Rondell P. Graham

Nanoceria: A Rare-Earth Nanoparticle as a Novel Anti-Angiogenic Therapeutic Agent in Ovarian Cancer

Ovarian cancer (OvCa) is the fifth most common cause of death from all cancers among women in United Sates and the leading cause of death from gynecological malignancies. While most OvCa patients initially respond to surgical debulking and chemotherapy, 75% of patients later succumb to the disease. Thus, there is an urgent need to test novel therapeutic agents to counteract the high mortality rate associated with OvCa. In this context, we have developed and engineered Nanoceria (NCe), nanoparticles of cerium oxide, possessing anti-oxidant properties, to be used as a therapeutic agent in OvCa. We show for the first time that NCe significantly inhibited production of reactive oxygen species (ROS) in A2780 cells, attenuated growth factor (SDF1, HB-EGF, VEGF165 and HGF) mediated cell migration and invasion of SKOV3 cells, without affecting the cell proliferation. NCe treatment also inhibited VEGF165 induced proliferation, capillary tube formation, activation of VEGFR2 and MMP2 in human umbilical vascular endothelial cells (HUVEC). NCe (0.1 mg/kg body weigh) treatment of A2780 ovarian cancer cells injected intra-peritoneally in nude mice showed significant reduction (p<0.002) in tumor growth accompanied by decreased tumor cell proliferation as evident from reduced tumor size and Ki67 staining. Accumulation of NCe was found in tumors isolated from treated group using transmission electron microscopy (TEM) and inductively coupled plasma mass spectroscopy (ICP-MS). Reduction of the tumor mass was accompanied by attenuation of angiogenesis, as observed by reduced CD31 staining and specific apoptosis of vascular endothelial cells. Collectively, these results indicate that cerium oxide based NCe is a novel nanoparticle that can potentially be used as an anti-angiogenic therapeutic agent in ovarian cancer.

Isocitrate dehydrogenase 1 and 2 mutations in cholangiocarcinoma

Somatic mutations in isocitrate dehydrogenase 1 and 2 genes are common in gliomas and help stratify patients with brain cancer into histologic and molecular subtypes. However, these mutations are considered rare in other solid tumors. The aims of this study were to determine the frequency of isocitrate dehydrogenase 1 and 2 mutations in cholangiocarcinoma and to assess histopathologic differences between specimens with and without an isocitrate dehydrogenase mutation. We sequenced 94 formalin-fixed, paraffin-embedded cholangiocarcinoma (67 intrahepatic and 27 extrahepatic) assessing for isocitrate dehydrogenase 1 (codon 132) and isocitrate dehydrogenase 2 (codons 140 and 172) mutations. Multiple histopathologic characteristics were also evaluated and compared with isocitrate dehydrogenase 1/2 mutation status. Of the 94 evaluated specimens, 21 (22%) had a mutation including 14 isocitrate dehydrogenase 1 and 7 isocitrate dehydrogenase 2 mutations. Isocitrate dehydrogenase mutations were more frequently observed in intrahepatic cholangiocarcinoma than in extrahepatic cholangiocarcinoma (28% versus 7%, respectively; P = .030). The 14 isocitrate dehydrogenase 1 mutations were R132C (n = 9), R132S (n = 2), R132G (n = 2), and R132L (n = 1). The 7 isocitrate dehydrogenase 2 mutations were R172K (n = 5), R172M (n = 1), and R172G (n = 1). Isocitrate dehydrogenase mutations were more frequently observed in tumors with clear cell change (P < .001) and poorly differentiated histology (P = .012). The results of this study show for the first time that isocitrate dehydrogenase 1 and 2 genes are mutated in cholangiocarcinoma. The results of this study are encouraging because it identifies a new potential target for genotype-directed therapeutic trials and may represent a potential biomarker for earlier detection of cholangiocarcinoma in a subset of cases.

