 5 passes during the EBUS-FNA procedure could achieve the similar adequacy rate compared to the overall ROSE evaluation of all the passes. CONCLUSIONS: To achieve the most benefits of ROSE and to reduce the procedure time for EBUS-FNA, we recommend performing ROSE for ≤5 passes depending on the adequacy, and save all additional passes for cell blocks preparation if more than 5 passes are attempted.

Invasive Lobular Carcinoma Mimicking Encapsulated Papillary Carcinoma with a Literature Review: A Rare Variant Detected Serendipitously.

Invasive lobular carcinoma with papillary features is a newly proposed variant which presents as a relatively well-circumscribed mass lesion with at least partial peripheral thickened fibrous capsule and central delicate fibrovascular cores, morphologically mimicking encapsulated papillary carcinoma or solid papillary carcinoma. There are only 5 cases described in literature. We here report an interesting example of invasive lobular carcinoma with papillary growth pattern surrounded by a thick fibrous capsule, mimicking encapsulated papillary carcinoma (EPC). The lobular nature of the tumor was confirmed by the absence of membranous expression of E-cadherin and p120 catenin. The lobular cytological features are subtle in this variant and may be overlooked. Differentiation of this variant from papillary carcinomas is crucial for correct tumor categorization, staging and most importantly, proper treatment.

NUT Carcinoma of the Submandibular Gland: A Case at This Uncommon Site with Review of the Literature.

Nuclear protein in testis (NUT) carcinoma is a rare, highly aggressive, undifferentiated carcinoma that harbors a characteristic rearrangement of the NUTM1 gene. The majority arise in adolescents and young adults especially from the midline structures of the thorax, head, and neck. Until the present, there have only been three reported cases of NUT carcinoma of the submandibular gland, two of which were reported in children and another one in an adult from Korea. Here, we report the first case of NUT carcinoma arising in the submandibular gland of an adult female in the United States, representing the fourth case worldwide. A fine needle aspiration and biopsy was performed, and the diagnosis was confirmed by NUT immunohistochemical staining and fusion of the BRD4 (19p13.12) and NUTM1 (15q14) gene loci by fluorescence in-situ hybridization on the resection specimen. Salivary gland is an unusual site for NUT carcinoma and is rarely described in submandibular gland. We reviewed the clinicopathologic features of this entity at this site along with role of NUTM1 gene rearrangements in NUT tumorigenesis.

Pleomorphic Lobular Carcinoma in Situ Diagnosed by Breast Core Biopsy: Clinicopathologic Features and Correlation With Subsequent Excision.

INTRODUCTION: Pleomorphic lobular carcinoma in situ (PLCIS) is a variant of LCIS with high-grade morphologic features. The number of case series studying PLCIS is limited, and clinical management of patients with PLCIS is controversial. We report a breast core biopsy (BCBx) series of PLCIS. MATERIALS AND METHODS: We reviewed 37 cases of PLCIS with or without microinvasion diagnosed by BCBx. PLCIS was defined as dyscohesive cells showing acinar expansion and loss of immunohistochemical membranous expression of e-cadherin or beta-catenin with nuclear pleomorphism with at least 2- to 3-fold variation in nuclear size, membrane irregularities, and variably prominent nucleoli. Clinical information and findings on excision were evaluated. RESULTS: Thirty-one (84%) patients presented with mammographic calcifications, 4 (11%) presented with ultrasound findings, 1 (3%) presented with magnetic resonance imaging enhancement, and 1 (3%) with combined imaging abnormality. The mean patient age was 62.3 years. Nineteen patients (51%) had a family history of breast cancer. Microinvasion was present on BCBx in 9 (24%) of the 37 patients. Excision, available in 34 patients, demonstrated invasive carcinoma in 24 (65%), which was multifocal in 11 (46%). Twenty-three patients with PLCIS without microinvasion on BCBx, and without known history of ipsilateral invasive cancer, underwent excision; 14 of these patients demonstrated invasive carcinoma, representing an upgrade to invasive carcinoma of 60%. CONCLUSION: We report the largest BCBx series of PLCIS and confirm its aggressive biology and frequent association with multifocal invasive lobular carcinoma, as well as frequent presentation in patients with a family history of breast cancer. Our results support excision to negative margins.

Expression and clinical significance of mammalian target of rapamycin/P70 ribosomal protein S6 kinase signaling pathway in human colorectal carcinoma tissue.

