Sushama Varma

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 ∼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.

BDNF overexpression increases dendrite complexity in hippocampal dentate gyrus

There is increasing evidence that brain-derived neurotrophic factor (BDNF) modulates synaptic and morphological plasticity in the developing and mature nervous system. Plasticity may be modulated partially by BDNFs effects on dendritic structure. Utilizing transgenic mice where BDNF overexpression was controlled by the beta-actin promoter, we evaluated the effects of long-term overexpression of BDNF on the dendritic structure of granule cells in the hippocampal dentate gyrus. BDNF transgenic mice provided the opportunity to investigate the effects of modestly increased BDNF levels on dendrite structure in the complex in vivo environment. While the elevated BDNF levels were insufficient to change levels of TrkB receptor isoforms or downstream TrkB signaling, they did increase dendrite complexity of dentate granule cells. These cells showed an increased number of first order dendrites, of total dendritic length and of total number of branch points. These results suggest that dendrite structure of granule cells is tightly regulated and is sensitive to modest increases in levels of BDNF. This is the first study to evaluate the effects of BDNF overexpression on dendrite morphology in the intact hippocampus and extends previous in vitro observations that BDNF influences synaptic plasticity by increasing complexity of dendritic arbors.

MYB Expression and Translocation in Adenoid Cystic Carcinomas and Other Salivary Gland Tumors With Clinicopathologic Correlation

Background Adenoid cystic carcinoma is a locally aggressive salivary gland neoplasm, which has a poor long-term prognosis. A chromosomal translocation involving the genes encoding the transcription factors, MYB and NFIB, has been recently discovered in these tumors. Methods MYB translocation and protein expression were studied in 37 adenoid cystic carcinomas, 112 other salivary gland neoplasms, and 409 nonsalivary gland neoplasms by fluorescence in situ hybridization and immunohistochemistry. MYB translocation and expression status in adenoid cystic carcinoma was correlated with clinicopathologic features including outcome, with a median follow-up of 77.1 months (range, 23.2 to 217.5 mo) for living patients. Results A balanced translocation between MYB and NFIB is present in 49% of adenoid cystic carcinomas but is not identified in other salivary gland tumors or nonsalivary gland neoplasms. There is no apparent translocation of MYB in 35% of the cases. Strong Myb immunostaining is very specific for adenoid cystic carcinomas but is only present in 65% of all cases. It is interesting to note that Myb immunostaining is confined to the basal cell component although the translocation is present in all the cells. Neoplasms with MYB translocation show a trend toward higher local relapse rates, but the results are not statistically significant with the current number of cases. Conclusions MYB translocation and expression are useful diagnostic markers for a subset of adenoid cystic carcinomas. The presence of the translocation may be indicative of local aggressive behavior, but a larger cohort may be required to show statistical significance.

Transcriptional profiling of long non-coding RNAs and novel transcribed regions across a diverse panel of archived human cancers

BackgroundMolecular characterization of tumors has been critical for identifying important genes in cancer biology and for improving tumor classification and diagnosis. Long non-coding RNAs, as a new, relatively unstudied class of transcripts, provide a rich opportunity to identify both functional drivers and cancer-type-specific biomarkers. However, despite the potential importance of long non-coding RNAs to the cancer field, no comprehensive survey of long non-coding RNA expression across various cancers has been reported.ResultsWe performed a sequencing-based transcriptional survey of both known long non-coding RNAs and novel intergenic transcripts across a panel of 64 archival tumor samples comprising 17 diagnostic subtypes of adenocarcinomas, squamous cell carcinomas and sarcomas. We identified hundreds of transcripts from among the known 1,065 long non-coding RNAs surveyed that showed variability in transcript levels between the tumor types and are therefore potential biomarker candidates. We discovered 1,071 novel intergenic transcribed regions and demonstrate that these show similar patterns of variability between tumor types. We found that many of these differentially expressed cancer transcripts are also expressed in normal tissues. One such novel transcript specifically expressed in breast tissue was further evaluated using RNA in situ hybridization on a panel of breast tumors. It was shown to correlate with low tumor grade and estrogen receptor expression, thereby representing a potentially important new breast cancer biomarker.ConclusionsThis study provides the first large survey of long non-coding RNA expression within a panel of solid cancers and also identifies a number of novel transcribed regions differentially expressed across distinct cancer types that represent candidate biomarkers for future research.