Fibroblast growth factor receptor 2 translocations in intrahepatic cholangiocarcinoma

Patients with cholangiocarcinoma often present with locally advanced or metastatic disease. There is a need for effective therapeutic strategies for advanced stage cholangiocarcinoma. Recently, FGFR2 translocations have been identified as a potential target for tyrosine kinase inhibitor therapies. This study evaluated 152 cholangiocarcinomas and 4 intraductal papillary biliary neoplasms of the bile duct for presence of FGFR2 translocations by fluorescence in situ hybridization and characterized the clinicopathologic features of cases with FGFR2 translocations. Thirteen (10 women, 3 men; 8%) of 156 biliary tumors harbored FGFR2 translocations, including 12 intrahepatic cholangiocarcinomas (12/96; 13%) and 1 intraductal papillary neoplasm of the bile duct. Histologically, cholangiocarcinomas with FGFR2 translocations displayed prominent intraductal growth (62%) or anastomosing tubular glands with desmoplasia (38%). Immunohistochemically, the tumors with FGFR2 translocations frequently showed weak and patchy expression of CK19 (77%). Markers of the stem cell phenotype in cholangiocarcinoma, HepPar1 and CK20, were negative in all cases. The median cancer-specific survival for patients whose tumors harbored FGFR2 translocations was 123 months compared to 37 months for cases without FGFR2 translocations (P = .039). This study also assessed 100 cholangiocarcinomas for ERBB2 amplification and ROS1 translocations. Of the cases tested, 3% and 1% were positive for ERBB2 amplification and ROS1 translocation, respectively. These results confirm that FGFR2, ERRB2, and ROS1 alterations are potential therapeutic targets for intrahepatic cholangiocarcinoma.

Ossifying Fibromyxoid Tumor of Soft Parts: A Clinicopathologic, Proteomic and Genomic Study

Ossifying fibromyxoid tumor (OFMTs) of soft parts is a rare soft tissue and bone tumor of borderline malignancy displaying an uncertain line of differentiation. The existence of fully malignant OFMT is controversial. To better understand the natural history and line of differentiation taken by OFMT, we studied 46 cases by light microscopic, immunohistochemical (IHC), genomic, proteomic, and fluorescence in situ hybridization (FISH) methods. Cases were classified according to the 2003 Folpe and Weiss system. Clinical and follow-up information was obtained. IHC for S-100 protein, desmin, epithelial membrane antigen (EMA), cytokeratins, smooth muscle actin (SMA), INI-1, neurofilament protein (NFP), CD56d excitatory amino acid transporter-4 (EAAT4), and MUC4 was performed on formalin-fixed, paraffin-embedded (FFPE) tissues. Gene expression profiling and proteomic studies were conducted on FFPE tissues from 13 and 5 cases, respectively. FISH for INI-1 was performed on 10 cases. The 46 tumors arose in 29 men and 17 women (median age, 52 y; range 39 to 63 y) and involved the proximal (N=17) and distal extremities (N=13), head and neck (N=9), and trunk (N=5). Median tumor size was 5.4 cm (range, 1.0 to 21.0 cm). Cases were classified as typical OFMT (26 of 46, 57%), atypical OFMT (5 of 46, 11%), and malignant OFMT (15 of 46 cases, 32%). Clinical follow-up (27 cases, median 55 months’ duration) showed all patients with typical and atypical OFMT to be alive without disease. Adverse events, including 3 local recurrences, 3 metastases, and 3 deaths, were seen only in malignant OFMT. IHC results were as follows: S-100 protein (30 of 41, 73%), desmin (15 of 39, 38%), cytokeratin (4 of 35, 11%), EMA (5 of 32, 16%), SMA (2 of 34, 6%), INI-1 (lost in mosaic pattern in 14 of 19, 74%), EAAT4 (31 of 39, 80%), MUC4 (3 of 14, 21%), NFP (8 of 10, 80%) and CD56 (6 of 14, 43%). Gene expression profiling showed typical and malignant OFMTs to cluster together, distinct from schwannian tumors. Proteomic study showed expression of various collagens, S-100 protein, and neuron-related proteins. FISH showed INI-1 deletion in 5 of 7 (71%) cases. We conclude that malignant OFMTs exist and may be recognized by the previously proposed criteria of Folpe and Weiss. Expression of neuron-related markers, in addition to Schwann cell and cartilage-associated markers, suggests a “scrambled” phenotype in OFMTs. Loss of INI-1 or other genes on 22q is likely important in the pathogenesis of these rare tumors.

Recurrent PAX3 - MAML3 fusion in biphenotypic sinonasal sarcoma

Biphenotypic sinonasal sarcoma (SNS) is a newly described tumor of the nasal and paranasal areas. Here we report a recurrent chromosomal translocation in SNS, t(2;4)(q35;q31.1), resulting in a PAX3-MAML3 fusion protein that is a potent transcriptional activator of PAX3 response elements. The SNS phenotype is characterized by aberrant expression of genes involved in neuroectodermal and myogenic differentiation, closely simulating the developmental roles of PAX3.