The activation of mammalian target of rapamycin (mTOR) has been reported in tumor development, but the role of mTOR in colorectal carcinomas remains unclear. The aim of the present study was to investigate the significance of mTOR and its downstream effector 70 kDa ribosomal protein S6 kinase (P70S6K) in human colorectal carcinomas. The phosphorylated (p-)mTOR and p-P70S6K proteins were examined by immunohistochemistry performed on tissue microarray containing tissue samples obtained from colorectal carcinoma (n=111), adenomatous polyps (n=40) and normal colonic mucosa (n=40), with a comparison between the expression of these proteins and the clinicopathological parameters of patients with carcinomas. The positive expression rates of p-mTOR and p-P70S6k were 60.4 and 65.8%, respectively, in colorectal carcinoma tissue, which was significantly increased compared with the tissue from adenomatous polyps (27.5 and 20%, respectively) and normal colonic mucosa (10.0 and 5.0%, respectively) (P<0.05). Overexpression of the p-mTOR and p-P70S6K proteins was significantly associated with the tumor-node-metastasis stage, the occurrence of distal and lymph node metastasis and the degree of differentiation. Aberrant expression of p-mTOR and p-P70S6K may contribute to the pathogenesis, growth, invasion and metastasis of colorectal carcinoma. The phosphorylation of these proteins was considered to be a promising marker to indicate the aggressive behaviors and prognosis of colorectal carcinomas. The overexpression of the mTOR/P70S6K signaling pathway may play an important role in colorectal carcinoma carcinogenesis. The expression of p-mTOR and p-P70S6K was considered as a promising marker to indicate the aggressive behaviors and prognosis of human colorectal carcinomas.

Mycoplasmal upper respiratory infection presenting as leukocytoclastic vasculitis.

Mycoplasma is a virulent organism that is known to primarily infect the respiratory tract; however, affection of the skin, nervous system, kidneys, heart and bloodstream has been observed in various forms, which include Stevens Johnson syndrome, erythema multiforme, toxic epidermal necrolysis, encephalitis, renal failure, conduction system abnormalities and hemolytic anemia. Small vessel vasculitis is a lesser-known complication of mycoplasma pneumonia infection. We report a case of mycoplasmal upper respiratory tract infection with striking cutaneous lesions as the presenting symptom. Mycoplasmal infection was confirmed by serology testing, skin biopsy was suggestive of leukocytoclastic vasculitis. This case brings forth an uncommon manifestation of mycoplasmal infection with extra-pulmonary affection, namely small vessel vasculitis.

Thrombocytosis portends adverse prognostic significance in patients with stage II colorectal carcinoma.

Thrombocytosis portends adverse prognostic significance in many types of cancers including ovarian and lung carcinoma. In this study, we determined the prevalence and prognostic significance of thrombocytosis (defined as platelet count in excess of 400 K/µl) in patients with colorectal cancer. We performed a retrospective analysis of 310 consecutive patients diagnosed at our institution between 2004 and 2013. The patients (48.7% male and 51.3% female) had a mean age of 69.9 years (+/- 12.7 years) at diagnosis. Thrombocytosis was found in a total of 25 patients, with a higher incidence in those with stage III and IV disease (14.4% of patients). Although the mean platelet count increased with the depth of tumor invasion (pT), its values remained within normal limits in the whole patient cohort. No patient with stage I cancer (n=57) had elevated platelet count at diagnosis. By contrast, five of the 78 patients (6.4%) with stage II cancer showed thrombocytosis, and four of these patients showed early recurrence and/or metastatic disease, resulting in shortened survival (they died within one year after surgery). The incidence of thrombocytosis increased to 12.2% and 20.6%, respectively, in patients with stage III and IV disease. The overall survival rate of patients with thrombocytosis was lower than those without thrombocytosis in the stage II and III disease groups, but this difference disappeared in patients with stage IV cancer who did poorly regardless of their platelet count. We concluded that thrombocytosis at diagnosis indicates adverse clinical outcome in colorectal cancer patients with stage II or III disease. This observation is especially intriguing in stage II patients because the clinical management of these patients is controversial. If our data are confirmed in larger studies, stage II colon cancer patients with thrombocytosis should be upstaged and treated as stage III/IV disease patients.

