Anti-KIT monoclonal antibody inhibits imatinib-resistant gastrointestinal stromal tumor growth.

Gastrointestinal stromal tumor (GIST) is the most common sarcoma of the gastrointestinal tract and arises from the interstitial cells of Cajal. It is characterized by expression of the receptor tyrosine kinase CD117 (KIT). In 70-80% of GIST cases, oncogenic mutations in KIT are present, leading to constitutive activation of the receptor, which drives the proliferation of these tumors. Treatment of GIST with imatinib, a small-molecule tyrosine kinase inhibitor, inhibits KIT-mediated signaling and initially results in disease control in 70-85% of patients with KIT-positive GIST. However, the vast majority of patients eventually develop resistance to imatinib treatment, leading to disease progression and posing a significant challenge in the clinical management of these tumors. Here, we show that an anti-KIT monoclonal antibody (mAb), SR1, is able to slow the growth of three human GIST cell lines in vitro. Importantly, these reductions in cell growth were equivalent between imatinib-resistant and imatinib-sensitive GIST cell lines. Treatment of GIST cell lines with SR1 reduces cell-surface KIT expression, suggesting that mAb-induced KIT down-regulation may be a mechanism by which SR1 inhibits GIST growth. Furthermore, we also show that SR1 treatment enhances phagocytosis of GIST cells by macrophages, indicating that treatment with SR1 may enhance immune cell-mediated tumor clearance. Finally, using two xenotransplantation models of imatinib-sensitive and imatinib-resistant GIST, we demonstrate that SR1 is able to strongly inhibit tumor growth in vivo. These results suggest that treatment with mAbs targeting KIT may represent an alternative, or complementary, approach for treating GIST.

Breakpoint analysis of transcriptional and genomic profiles uncovers novel gene fusions spanning multiple human cancer types.

Gene fusions, like BCR/ABL1 in chronic myelogenous leukemia, have long been recognized in hematologic and mesenchymal malignancies. The recent finding of gene fusions in prostate and lung cancers has motivated the search for pathogenic gene fusions in other malignancies. Here, we developed a "breakpoint analysis" pipeline to discover candidate gene fusions by tell-tale transcript level or genomic DNA copy number transitions occurring within genes. Mining data from 974 diverse cancer samples, we identified 198 candidate fusions involving annotated cancer genes. From these, we validated and further characterized novel gene fusions involving ROS1 tyrosine kinase in angiosarcoma (CEP85L/ROS1), SLC1A2 glutamate transporter in colon cancer (APIP/SLC1A2), RAF1 kinase in pancreatic cancer (ATG7/RAF1) and anaplastic astrocytoma (BCL6/RAF1), EWSR1 in melanoma (EWSR1/CREM), CDK6 kinase in T-cell acute lymphoblastic leukemia (FAM133B/CDK6), and CLTC in breast cancer (CLTC/VMP1). Notably, while these fusions involved known cancer genes, all occurred with novel fusion partners and in previously unreported cancer types. Moreover, several constituted druggable targets (including kinases), with therapeutic implications for their respective malignancies. Lastly, breakpoint analysis identified new cell line models for known rearrangements, including EGFRvIII and FIP1L1/PDGFRA. Taken together, we provide a robust approach for gene fusion discovery, and our results highlight a more widespread role of fusion genes in cancer pathogenesis.

ROR2 is a novel prognostic biomarker and a potential therapeutic target in leiomyosarcoma and gastrointestinal stromal tumour.

Soft-tissue sarcomas are a group of malignant tumours whose clinical management is complicated by morphological heterogeneity, inadequate molecular markers and limited therapeutic options. Receptor tyrosine kinases (RTKs) have been shown to play important roles in cancer, both as therapeutic targets and as prognostic biomarkers. An initial screen of gene expression data for 48 RTKs in 148 sarcomas showed that ROR2 was expressed in a subset of leiomyosarcoma (LMS), gastrointestinal stromal tumour (GIST) and desmoid-type fibromatosis (DTF). This was further confirmed by immunohistochemistry (IHC) on 573 tissue samples from 59 sarcoma tumour types. Here we provide evidence that ROR2 expression plays a role in the invasive abilities of LMS and GIST cells in vitro. We also show that knockdown of ROR2 significantly reduces tumour mass in vivo using a xenotransplantation model of LMS. Lastly, we show that ROR2 expression, as measured by IHC, predicts poor clinical outcome in patients with LMS and GIST, although it was not independent of other clinico-pathological features in a multivariate analysis, and that ROR2 expression is maintained between primary tumours and their metastases. Together, these results show that ROR2 is a useful prognostic indicator in the clinical management of these soft-tissue sarcomas and may represent a novel therapeutic target.

