Emerging Entities in Salivary Pathology: A Practical Review of Sclerosing Microcystic Adenocarcinoma, Microsecretory Adenocarcinoma, and Secretory Myoepithelial Carcinoma.

In recent years, increased molecular testing and improved immunohistochemical panels have facilitated more specific classification of salivary gland carcinomas, leading to recognition of several novel tumor types and unique histologic variants. Sclerosing microcystic adenocarcinoma, microsecretory adenocarcinoma, and secretory myoepithelial carcinoma are three such recently described entities that demonstrate low-grade cytology, production of prominent secretory material, and variable amounts of sclerotic stroma. This review provides a practical overview of these important and overlapping emerging entities in salivary gland pathology with a focus on distinctive histologic features and helpful ancillary studies that differentiate them from a wide range of familiar morphologic mimics.

Sclerosing Polycystic Adenoma.

Sclerosing polycystic adenoma (SPA) is the more appropriate name for sclerosing polycystic adenosis. SPA is an uncommon salivary gland lesion with a constellation of unusual histologic findings that were originally interpreted as analogous to breast fibrocystic changes. The histologic findings in SPA include fibrosis, cystic alterations, apocrine metaplasia, and proliferations of ducts, acini, and myoepithelial cells in variable proportions. Because of its unusual mixed histology, SPA may be confused with a variety of lesions, ranging from reactive conditions to benign or even malignant neoplasms. The features of SPA are reviewed, with an emphasis on resolving its differential diagnosis.

Histopathological evaluation of minor salivary gland papillary-cystic tumours: focus on genetic alterations in sialadenoma papilliferum and intraductal papillary mucinous neoplasm.

AIMS: Minor salivary gland tumours showing a predominant papillary-cystic structure are rare, and constitute a mixture of various types of neoplasm; thus, the histopathological assessment of these tumours poses a significant diagnostic challenge. We aimed to delineate the histological characteristics of these tumours and further mutational aspects with a particular focus on sialadenoma papilliferum (SP) and intraductal papillary mucinous neoplasm (IPMN). METHODS AND RESULTS: We retrieved 28 papillary-cystic tumours of the minor salivary glands, and performed histological re-evaluation and mutation analyses of several key oncogenes. The histological classifications were as follows: SP (n = 10), SP-like intraductal papillary tumour (SP-IPT) (n = 2), IPMN (n = 9), intraductal papilloma, cystadenoma, and cystadenocarcinoma (two, three and two respectively). Whereas SP typically consisted of a combination of exophytic squamous epithelium and endophytic intraductal papillary infoldings, SP-IPT lacked the exophytic component. SP and SP-IPT frequently harboured BRAF V600E mutations (75.0%), which were identified in both squamous and ductal components. IPMN was characterised by a well-demarcated cystic lesion filled exclusively with a papillary proliferation of mucinous cells and a high rate of AKT1 E17K mutations (88.9%). Intraductal papillomas were unilocular cystic lesions with intraluminal papillary growth of bland columnar cells. In contrast, both cystadenomas and cystadenocarcinomas showed a multicystic appearance with a papillary configuration. Cystadenocarcinomas invaded the surrounding tissue and were composed of markedly atypical tumour cells. CONCLUSION: The appropriate interpretation of histological findings and specific genetic alterations (e.g. BRAF V600E and AKT1 E17K in SP and IPMN) would be useful for the correct diagnosis of minor salivary gland papillary-cystic tumours.

Mechanism of cytokinesis failure in ovarian cystadenomas with defective BRCA1 and P53 pathways.

We previously described an in vitro model in which serous ovarian cystadenomas were transfected with SV40 large T antigen, resulting in loss of RB and P53 functions and thus mimicking genetic defects present in early high-grade serous extra-uterine Müllerian (traditionally called high-grade serous ovarian) carcinomas including those associated with the BRCA1 mutation carrier state. We showed that replicative aging in this cell culture model leads to a mitotic arrest at the spindle assembly checkpoint. Here we show that this arrest is due to a reduction in microtubule anchoring that coincides with decreased expression of the BUB1 kinase and of the phosphorylated form of its substrate, BUB3. The ensuing prolonged mitotic arrest leads to cohesion fatigue resulting in cell death or, in cells that recover from this arrest, in cytokinesis failure and polyploidy. Down-regulation of BRCA1 to levels similar to those present in BRCA1 mutation carriers leads to increased and uncontrolled microtubule anchoring to the kinetochore resulting in overcoming the spindle assembly checkpoint. Progression to anaphase under those conditions is associated with formation of chromatin bridges between chromosomal plates due to abnormal attachments to the kinetochore, significantly increasing the risk of cytokinesis failure. The dependence of this scenario on accelerated replicative aging can, at least in part, account for the site specificity of the cancers associated with the BRCA1 mutation carrier state, as epithelia of the mammary gland and of the reproductive tract are targets of cell-nonautonomous consequences of this carrier state on cellular proliferation associated with menstrual cycle progressions.

Integrated transcriptomic and epigenomic analysis of ovarian cancer reveals epigenetically silenced GULP1.