Triptolide induces apoptosis of breast cancer cells via a mechanism associated with the Wnt/ß-catenin signaling pathway.

Triptolide is a diterpene triepoxide compound extracted from the medicinal plant, Tripterygium wilfordii Hook F. The aim of the present study was to determine whether triptolide inhibits the proliferation of breast cancer cells and to further investigate the associated molecular mechanisms. The effects of triptolide on the cell viability of three breast cancer cell lines, specifically, highly metastatic MDA-MB-231, human epidermal growth factor receptor 2-positive BT-474 and estrogen receptor-positive MCF7 cells, were measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and apoptosis assays. Western blot analysis was performed to investigate the expression levels of ß-catenin in the control and triptolide-treated cells. The results demonstrated that triptolide treatment caused cell death in the three types of malignant cell lines. Treatment with 25 nM triptolide for 48 h exhibited marked inhibitory effects on the cell viability of the three types of cells, with greater effects observed in BT-474 cells compared with the other two cell types. When compared with the cells not treated with triptolide, 50 nM triptolide treatment resulted in apoptosis of MDA-MB-231, BT-474 and MCF7 cells with apoptotic rates of ~80%. Western blot analysis indicated that triptolide treatment of MDA-MB-231, BT-474 and MCF7 cells decreased the expression levels of ß-catenin to 5-10% of the levels observed in the cells treated with dimethyl sulfoxide only. Therefore, the results of the present study indicate that triptolide induces the apoptosis of breast cancer cells via a mechanism associated with the Wnt/ß-catenin signaling pathway.

CD133 and CD44 are universally overexpressed in GIST and do not represent cancer stem cell markers.

Although imatinib mesylate has been a major breakthrough in the treatment of advanced gastrointestinal stromal tumors (GIST), complete responses are rare and most patients eventually develop resistance to the drug. Thus, the possibility of an imatinib-insensitive cell subpopulation within GIST tumors, harboring stem cell characteristics, may be responsible for the clinical failures. However, the existence of a cancer stem cell component in GIST has not been yet established. This study was aimed to determine whether expression of commonly used stem cell markers in other malignancies, that is, CD133 and CD44, might identify cells with characteristics of cancer stem/progenitor cells in human GIST. CD133 and CD44 expression in GIST explants was analyzed by flow cytometry, immunofluorescence, and gene expression. Their transcription levels were correlated with clinical and molecular factors in a large, well-annotated cohort of GIST patients. FACS sorted GIST cells based on CD133 and CD44 expression were isolated and used to assess phenotypic characteristics, ability to maintain their surface expression, sensitivity to imatinib, and expression signature. The enrichment in CD133/CD44 cells in the side population (SP) assay was also investigated. CD133 expression was consistently found in GIST. CD133(-) cells formed more colonies, were more invasive in a matrigel assay, and showed enrichment in the SP cells, compared to CD133(+) cells. CD133 expression was also detected in the two imatinib-sensitive GIST cell lines, while was absent in the imatinib-resistant lines. Our results show that CD133 and CD44 are universally expressed in GIST, and may represent a lineage rather than a cancer stem cell marker.

Imatinib potentiates antitumor T cell responses in gastrointestinal stromal tumor through the inhibition of Ido.

Imatinib mesylate targets mutated KIT oncoproteins in gastrointestinal stromal tumor (GIST) and produces a clinical response in 80% of patients. The mechanism is believed to depend predominantly on the inhibition of KIT-driven signals for tumor-cell survival and proliferation. Using a mouse model of spontaneous GIST, we found that the immune system contributes substantially to the antitumor effects of imatinib. Imatinib therapy activated CD8(+) T cells and induced regulatory T cell (T(reg) cell) apoptosis within the tumor by reducing tumor-cell expression of the immunosuppressive enzyme indoleamine 2,3-dioxygenase (Ido). Concurrent immunotherapy augmented the efficacy of imatinib in mouse GIST. In freshly obtained human GIST specimens, the T cell profile correlated with imatinib sensitivity and IDO expression. Thus, T cells are crucial to the antitumor effects of imatinib in GIST, and concomitant immunotherapy may further improve outcomes in human cancers treated with targeted agents.

Consistent MYC and FLT4 gene amplification in radiation-induced angiosarcoma but not in other radiation-associated atypical vascular lesions.