Distinctive contact between CD34+ hematopoietic progenitors and CXCL12+ CD271+ mesenchymal stromal cells in benign and myelodysplastic bone marrow.

Mesenchymal stromal cells (MSCs) support hematopoiesis and are cytogenetically and functionally abnormal in myelodysplastic syndrome (MDS), implying a possible pathophysiologic role in MDS and potential utility as a diagnostic or risk-stratifying tool. We have analyzed putative MSC markers and their relationship to CD34+ hematopoietic stem/progenitor cells (HSPCs) within intact human bone marrow in paraffin-embedded bone marrow core biopsies of benign, MDS and leukemic (AML) marrows using tissue microarrays to facilitate scanning, image analysis and quantitation. We found that CD271+, ALP+ MSCs formed an extensive branching perivascular, periosteal and parenchymal network. Nestin was brightly positive in capillary/arteriolar endothelium and occasional subendothelial cells, whereas CD146 was most brightly expressed in SMA+ vascular smooth muscle/pericytes. CD271+ MSCs were distinct by double immunofluorescence from CD163+ macrophages and were in close contact with but distinct from brightly nestin+ and from brightly CD146+ vascular elements. Double immunofluorescence revealed an intimate spatial relationship between CD34+ HSPCs and CD271+ MSCs; remarkably, 86% of CD34+ HSPCs were in direct contact with CD271+ MSCs across benign, MDS and AML marrows, predominantly in a perivascular distribution. Expression of the intercrine chemokine CXCL12 was strong in the vasculature in both benign and neoplastic marrow, but was also present in extravascular parenchymal cells, particularly in MDS specimens. We identified these parenchymal cells as MSCs by ALP/CXCL12 and CD271/CXCL12 double immunofluorescence. The area covered by CXCL12+ ALP+ MSCs was significantly greater in MDS compared with benign and AML marrow (P=0.021, Kruskal-Wallis test). The preservation of direct CD271+ MSC/CD34+ HSPC contact across benign and neoplastic marrow suggests a physiologically important role for the CD271+ MSC/CD34+ HSPC relationship and possible abnormal exposure of CD34+ HSPCs to increased MSC CXCL12 expression in MDS.

Phosphatidylinositol-3-kinase pathway mutations are common in breast columnar cell lesions.

The phosphatidylinositol-3-kinase pathway is one of the most commonly mutated pathways in invasive breast carcinoma, with PIK3CA mutations in ∼25% of invasive carcinomas, and AKT1 mutations in up to 5%. Ductal carcinoma in situ, and benign papillomas harbor similar mutations. However, activating point mutations in breast columnar cell lesions have been infrequently studied. Twenty-three breast resection specimens containing columnar cell lesions were identified; 14 with associated invasive carcinoma or carcinoma in situ. DNA extracts were prepared from formalin-fixed paraffin-embedded tissue and screened for a panel of point mutations (321 mutations in 30 genes) using a multiplex PCR panel with mass-spectroscopy readout. PIK3CA mutations were identified in 13/24 columnar cell lesions (54%) and 3/8 invasive carcinomas (37%). The mutation status of columnar cell lesions and associated carcinoma was frequently discordant. Of the 14 cases, only 5 demonstrated the same genotype in matched samples of columnar cell lesions and carcinoma (4 wild type, 1 PIK3CA H1047R). Interestingly, five patients had mutations in columnar cell lesions with wild-type carcinoma; two patients had different point mutations in columnar cell lesions and carcinoma. Only three cases had wild-type columnar cell lesion and mutated carcinoma. The 50% PIK3CA mutation prevalence in columnar cell lesions is greater than reported in most studies of invasive breast cancer. Further, columnar cell lesions and carcinoma were frequently discordant for PIK3CA/AKT1 mutation status. These findings raise interesting questions about the role of PIK3CA/AKT pathway in breast carcinogenesis, and the biologic/precursor potential of columnar cell lesions.