DNAJB1-PRKACA is specific for fibrolamellar carcinoma

Fibrolamellar carcinoma is a distinct subtype of hepatocellular carcinoma that predominantly affects young patients without underlying cirrhosis. A recurrent DNAJB1-PRKACA fusion has recently been reported in fibrolamellar carcinomas. To determine the specificity of this fusion and to develop routinely available clinical methods of detection, we developed an RT-PCR assay for paraffin-embedded tissues and a FISH probe for detection of the rearrangements of the PRKACA locus. We also developed an RNA in situ hybridization assay to assess expression levels of the total chimeric transcript and wild-type transcripts. A total of 106 primary liver tumors were studied by RT-PCR, including 26 fibrolamellar carcinomas (4 of which were metastases to the abdominal wall or lymph nodes), 25 conventional hepatocellular carcinomas, 25 cholangiocarcinomas, 25 hepatic adenomas, and 5 hepatoblastomas. RT-PCR was successful in 92% of tested fibrolamellar carcinoma cases (24/26) and the DNAJB1-PRKACA fusion transcript was found in all fibrolamellar carcinomas but not in other tumor types. FISH was tested in 19 fibrolamellar carcinomas and in 6 scirrhous hepatocellular carcinomas, which can closely mimic fibrolamellar carcinoma. Rearrangements of the PRKACA locus was seen in all 19 fibrolamellar carcinoma specimens, but in none of the scirrhous hepatocellular carcinomas. Finally, a RNA in situ hybridization strategy was positive in 7/7 successfully hybridized cases, and showed mRNA over-expression in all of the fibrolamellar carcinomas. In addition, the stromal cells embedded in the characteristic intratumoral fibrosis of fibrolamellar carcinomas and the background liver tissues were negative for the DNAJB1-PRKACA fusion by all tested methods. In conclusion, detection of DNAJB1-PRKACA is a very sensitive and specific finding in support of the diagnosis of fibrolamellar carcinoma.

PHF1 rearrangements in ossifying fibromyxoid tumors of soft parts: A fluorescence in situ hybridization study of 41 cases with emphasis on the malignant variant.

Ossifying fibromyxoid tumor of soft parts (OFMT) is a rare soft tissue neoplasm of uncertain differentiation. Very recently recurrent rearrangements of the PHF1 gene have been reported in OFMT, including typical, atypical, and malignant variants. We sought to validate and extend these findings in a larger series of well-characterized OFMT, in particular malignant variants. Slides and blocks from 41 OFMT were retrieved, rereviewed, and classified as typical, atypical, and malignant using previously published criteria. Interphase fluorescence in situ hybridization (FISH) was performed on paraffin-embedded sections of each case using a break-apart probe strategy, with direct-labeled FISH probes designed from bacterial artificial chromosomes. The 41 tumors occurred in 23 men and 18 women with a mean age of 55 years and involved the head and neck, trunk, and upper and lower limbs. The tumors were classified as typical (n=14), atypical (n=6) and malignant (n=21). PHF1 rearrangements were detected in 20 of 41 cases (49%) including 43% typical, 50% atypical, and 52% malignant cases. The results of our study confirm previous findings, with PHF1 rearrangements present in nearly 50% of OFMT, including roughly similar percentages of typical, atypical, and malignant tumors. These results support our previous hypothesis that OFMT might represent a translocation-associated tumor, underscore the likely importance of PHF1 rearrangements in the pathogenesis of these lesions, confirm the relationship between typical and malignant OFMT, and suggest a role for PHF1 FISH in the diagnosis of morphologically challenging cases.