Angiosarcoma (AS) is a distinct group of sarcomas characterized by upregulation of vascular-specific receptor tyrosine kinases, including TIE1, KDR, TEK, and FLT1. In keeping with the clinical heterogeneity, gene-expression profiling distinguishes two AS genomic clusters, which correlate with anatomical location and prior exposure to radiation. Furthermore, a high percentage of secondary AS, but not primary AS, shows distinct 8q24 chromosomal gains, due to MYC amplification. In this study, we mined the transcriptional output of 10 secondary and 11 primary AS to better define the dichotomy in the pathogenesis of these two clinical subsets. The oncogenic role of MYC was investigated further in secondary AS as well as in radiation-induced atypical vascular lesions (AVL) and other radiation-associated sarcomas. High-level MYC amplification was found in 100% of secondary AS, but in none of the AVL or other radiation-associated sarcomas. Coamplification of FLT4 (encoding VEGFR3) was identified in 25% of secondary AS, but not in other types. Our findings reinforce the distinct pathogenesis of AS subtypes, with MYC amplification being an early, but necessary event in secondary AS. Secondary genetic hits, such as FLT4 gene coamplification or KDR mutations, may play a role in tumor progression as well as potential therapeutic targeting.

Comprehensive genomic analysis reveals clinically relevant molecular distinctions between thymic carcinomas and thymomas.

PURPOSE: Thymomas and thymic carcinomas are rare intrathoracic malignancies that can be invasive and refractory to conventional treatment. Because these tumors both originate from the thymus, they are often grouped together clinically. However, whether the underlying biology of these tumors warrants such clustering is unclear, and the optimum treatment of either entity is unknown. EXPERIMENTAL DESIGN: All thymic tumors were profiled for mutations in genes encoding components of the EGFR and KIT signaling pathways, assessed for EGFR and KIT expression by immunohistochemistry, and analyzed by array-based comparative genomic hybridization. Previously untreated tumors were subjected to global gene expression arrays. RESULTS: We analyzed 45 thymic tumors [thymoma, n = 38 (type A, n = 8; type B2, n = 22; type B3, n = 8); thymic carcinoma, n = 7]. One thymoma and one thymic carcinoma harbored KRAS mutations (G12A and G12V, respectively), and one thymoma had a G13V HRAS mutation. Three tumors displayed strong KIT staining. Two thymic carcinomas harbored somatic KIT mutations (V560del and H697Y). In cell viability assays, the V560del mutant was associated with similar sensitivities to imatinib and sunitinib, whereas the H697Y mutant displayed greater sensitivity to sunitinib. Genomic profiling revealed distinct differences between type A to B2 thymomas versus type B3 and thymic carcinomas. Moreover, array-based comparative genomic hybridization could readily distinguish squamous cell carcinomas of the thymus versus the lung, which can often present a diagnostic challenge. CONCLUSIONS: Comprehensive genomic analysis suggests that thymic carcinomas are molecularly distinct from thymomas. These data have clinical, pathologic, and therapeutic implications for the treatment of thymic malignancies.

Mechanisms of sunitinib resistance in gastrointestinal stromal tumors harboring KITAY502-3ins mutation: an in vitro mutagenesis screen for drug resistance.

PURPOSE: Although tyrosine kinase inhibitors have improved survival in advanced gastrointestinal stromal tumor (GIST), complete response is rare and most patients eventually fail the first-line treatment with imatinib. Sunitinib malate is the only approved second-line therapy for patients with imatinib-resistant or imatinib-intolerant GIST. The clinical benefit of sunitinib is genotype-dependent in regards to both primary and secondary mutations, with GIST patients harboring the KIT(AY502-3ins) exon 9 mutation being the most sensitive. EXPERIMENTAL DESIGN: As sunitinib resistance is now emerging, our goal was to investigate mechanisms of progression and to test the efficacy of novel tyrosine kinase inhibitor on these resistant mutants in vitro. N-ethyl-N-nitrosourea mutagenesis of Ba/F3 cells expressing the KIT(AY502-3ins) mutant was used to investigate novel patterns of resistant mutations evolving in the presence of sunitinib. RESULTS: Tumors from patients who developed sunitinib resistance after at least 1 year of radiographic response were analyzed, showing similar findings of a primary KIT(AY502-3ins) mutation and a secondary mutation in the KIT activation loop. Ba/F3 cells expressing these sunitinib-resistant double mutants showed sensitivity to both dasatinib and nilotinib. CONCLUSIONS: Sunitinib resistance in GIST shares similar pathogenetic mechanisms identified in imatinib failure, with acquisition of secondary mutations in the activation domain after an extended initial response to the drug. Moreover, in vitro mutagenesis with or without N-ethyl-N-nitrosourea of Ba/F3 cells expressing KIT(AY502-3ins) showed acquisition of secondary mutations restricted to the second kinase domain of KIT. In contrast, in vitro resistance to imatinib produces a broader spectrum of secondary mutations including mutations in both KIT kinase domains.