CSF1 expression in nongynecological leiomyosarcoma is associated with increased tumor angiogenesis.

Leiomyosarcoma (LMS) is a malignant tumor of smooth muscle cells for which few effective therapies exist. A subset of LMS cases express macrophage colony-stimulating factor (CSF1) and the resultant tumor-associated macrophage (TAM) infiltration predicts poor clinical outcome. Further, TAMs have been shown to increase tumor angiogenesis. Here, we analyzed 149 LMS cases by immunohistochemistry for vascular marker CD34 and show that high microvessel density (MVD) in nongynecological LMS cases significantly predicts poor patient outcome. The majority of high MVD cases were also CSF1-positive, and when combining high MVD with CSF1 expression, an even stronger prognostic correlation with patient outcome was obtained. Gene expression profiling revealed that MVD has a stronger correlation with CSF1 expression than with expression of vascular endothelial growth factor isoforms, which have traditionally been used as markers of angiogenesis and as anti-angiogenic therapeutic targets. Finally, patterns of CSF1 expression and TAM recruitment remained consistent between primary tumors and their metastases, and between primary tumors and those grown as xenografts in mice, highlighting the stability of these features to the biology of LMS tumors. Together, these findings suggest an important role for CSF1 and the resulting TAM infiltration in the pathological neovascularization of LMS tumors and provide a rationale for CSF1-targeted therapies in LMS.

Computational prediction and experimental validation associating FABP-1 and pancreatic adenocarcinoma with diabetes.

BACKGROUND: Pancreatic cancer, composed principally of pancreatic adenocarcinoma (PaC), is the fourth leading cause of cancer death in the United States. PaC-associated diabetes may be a marker of early disease. We sought to identify molecules associated with PaC and PaC with diabetes (PaC-DM) using a novel translational bioinformatics approach. We identified fatty acid binding protein-1 (FABP-1) as one of several candidates. The primary aim of this pilot study was to experimentally validate the predicted association between FABP-1 with PaC and PaC with diabetes. METHODS: We searched public microarray measurements for genes that were specifically highly expressed in PaC. We then filtered for proteins with known involvement in diabetes. Validation of FABP-1 was performed via antibody immunohistochemistry on formalin-fixed paraffin embedded pancreatic tissue microarrays (FFPE TMA). FFPE TMA were constructed using 148 cores of pancreatic tissue from 134 patients collected between 1995 and 2002 from patients who underwent pancreatic surgery. Primary analysis was performed on 21 normal and 60 pancreatic adenocarcinoma samples, stratified for diabetes. Clinical data on samples was obtained via retrospective chart review. Serial sections were cut per standard protocol. Antibody staining was graded by an experienced pathologist on a scale of 0-3. Bivariate and multivariate analyses were conducted to assess FABP-1 staining and clinical characteristics. RESULTS: Normal samples were significantly more likely to come from younger patients. PaC samples were significantly more likely to stain for FABP-1, when FABP-1 staining was considered a binary variable. Compared to normals, there was significantly increased staining in diabetic PaC samples (p = 0.004) and there was a trend towards increased staining in the non-diabetic PaC group (p = 0.07). In logistic regression modeling, FABP-1 staining was significantly associated with diagnosis of PaC (OR 8.6 95% CI 1.1-68, p = 0.04), though age was a confounder. CONCLUSIONS: Compared to normal controls, there was a significant positive association between FABP-1 staining and PaC on FFPE-TMA, strengthened by the presence of diabetes. Further studies with closely phenotyped patient samples are required to understand the true relationship between FABP-1, PaC and PaC-associated diabetes. A translational bioinformatics approach has potential to identify novel disease associations and potential biomarkers in gastroenterology.

Variations in stromal signatures in breast and colorectal cancer metastases.