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.

BDNF overexpression produces a long-term increase in myelin formation in the peripheral nervous system

The neurotrophin brain‐derived neurotrophic factor (BDNF) is an endogenous regulator of the myelination process during development in the peripheral nervous system. Enhancement of myelin formation by BDNF is mediated by the neurotrophin receptor p75NTR. Although this neurotrophin is a positive modulator of myelination during early development, the final effects of BDNF on myelin sheaths after active myelination is completed are largely unknown. Using BDNF transgenic mice, we examined the long‐term effects of BDNF on myelination of the peripheral nervous system in vivo. Elevation of BDNF levels in the transgenic mice produced an increase in both the rate and extent of the myelination process. BDNF enhanced and accelerated myelin formation during early development and this increase in myelin content and thickness was maintained in adulthood. Besides enhanced myelination, BDNF also influenced axon caliber size but to a lesser extent. This lagging increase in axon caliber compared to myelin suggests that the axon size is not the only determinant of myelin thickness.

Genome evolution during progression to breast cancer.

Cancer evolution involves cycles of genomic damage, epigenetic deregulation, and increased cellular proliferation that eventually culminate in the carcinoma phenotype. Early neoplasias, which are often found concurrently with carcinomas and are histologically distinguishable from normal breast tissue, are less advanced in phenotype than carcinomas and are thought to represent precursor stages. To elucidate their role in cancer evolution we performed comparative whole-genome sequencing of early neoplasias, matched normal tissue, and carcinomas from six patients, for a total of 31 samples. By using somatic mutations as lineage markers we built trees that relate the tissue samples within each patient. On the basis of these lineage trees we inferred the order, timing, and rates of genomic events. In four out of six cases, an early neoplasia and the carcinoma share a mutated common ancestor with recurring aneuploidies, and in all six cases evolution accelerated in the carcinoma lineage. Transition spectra of somatic mutations are stable and consistent across cases, suggesting that accumulation of somatic mutations is a result of increased ancestral cell division rather than specific mutational mechanisms. In contrast to highly advanced tumors that are the focus of much of the current cancer genome sequencing, neither the early neoplasia genomes nor the carcinomas are enriched with potentially functional somatic point mutations. Aneuploidies that occur in common ancestors of neoplastic and tumor cells are the earliest events that affect a large number of genes and may predispose breast tissue to eventual development of invasive carcinoma.

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.

Gene expression profiling identifies p63 as a diagnostic marker for giant cell tumor of the bone

Giant cell tumor of the bone (GCTOB) is a primary bone tumor that occurs mainly in young adults and is capable of locally aggressive growth. Its histologic appearance can resemble a number of benign and malignant tumors but no useful diagnostic marker is known currently. To identify diagnostic markers for this tumor, global gene expression profiling using cDNA microarray was performed on 6 fresh-frozen GCTOB, 3 aneurysmal bone cysts, 4 fibrous dysplasias and 12 giant cell tumors of tendon sheath/diffuse-type giant cell tumors. Unsupervised hierarchical clustering separated the tumors based on their histopathologic types, and significance analysis of microarray identified several genes including TP73L (encoding the p63 protein) that are significantly highly expressed in GCTOB relative to these other tumors. The diagnostic utility of p63 was subsequently confirmed using anti-p63 antibody on a series of 26 GCTOB, 25 aneurysmal bone cysts, 15 chondroblastomas, 13 giant cell reparative granulomas, 13 chondromyxoid fibromas, 4 brown tumors, 4 fibrous dysplasias, 53 giant cell tumors of tendon sheath/diffuse-type giant cell tumors and 385 additional mesenchymal tumors in tissue microarrays. Strong p63 nuclear staining was present in 18 of 26 (69%) GCTOB, 3 of 15 (20%) chondroblastomas and in 1 of 25 (4%) aneurysmal bone cysts while none of the other tumors commonly considered in the differential diagnosis of GCTOB showed any detectable p63 staining. Strong p63 staining is rare in bone and soft-tissue tumors in general. In contrast to the pattern of p63 staining, the majority of the chondroblastomas (70%) demonstrated S-100 immunoreactivity while only a minority of the GCTOB (8%) was immunoreactive for S-100. These findings altogether show that p63 can be used as a diagnostic marker to aid the clinical diagnosis of GCTOB.

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.