Chordoma and chondrosarcoma gene profile: implications for immunotherapy.

Chordoma and chondrosarcoma are malignant bone tumors characterized by the abundant production of extracellular matrix. The resistance of these tumors to conventional therapeutic modalities has prompted us to delineate the gene expression profile of these two tumor types, with the expectation to identify potential molecular therapeutic targets. Furthermore the transcriptional profile of chordomas and chrondrosarcomas was compared to a wide variety of sarcomas as well as to that of normal tissues of similar lineage, to determine whether they express unique gene signatures among other tumors of mesenchymal origin, and to identify changes associated with malignant transformation. A HG-U133A Affymetrix Chip platform was used to determine the gene expression signature in 6 chordoma and 14 chondrosarcoma lesions. Validation of selected genes was performed by qPCR and immunohistochemistry (IHC) on an extended subset of tumors. By unsupervised clustering, chordoma and chondrosarcoma tumors grouped together in a genomic cluster distinct from that of other sarcoma types. They shared overexpression of many extracellular matrix genes including aggrecan, type II & X collagen, fibronectin, matrillin 3, high molecular weight-melanoma associated antigen (HMW-MAA), matrix metalloproteinase MMP-9, and MMP-19. In contrast, T Brachyury and CD24 were selectively expressed in chordomas, as were Keratin 8,13,15,18 and 19. Chondrosarcomas are distinguished by high expression of type IX and XI collagen. Because of its potential usefulness as a target for immunotherapy, the expression of HMW-MAA was analyzed by IHC and was detected in 62% of chordomas and 48% of chondrosarcomas, respectively. Furthermore, western blotting analysis showed that HMW-MAA synthesized by chordoma cell lines has a structure similar to that of the antigen synthesized by melanoma cells. In conclusion, chordomas and chondrosarcomas share a similar gene expression profile of up-regulated extracellular matrix genes. HMW-MAA represents a potential useful target to apply immunotherapy to these tumors.

Novel V600E BRAF mutations in imatinib-naive and imatinib-resistant gastrointestinal stromal tumors.

BRAF and NRAS are commonly mutated in cancer and represent the most frequent genetic events in malignant melanoma. More recently, a subset of melanomas was shown to overexpress KIT and harbor KIT mutations. Although most gastrointestinal stromal tumors (GISTs) exhibit activating mutations in either KIT or PDGFRA, about 10% of the cases lack mutations in these genes. It is our hypothesis following the melanoma model that mutations in BRAF or NRAS may play a role in wild-type GIST pathogenesis. Alterations in RAS/MEK/ERK pathway may also be involved in development of imatinib resistance in GIST, particularly in tumors lacking secondary KIT or PDGFRA mutations. Imatinib-naive wild-type GISTs from 61 patients, including 15 children and 28 imatinib-resistant tumors without secondary KIT mutations were analyzed. Screening for hot spots mutations in BRAF (exons 11 and 15) and NRAS (exons 2 and 3) was performed. A BRAF exon 15 V600E was identified in 3 of 61 GIST patients, who shared similar clinical features, being 49- to 55-years-old females and having their tumors located in the small bowel. The tumors were strongly KIT immunoreactive and had a high risk of malignancy. An identical V600E BRAF mutation was also identified in one of 28 imatinib resistant GIST lacking a defined mechanism of drug resistance. In conclusion, we identified a primary BRAF V600E mutations in 7% of adult GIST patients, lacking KIT/PDGFRA mutations. The BRAF-mutated GISTs show predilection for small bowel location and high risk of malignancy. A secondary V600E BRAF mutation could represent an alternative mechanism of imatinib resistance. Kinase inhibitors targeting BRAF may be effective therapeutic options in this molecular GIST subset.

Molecular characterization of pediatric gastrointestinal stromal tumors.