The tumour microenvironment (TME) plays an important role in tumour survival and growth, but little is known about the degree of preservation between different stromal response patterns found in primary tumours and their metastases. We have previously identified gene expression profiles for two distinct stromal signatures in breast carcinoma of fibroblast (aka DTF) and macrophage (aka CSF1) response and found them to be correlated with clinicopathological features, including outcome. In this study, we compare the DTF fibroblast and CSF1 macrophage stromal response patterns in primary breast and colorectal cancers to their matched lymph node metastases. In both breast and colorectal cancer, there was a significant positive correlation between the CSF1 macrophage signature in the primary tumours and the matched lymph node metastases, as assessed by immunohistochemical markers. No such correlation was observed for the DTF fibroblast signature. A similar result was seen in independent analysis of two published gene expression microarray datasets. The variations of these stromal reaction patterns from the primary to the metastasis shed light on the relationship between the neoplastic cells and the non-neoplastic cells in the TME. The preservation of the CSF1 macrophage response pattern in metastases lends support to targeting the CSF1 pathway in cancer.

Genome-wide transcriptome analyses reveal p53 inactivation mediated loss of miR-34a expression in malignant peripheral nerve sheath tumours.

Malignant peripheral nerve sheath tumours (MPNSTs) are aggressive soft tissue tumours that occur either sporadically or in patients with neurofibromatosis type 1. The malignant transformation of the benign neurofibroma to MPNST is incompletely understood at the molecular level. We have determined the gene expression signature for benign and malignant PNSTs and found that the major trend in malignant transformation from neurofibroma to MPNST consists of the loss of expression of a large number of genes, rather than widespread increase in gene expression. Relatively few genes are expressed at higher levels in MPNSTs and these include genes involved in cell proliferation and genes implicated in tumour metastasis. In addition, a gene expression signature indicating p53 inactivation is seen in the majority of MPNSTs. Subsequent microRNA profiling of benign and malignant PNSTs indicated a relative down-regulation of miR-34a in most MPNSTs compared to neurofibromas. In vitro studies using the cell lines MPNST-14 (NF1 mutant) and MPNST-724 (from a non-NF1 individual) show that exogenous expression of p53 or miR-34a promotes apoptotic cell death. In addition, exogenous expression of p53 in MPNST cells induces miR-34a and other miRNAs. Our data show that p53 inactivation and subsequent loss of expression of miR-34a may significantly contribute to the MPNST development. Collectively, our findings suggest that deregulation of miRNAs has a potential role in the malignant transformation process in peripheral nerve sheath tumours.

Genomic profiling identifies GATA6 as a candidate oncogene amplified in pancreatobiliary cancer.

Pancreatobiliary cancers have among the highest mortality rates of any cancer type. Discovering the full spectrum of molecular genetic alterations may suggest new avenues for therapy. To catalogue genomic alterations, we carried out array-based genomic profiling of 31 exocrine pancreatic cancers and 6 distal bile duct cancers, expanded as xenografts to enrich the tumor cell fraction. We identified numerous focal DNA amplifications and deletions, including in 19% of pancreatobiliary cases gain at cytoband 18q11.2, a locus uncommonly amplified in other tumor types. The smallest shared amplification at 18q11.2 included GATA6, a transcriptional regulator previously linked to normal pancreas development. When amplified, GATA6 was overexpressed at both the mRNA and protein levels, and strong immunostaining was observed in 25 of 54 (46%) primary pancreatic cancers compared to 0 of 33 normal pancreas specimens surveyed. GATA6 expression in xenografts was associated with specific microarray gene-expression patterns, enriched for GATA binding sites and mitochondrial oxidative phosphorylation activity. siRNA mediated knockdown of GATA6 in pancreatic cancer cell lines with amplification led to reduced cell proliferation, cell cycle progression, and colony formation. Our findings indicate that GATA6 amplification and overexpression contribute to the oncogenic phenotypes of pancreatic cancer cells, and identify GATA6 as a candidate lineage-specific oncogene in pancreatobiliary cancer, with implications for novel treatment strategies.