Eosinophilia and Allergic Disorders in Autoimmune Pancreatitis

OBJECTIVES:In autoimmune pancreatitis (AIP), the prevalence, interrelationships, and significance of peripheral eosinophilia, allergic disorders, and eosinophil infiltration in the pancreas remain unclear.METHODS:From medical records, we obtained data on peripheral eosinophil counts at presentation and follow-up, and clinical diagnoses of allergic disorders in 97 AIP patients (78 type 1 and 19 type 2), which were compared with matched healthy controls. Available pancreatic histologic specimens were graded for eosinophils. Peripheral eosinophilia was defined as counts >0.5 × 109 per liter. We examined nature of and association between these parameters in AIP.RESULTS:Among 78 type 1 AIP patients (mean age 62±14 years, 77% men), peripheral eosinophilia at presentation was diagnosed in 12% and allergic disorders in 15% (vs. 0 and 4% in controls, P=0.0004 and 0.006, respectively). Allergic disorders were observed in 27 and 11% of type 1 AIP with and without eosinophilia, respectively (P=0.08). Patients with and without peripheral eosinophilia were similar in clinical profile. Moderate-to-severe eosinophil infiltration was present in 67% of pancreas resection specimens and did not correlate with peripheral eosinophilia. Type 2 AIP did not differ from type 1 AIP in any of these parameters.CONCLUSIONS:Peripheral eosinophilia, allergic disorders, and pancreatic eosinophil infiltration are associated with AIP. Eosinophilia in AIP may not reflect an allergic phenomenon, but appears to be consistent with autoimmune mechanism.

Islet-1 is a sensitive but not entirely specific marker for pancreatic neuroendocrine neoplasms and their metastases.

Islet-1 (Isl1) is a transcription factor involved in the embryogenesis of islets of Langerhans. Immunohistochemically, Isl1 is a sensitive lineage-specific marker for pancreatic neuroendocrine neoplasms (NENs) and their metastases. Its specificity has not been documented, nor have large numbers of NENs from other parts of the gut or other organs been studied. We examined Isl1 expression in 203 primary NENs (gastroenteropancreatic, lung, breast, and ovarian neoplasms) and 40 hepatic NEN metastases (enteropancreatic and lung neoplasms) from known primaries. The correlation between Isl1 and CDX2 expression was studied using a tissue microarray containing 46 pancreatic NENs. Immunostaining for Isl1 and CDX2 was also performed in selected NENs from other sites. Isl1 was positive in 90% of pancreatic, 89% of duodenal, 100% of rectal, 38% of colonic, 14% of appendiceal, and 6% of ileal primaries. Isl1 was negative in all other NENs. Among metastatic neoplasms, 76% of pancreatic and 2 of 2 rectal NEN metastases were Isl1 positive, whereas all other tested metastases were negative. The overall sensitivity and specificity of Isl1 in identifying primary pancreatic NENs was 88% and 80%, respectively. Thirty-six of 46 pancreatic NENs in the tissue microarray were Isl1 positive; 4 were Isl1 negative but CDX2 positive. Our findings confirm that Isl1 is a sensitive marker of pancreatic origin in cases of metastatic NEN. However, Isl1 does not distinguish pancreatic NEN from duodenal and colorectal NEN, even when used in association with CDX2.

Isolated IgG4-related sclerosing cholangitis: A report of 9 cases

Extrahepatic bile ducts are the most commonly involved extrapancreatic organ site in patients with type 1 autoimmune pancreatitis. IgG4-related sclerosing cholangitis (IgG4-SC) alone, without evidence of pancreatic or other organ involvement, is uncommon and is difficult to distinguish from cholangiocarcinoma preoperatively. We describe 9 patients with isolated IgG4-SC over an approximate 10-year period, each clinically suspected to have cholangiocarcinoma. We examined the clinical, radiological, cytologic (including fluorescence in situ hybridization results), and histologic features. IgG and IgG4 immunohistochemistry were performed. All 9 patients were middle-aged men who presented with obstructive jaundice. The biliary strictures involved all parts of the extrahepatic biliary tree. Serum IgG4 was slightly elevated in three of eight patients. Cytologic findings were interpreted as negative in six, atypical in one, and suspicious for adenocarcinoma in one. Fluorescence in situ hybridization revealed aneuploidy in one and was equivocal (trisomy 7) in 2. Eight of 9 patients underwent radical resection for suspected cholangiocarcinoma. There was only one case diagnosed with IgG4-SC preoperatively based on biopsy. Histologic sections revealed a prominent lymphoplasmacytic infiltrate with storiform fibrosis and marked increased IgG4-positive plasma cells (≥30/high-power field) in all specimens. Fifty percent of cases (4/8) had IgG4/IgG plasma cell ratio >40%. On median follow-up of 2.8 years, no relapse has occurred in any patient. Extrahepatic IgG4-SC may present as an isolated lesion mimicking cholangiocarcinoma. The diagnosis can be challenging. Clinicians and pathologists should recognize this to avoid major surgery.