PURPOSE: Pediatric gastrointestinal stromal tumors (GIST) are rare and occur preferentially in females as multifocal gastric tumors, typically lacking mutations in KIT and PDGFRA. As KIT oncoprotein is consistently overexpressed in pediatric GIST, we sought to investigate the activation of KIT downstream targets and alterations of KIT/PDGFRA gene copy number, mine novel therapeutic targets by gene expression, and test tyrosine kinase receptor activation by proteomic profiling. EXPERIMENTAL DESIGN: Seventeen pediatric GISTs were investigated for KIT/PDGFRA genotype and biochemical activation of KIT downstream targets. The transcriptional profile of 13 nodules from 8 pediatric patients was compared with 8 adult wild-type (WT) GISTs, including 3 young adults. The drug sensitivity of second-generation kinase inhibitors was tested in murine Ba/F3 cells expressing human WT KIT, as well as in short-term culture of explants of WT GIST cells. RESULTS: A KIT/PDGFRA WT genotype was identified in all 12 female patients, whereas two of five males had either a KIT exon 11 or PDGFRA exon 18 mutation. KIT downstream targets were consistently activated. Pediatric GISTs showed a distinct transcriptional signature, with overexpression of BAALC, PLAG1, IGF1R, FGF4, and NELL1. In vitro studies showed that nilotinib, sunitinib, dasatinib, and sorafenib are more effective than imatinib against WT KIT. CONCLUSIONS: Rare cases of pediatric GIST may occur in male patients and harbor activating KIT/PDGFRA mutations. Pediatric GISTs show distinct transcriptional signature, suggesting a different biology than WT GIST in adults. In vitro drug screening showed that second-generation kinase inhibitors may provide greater clinical benefit in pediatric GIST.

Sorafenib inhibits the imatinib-resistant KITT670I gatekeeper mutation in gastrointestinal stromal tumor.

PURPOSE: Resistance is commonly acquired in patients with metastatic gastrointestinal stromal tumor who are treated with imatinib mesylate, often due to the development of secondary mutations in the KIT kinase domain. We sought to investigate the efficacy of second-line tyrosine kinase inhibitors, such as sorafenib, dasatinib, and nilotinib, against the commonly observed imatinib-resistant KIT mutations (KIT(V654A), KIT(T670I), KIT(D820Y), and KIT(N822K)) expressed in the Ba/F3 cellular system. EXPERIMENTAL DESIGN: In vitro drug screening of stable Ba/F3 KIT mutants recapitulating the genotype of imatinib-resistant patients harboring primary and secondary KIT mutations was investigated. Comparison was made to imatinib-sensitive Ba/F3 KIT mutant cells as well as Ba/F3 cells expressing only secondary KIT mutations. The efficacy of drug treatment was evaluated by proliferation and apoptosis assays, in addition to biochemical inhibition of KIT activation. RESULTS: Sorafenib was potent against all imatinib-resistant Ba/F3 KIT double mutants tested, including the gatekeeper secondary mutation KIT(WK557-8del/T670I), which was resistant to other kinase inhibitors. Although all three drugs tested decreased cell proliferation and inhibited KIT activation against exon 13 (KIT(V560del/V654A)) and exon 17 (KIT(V559D/D820Y)) double mutants, nilotinib did so at lower concentrations. CONCLUSIONS: Our results emphasize the need for tailored salvage therapy in imatinib-refractory gastrointestinal stromal tumors according to individual molecular mechanisms of resistance. The Ba/F3 KIT(WK557-8del/T670I) cells were sensitive only to sorafenib inhibition, whereas nilotinib was more potent on imatinib-resistant KIT(V560del/V654A) and KIT(V559D/D820Y) mutant cells than dasatinib and sorafenib.

L576P KIT mutation in anal melanomas correlates with KIT protein expression and is sensitive to specific kinase inhibition.