Coordinate expression of colony-stimulating factor-1 and colony-stimulating factor-1-related proteins is associated with poor prognosis in gynecological and nongynecological leiomyosarcoma.

Previously, we showed that the presence of high numbers of macrophages correlates with poor prognosis in nongynecological leiomyosarcoma (LMS). In gynecological LMS, a similar trend was noted but did not reach statistical significance. Colony-stimulating factor-1 (CSF1) is a major chemoattractant for macrophages. Here we show that in a subset of LMS cases, CSF1 is expressed by the malignant cells. Previously, we found that CSF1 is translocated and highly expressed in tenosynovial giant cell tumors (TGCTs), and this observation allowed us to identify genes that showed a coordinate expression with CSF1. Here, we evaluated the expression of CSF1 and TGCT-associated proteins in 149 cases of LMS. The coordinate expression of CSF1 and three TGCT-associated proteins (CD163, FCGR3a, and CTSL1) identified cases with poor prognosis in both gynecological LMS (P = 0.00006) and nongynecological LMS (P = 0.03). In gynecological LMS, the coordinate expression of these four markers was the only independent prognosticator in multivariate analysis (hazard ratio, 4.2; 95% CI, 1.12 to 16; P = 0.03). Our findings indicate that CSF1 may play an important role in the clinical behavior of LMS that may open a window for new therapeutic reagents.

The macrophage colony-stimulating factor 1 response signature in breast carcinoma.

PURPOSE: Macrophages play an important role in breast carcinogenesis. The pathways that mediate the macrophage contribution to breast cancer and the heterogeneity that exists within macrophages are incompletely understood. Macrophage colony-stimulating factor 1 (CSF1) is the primary regulator of tissue macrophages. The purpose of this study was to define a novel CSF1 response signature and to evaluate its clinical and biological significance in breast cancer. EXPERIMENTAL DESIGN: We defined the CSF1 response signature by identifying genes overexpressed in tenosynovial giant cell tumor and pigmented villonodular synovitis (tumors composed predominantly of macrophages recruited in response to the overexpression of CSF1) compared with desmoid-type fibromatosis and solitary fibrous tumor. To characterize the CSF1 response signature in breast cancer, we analyzed the expression of CSF1 response signature genes in eight published breast cancer gene expression data sets (n = 982) and did immunohistochemistry and in situ hybridization for CSF1 response genes on a breast cancer tissue microarray (n = 283). RESULTS: In both the gene microarray and tissue microarray analyses, a consistent subset (17-25%) of breast cancers shows the CSF1 response signature. The signature is associated with higher tumor grade, decreased expression of estrogen receptor, decreased expression of progesterone receptor, and increased TP53 mutations (P < 0.001). CONCLUSIONS: Our data show that the CSF1 response signature is consistently seen in a subset of breast carcinomas and correlates with biological features of the tumor. Our findings provide insight into macrophage biology and may facilitate the development of personalized therapy for patients most likely to benefit from CSF1-targeted treatments.

The Stanford Tissue Microarray Database.

The Stanford Tissue Microarray Database (TMAD; http://tma.stanford.edu) is a public resource for disseminating annotated tissue images and associated expression data. Stanford University pathologists, researchers and their collaborators worldwide use TMAD for designing, viewing, scoring and analyzing their tissue microarrays. The use of tissue microarrays allows hundreds of human tissue cores to be simultaneously probed by antibodies to detect protein abundance (Immunohistochemistry; IHC), or by labeled nucleic acids (in situ hybridization; ISH) to detect transcript abundance. TMAD archives multi-wavelength fluorescence and bright-field images of tissue microarrays for scoring and analysis. As of July 2007, TMAD contained 205 161 images archiving 349 distinct probes on 1488 tissue microarray slides. Of these, 31 306 images for 68 probes on 125 slides have been released to the public. To date, 12 publications have been based on these raw public data. TMAD incorporates the NCI Thesaurus ontology for searching tissues in the cancer domain. Image processing researchers can extract images and scores for training and testing classification algorithms. The production server uses the Apache HTTP Server, Oracle Database and Perl application code. Source code is available to interested researchers under a no-cost license.

A panel of antibodies to determine site of origin and malignancy in smooth muscle tumors.

Leiomyosarcomas are malignant smooth muscle tumors that occur most commonly in the gynecologic tract and soft tissue. There are different diagnostic criteria of malignancy for smooth muscle tumors arising at gynecologic and soft tissue sites and they may be managed differently but determining the primary site of a smooth muscle tumor can be difficult in some cases. In addition, the distinction between malignant and benign gynecologic tract smooth muscle tumors on morphologic grounds can be challenging. Using a series of tissue microarrays that contain 245 cases of leiomyosarcomas (102 gynecologic) with survival data, and 49 cases of uterine leiomyoma, we examined the ability of selected immune-markers (estrogen receptor (ER) and WT1) to distinguish between leiomyosarcomas of gynecologic and nongynecologic origin. In addition, we examined whether immunostains for p16, p53 and Ki-67 could distinguish between malignant and benign gynecologic smooth muscle tumors. ER nuclear positivity was observed in 3 and 50% of the nongynecologic and gynecologic leiomyosarcomas, respectively (P<0.001). Nuclear WT1 positivity was seen in 0 and 8% of the nongynecologic and gynecologic leiomyosarcomas, respectively (P<0.001). 87% of primary gynecologic leiomyosarcomas and 2% of uterine leiomyomas showed diffuse (>or=50% of cells) p16 staining (P<0.001). 23% of gynecologic leiomyosarcomas showed p53 immunopositivity (>or=50% of cells) whereas none of the leiomyomas were positive for p53 (P<0.001). 65% of the gynecologic leiomyosarcomas and 0% of the leiomyomas exhibited >10% Ki-67 proliferation index (P<0.001). Diffuse p16 and p53 immunopositivity and high Ki-67 proliferation index, singly or in combination, yielded an overall sensitivity of 92% and specificity of 98% for distinguishing between gynecologic leiomyosarcomas and leiomyomas and can be used as indicators of malignancy for gynecologic smooth muscle tumors. Although ER positivity can be used to support the gynecologic origin of a leiomyosarcomas, nuclear WT1 immunostaining is of little use.

Inter-observer reproducibility of HER2 immunohistochemical assessment and concordance with fluorescent in situ hybridization (FISH): pathologist assessment compared to quantitative image analysis.

BACKGROUND: In breast cancer patients, HER2 overexpression is routinely assessed by immunohistochemistry (IHC) and equivocal cases are subject to fluorescent in situ hybridization (FISH). Our study compares HER2 scoring by histopathologists with automated quantitation of staining, and determines the concordance of IHC scores with FISH results. METHODS: A tissue microarray was constructed from 1,212 invasive breast carcinoma cases with linked treatment and outcome information. IHC slides were semi-quantitatively scored by two independent pathologists on a range of 0 to 3+, and also analyzed with an Ariol automated system by two operators. 616 cases were scorable by both IHC and FISH. RESULTS: Using data from unequivocal positive (3+) or negative (0, 1+) results, both visual and automated scores were highly consistent: there was excellent concordance between two pathologists (kappa = 1.000, 95% CI: 1-1), between two machines (kappa = 1.000, 95% CI: 1-1), and between both visual and both machine scores (kappa = 0.898, 95% CI: 0.775-0.979). Two pathologists successfully distinguished negative, positive and equivocal cases (kappa = 0.929, 95% CI: 0.909-0.946), with excellent agreement with machine 1 scores (kappa = 0.835, 95% CI: 0.806-0.862; kappa = 0.837, 95% CI: 0.81-0.862), and good agreement with machine 2 scores (kappa = 0.698, 95% CI: 0.6723-0.723; kappa = 0.709, 95% CI: 0.684-0.732), whereas the two machines showed good agreement (kappa = 0.806, 95% CI: 0.785-0.826). When comparing categorized IHC scores and FISH results, the agreement was excellent for visual 1 (kappa = 0.814, 95% CI: 0.768-0.856), good for visual 2 (kappa = 0.763, 95% CI: 0.712-0.81) and machine 1 (kappa = 0.665, 95% CI: 0.609-0.718), and moderate for machine 2 (kappa = 0.535, 95% CI: 0.485-0.584). CONCLUSION: A fully automated image analysis system run by an experienced operator can provide results consistent with visual HER2 scoring. Further development of such systems will likely improve the accuracy of detection and categorization of membranous staining, making this technique suitable for use in quality assurance programs and eventually in clinical practice.