Activating mutations in either BRAF or NRAS are seen in a significant number of malignant melanomas, but their incidence appears to be dependent to ultraviolet light exposure. Thus, BRAF mutations have the highest incidence in non-chronic sun damaged (CSD), and are uncommon in acral, mucosal and CSD melanomas. More recently, activating KIT mutations have been described in rare cases of metastatic melanoma, without further reference to their clinical phenotypes. This finding is intriguing since KIT expression is downregulated in most melanomas progressing to more aggressive lesions. In this study, we investigated a group of anal melanomas for the presence of BRAF, NRAS, KIT and PDGFRA mutations. A heterozygous KIT exon 11 L576P substitution was identified in 3 of 20 cases tested. The 3 KIT mutation-carrying tumors were strongly immunopositive for KIT protein. No KIT mutations were identified in tumors with less than 4+ KIT immunostaining. NRAS mutation was identified in one tumor. No BRAF or PDGFRA mutations were identified in either KIT positive or negative anal melanomas. In vitro drug testing of stable transformant Ba/F3 KIT(L576P) mutant cells showed sensitivity for dasatinib (previously known as BMS-354825), a dual SRC/ABL kinase inhibitor, and imatinib. However, compared to an imatinib-sensitive KIT mutant, dasatinib was potent at lower doses than imatinib in the KIT(L576P) mutant. These results suggest that a subset of anal melanomas show activating KIT mutations, which are susceptible for therapy with specific kinase inhibitors.

Pathologic and molecular heterogeneity in imatinib-stable or imatinib-responsive gastrointestinal stromal tumors.

PURPOSE: Gastrointestinal stromal tumor (GIST) is the most common sarcoma of the intestinal tract. Nearly all tumors express KIT protein, and most have an activating mutation in either KIT or PDGFRA. Therapy with selective tyrosine kinase inhibitors achieves a partial response or stable disease in approximately 80% of patients with advanced GIST. However, after an initial clinical response, some patients develop imatinib resistance. Our goal was to investigate the spectrum of pathologic response and molecular alterations in a group of GIST patients, clinically defined as having imatinib-stable/imatinib-responsive lesions, who underwent surgical resection. EXPERIMENTAL DESIGN: Forty-three tumor nodules from 28 patients were available for pathologic and molecular analysis, which included genotyping for primary and secondary KIT/PDGFRA-mutations, cell cycle alterations, and biochemical activation status of KIT and downstream targets. The transcriptional changes of a subset of these tumors were compared with a group of imatinib-naive GISTs on a U133A Affymetrix expression platform. RESULTS: The histologic response did not correlate with imatinib therapy duration or with proliferative activity. Second-site KIT mutation was identified in only one tumor nodule. Activation of KIT and downstream targets was consistent in all tumors analyzed. Ultrastructurally, a subset of tumors showed a smooth muscle phenotype, which correlated with overexpression of genes involved in muscle differentiation and function. CONCLUSIONS: The histologic response to imatinib is heterogeneous and does not correlate well with clinical response. Second-site KIT mutations are rare in imatinib-responsive GISTs compared with imatinib-resistant tumors. The gene signature of imatinib-response in GISTs showed alterations of cell cycle control as well as up-regulation of genes involved in muscle differentiation and function.

Skeletal metastases in myxoid liposarcoma: an unusual pattern of distant spread.

BACKGROUND: Myxoid liposarcoma (MLS), the second most common subtype of liposarcoma, occurs predominantly in the extremities of young adults and has a disproportionately high tendency to metastasize to unusual soft tissue locations, before disseminated spread or pulmonary metastases. Anecdotal evidence, mainly supported by isolated case reports, suggests that a subset of these patients also develop bone metastasis, especially within the spine, which was previously under-appreciated. STUDY DESIGN: In this study we investigate the incidence of osseous metastases in a well annotated sarcoma database and correlate this endpoint with clinicopathologic and molecular findings. RESULTS: From a total of 230 patients with MLS diagnosis confirmed histologically, who were managed and followed prospectively at MSKCC, 40 (17%) developed skeletal metastases, comprising 56% of all metastatic events. A significant number of these bone metastases were identified early in the disease course, before the manifestation of disease in sites where sarcomas usually metastasize, such as lung. From the time of 1st metastasis, the 5 years median survival was 16%. The majority (78%) of MLS patients developing bone metastases had a histologic high grade primary tumor. The median overall survival for the high grade tumors was 55 months, as compared to 105 months for low grade cases. Eleven (84%) of 13 cases tested by RT-PCR demonstrated a type II TLS-CHOP fusion transcript. CONCLUSION: These findings suggest that MLS has a high incidence of osseous metastases, with predilection to spine, and often associated with the most common type of TLS-CHOP transcript. Screening should include images of the spine in high-risk MLS patients to exclude spinal metastases.