Prognostic significance of macrophage infiltration in leiomyosarcomas.

PURPOSE: Macrophages are migratory cells that are frequently recruited to the site of tumors. Their presence is associated with poor clinical outcome in a variety of epithelial malignancies. The aim of this study is to examine the prognostic significance of tumor-associated macrophages in sarcomas. EXPERIMENTAL DESIGN: Global gene expression profiling data of a series of soft tissue tumors were analyzed for macrophage-associated gene expression. Immunohistochemistry on tissue microarrays containing leiomyosarcoma cases with known clinical outcome was used to verify the presence of macrophages and to examine the relationship between tumor-associated macrophages and clinical outcome. RESULTS: Gene expression profiling revealed high-level expression of several macrophage-associated genes such as CD163 and CD68 in a subset of leiomyosarcomas, indicating the presence of variable numbers of tumor-infiltrating macrophages. This was confirmed by CD68 and CD163 immunostaining of a tissue microarray containing 149 primary leiomyosarcomas. Kaplan-Meier survival analysis showed that high density of tumor-infiltrating macrophages as identified by CD163 or CD68 staining is associated with a significantly worse disease-specific survival in nongynecologic leiomyosarcomas, whereas leiomyosarcomas arising from the gynecologic tract showed no significant association between macrophage infiltration and survival. The presence of tumor necrosis did not correlate significantly with outcome. CONCLUSIONS: An increased density of CD163- or CD68-positive tumor-infiltrating macrophages is associated with poor outcome in nongynecologic leiomyosarcomas. This may help the clinical management of patients with leiomyosarcomas.

hCAP-D3 expression marks a prostate cancer subtype with favorable clinical behavior and androgen signaling signature.

Growing evidence suggests that only a fraction of prostate cancers detected clinically are potentially lethal. An important clinical issue is identifying men with indolent cancer who might be spared aggressive therapies with associated morbidities. Previously, using microarray analysis we defined 3 molecular subtypes of prostate cancer with different gene-expression patterns. One, subtype-1, displayed features consistent with more indolent behavior, where an immunohistochemical marker (AZGP1) for subtype-1 predicted favorable outcome after radical prostatectomy. Here we characterize a second candidate tissue biomarker, hCAP-D3, expressed in subtype-1 prostate tumors. hCAP-D3 expression, assayed by RNA in situ hybridization on a tissue microarray comprising 225 cases, was associated with decreased tumor recurrence after radical prostatectomy (P=0.004), independent of pathologic tumor stage, Gleason grade, and preoperative prostate-specific antigen levels. Simultaneous assessment of hCAP-D3 and AZGP1 expression in this tumor set improved outcome prediction. We have previously demonstrated that hCAP-D3 is induced by androgen in prostate cells. Extending this finding, Gene Set Enrichment Analysis revealed enrichment of androgen-responsive genes in subtype-1 tumors (P=0.019). Our findings identify hCAP-D3 as a new biomarker for subtype-1 tumors that improves prognostication, and reveal androgen signaling as an important biologic feature of this potentially clinically favorable molecular subtype.

A novel monoclonal antibody against DOG1 is a sensitive and specific marker for gastrointestinal stromal tumors.

Gastrointestinal stromal tumors (GIST) occur primarily in the wall of the intestine and are characterized by activating mutations in the receptor tyrosine kinases genes KIT or PDGFRA. The diagnosis of GIST relies heavily on the demonstration of KIT/CD117 protein expression by immunohistochemistry. However, KIT expression is absent in approximately 4% to 15% of GIST and this can complicate the diagnosis of GIST in patients who may benefit from treatment with receptor tyrosine kinase inhibitors. We previously identified DOG1/TMEM16A as a novel marker for GIST using a conventional rabbit antipeptide antiserum and an in situ hybridization probe. Here, we describe 2 new monoclonal antibodies against DOG1 (DOG1.1 and DOG1.3) and compare their staining profiles with KIT and CD34 antibodies on 447 cases of GIST. These included 306 cases with known mutational status for KIT and PDGFRA from a molecular consultation service. In addition, 935 other mesenchymal tumors and 432 nonsarcomatous tumors were studied. Both DOG1 antibodies showed high sensitivity and specificity for GIST, with DOG1.1 showing some advantages. This antibody yielded positive staining in 370 of 425 (87%) scorable GIST, whereas CD117 was positive in 317 of 428 (74%) GIST and CD34 in 254 of 430 (59%) GIST. In GIST with mutations in PDGFRA, 79% (23/29) showed DOG1.1 immunoreactivity while only 9% (3/32) and 27% (9/33) stained for CD117 and CD34, respectively. Only 1 of 326 (0.3%) leiomyosarcomas and 1 of 39 (2.5%) synovial sarcomas among the 935 soft tissue tumors examined showed positive immunostaining for DOG1.1. In addition, DOG1.1 immunoreactivity was seen in fewer cases of carcinoma, melanoma, and seminoma as compared with KIT.

Characterization of a novel anti-fatty acid synthase (FASN) antiserum in breast tissue.

Fatty acid synthase (FASN) expression has been reported in many different tumors, including breast cancer. In gene microarray studies, the fatty acid synthase gene co-clustered with cytokeratins 5 and 17 and other genes that defined the basal-like subset of breast cancers. To define the use of this marker in breast pathology, a rabbit polyclonal antiserum (S143) to a peptide fragment of this gene was produced and compared with a commercially available monoclonal antibody by immunohistochemistry on various tissue microarrays and whole tissue sections. The tissue microarrays included 1090 breast cancers and 244 normal breast tissues. Whole tissue sections consisted of benign and malignant tissues from breast resection specimens. In contrast to other 'basal' markers identified by gene expression profiling data, the fatty acid synthase (FASN) expression pattern in normal breast was notable for its expression in only a small subset of basal and suprabasal cells. Dual staining experiments revealed that the subpopulation of cells labeling with FASN did not coexpress myoepithelial markers CK5/6 or p63, but did coexpress e-cadherin. In addition to staining a subset of basal and suprabasal cells, the antiserum highlighted apocrine differentiation, and stained 106/144 (74%) cases of columnar cell lesions and five of five cases of flat epithelial atypia. Despite its association with basal keratins in gene array studies, FASN expression did not correlate significantly with the outcome in breast cancer. We describe an expression pattern that highlights only a subset of basal and suprabasal cells in normal breast ducts and we show by dual expression studies that this subset of cells is different from myoepithelial and basal cytokeratin-positive cells. In addition, FASN expression is described in apocrine metaplasia, columnar cell lesions, and flat epithelial atypia.

Determination of stromal signatures in breast carcinoma.

Many soft tissue tumors recapitulate features of normal connective tissue. We hypothesize that different types of fibroblastic tumors are representative of different populations of fibroblastic cells or different activation states of these cells. We examined two tumors with fibroblastic features, solitary fibrous tumor (SFT) and desmoid-type fibromatosis (DTF), by DNA microarray analysis and found that they have very different expression profiles, including significant differences in their patterns of expression of extracellular matrix genes and growth factors. Using immunohistochemistry and in situ hybridization on a tissue microarray, we found that genes specific for these two tumors have mutually specific expression in the stroma of nonneoplastic tissues. We defined a set of 786 gene spots whose pattern of expression distinguishes SFT from DTF. In an analysis of DNA microarray gene expression data from 295 previously published breast carcinomas, we found that expression of this gene set defined two groups of breast carcinomas with significant differences in overall survival. One of the groups had a favorable outcome and was defined by the expression of DTF genes. The other group of tumors had a poor prognosis and showed variable expression of genes enriched for SFT type. Our findings suggest that the host stromal response varies significantly among carcinomas and that gene expression patterns characteristic of soft tissue tumors can be used to discover new markers for normal connective tissue cells.