Many epigenetically inactivated genes involved in ovarian cancer (OC) development and progression remain to be identified. In this study we undertook an integrated approach that consisted of identification of genome-wide expression patterns of primary OC samples and normal ovarian surface epithelium along with a pharmacologic unmasking strategy using 3 OC and 3 immortalized normal ovarian epithelial cell lines. Our filtering scheme identified 43 OC specific methylated genes and among the 5 top candidates (GULP1, CLIP4, BAMBI, NT5E, TGFß2), we performed extended studies of GULP1. In a training set, we identified GULP1 methylation in 21/61 (34%) of cases with 100% specificity. In an independent cohort, the observed methylation was 40% (146/365) in OC, 12.5% (2/16) in borderline tumors, 11% (2/18) in cystadenoma and 0% (0/13) in normal ovarian epithelium samples. GULP1 methylation was associated with clinicopathological parameters such as stage III/IV (p = 0.001), poorly differentiated grade (p = 0.033), residual disease (p < 0.0003), worse overall (p = 0.02) and disease specific survival (p = 0.01). Depletion of GULP1 in OC cells led to increased pro-survival signaling, inducing survival and colony formation, whereas reconstitution of GULP1 negated these effects, suggesting that GULP1 is required for maintaining cellular growth control.

Impact of next-generation sequencing on the clinical diagnosis of pancreatic cysts.

BACKGROUND AND AIMS: The value of next-generation sequencing (NGS) of pancreatic cyst fluid relative to the clinical and imaging impression has not been well-studied. The aim of this study was to assess the impact of NGS on the clinical diagnosis from imaging and carcinoembryonic antigen (CEA) and thus the management of pancreatic cysts. METHODS: Ninety-two pancreatic cyst fluids from 86 patients were analyzed by cytology, CEA, and targeted NGS. Cysts were classified by imaging as nonmucinous, mucinous, or not specified. NGS results were compared with the imaging impression stratified by CEA and cytology. RESULTS: NGS impacted the clinical diagnosis by defining a cyst as mucinous in 48% of cysts without elevated CEA levels. The VHL gene in 2 intraductal papillary mucinous neoplasms (IPMNs) supported a serous cystadenoma. Twenty percent of cysts that were nonmucinous by imaging were mucinous by NGS. Of the 14 not-specific cysts, CEA levels were not elevated in 12 (86%), and NGS established a mucinous etiology in 3 (25%). A KRAS or GNAS mutation supported an IPMN with nonmucinous CEA in 71%. A KRAS mutation reclassified 19% of nonneoplastic cysts with nonmucinous CEA as mucinous. Seven cyst fluids (8%) had either a TP53 mutation or loss of CDKN2A or SMAD4 in addition to KRAS and/or GNAS mutations; 5 of 7 (71%) were clinically malignant, and high-grade cytology was detected in all 5. Overall, CEA was more specific for a mucinous etiology (100%), but NGS was more sensitive (86% vs 57%). CONCLUSIONS: NGS of pancreatic cyst fluid impacts clinical diagnosis and patient management by defining, supporting, or changing the clinical diagnosis based on imaging and CEA. NGS was most valuable in identifying mucinous cysts with nonmucinous CEA. An added benefit is the potential to detect mutations late in the progression to malignancy that may increase the risk classification of the cyst based on imaging and cytology.

Granulosa Cell-Specific Brca1 Loss Alone or Combined with Trp53 Haploinsufficiency and Transgenic FSH Expression Fails to Induce Ovarian Tumors.

BRCA1 mutations are associated with ovarian cancer. Previous studies reported that murine granulosa cell (GC) Brca1 loss caused ovarian-uterine tumors resembling serous cystadenomas, but the pathogenesis of these tumors may have been confounded by ectopic Brca1 expression and altered estrous cycling. We have used Tg.AMH.Cre conferring proven ovarian and GC-specific Cre activity to selectively target Brca1 disruption, denoted Brca1(GC-/-). Furthermore, ovary-specific Brca1(GC-/-) was combined with global Trp53 haploinsufficiency (Trp53(+/-)) and transgenic follicle-stimulating hormone (Tg.FSH) overexpression as a multi-hit strategy to investigate additional genetic and hormonal ovarian tumorigenesis mechanisms. However, 12-month-old Brca1(GC-/-) mice had no detectable ovarian or uterine tumors. Brca1(GC-/-) mice had significantly increased ovary weights, follicles exhibiting more pyknotic granulosa cells, and fewer corpora lutea with regular estrous cycling compared to controls. Isolated Brca1(GC-/-) mutation lengthened the estrous cycle and proestrus stage; however, ovarian cystadenomas were not observed, even when Brca1(GC-/-) was combined with Trp53(+/-) and overexpressed Tg.FSH. Our Brca1(GC-/-) models reveal that specific intra-follicular Brca1 loss alone, or combined with cancer-promoting genetic (Trp53 loss) and endocrine (high serum FSH) changes, was not sufficient to cause ovarian tumors. Our findings show that the ovary is remarkably resistant to oncogenesis, and support the emerging view of an extragonadal, multi-hit origin for ovarian tumorigenesis.

GNAS mutation is a frequent event in pancreatic intraductal papillary mucinous neoplasms and associated adenocarcinomas.

In contrast to pancreatic ductal adenocarcinomas (PDAs), intraductal papillary mucinous neoplasms (IPMNs) frequently harbour GNAS mutations. To characterise GNAS-mutated pancreatic carcinomas, we examined mutations of GNAS and KRAS in 290 pancreatic adenocarcinomas and 77 pancreatic intraepithelial neoplasias (PanINs). In 64 % (39/61) of IPMNs and 37 % (11/30) of IPMN-associated adenocarcinomas, a GNAS mutation was found. GNAS mutations were frequent (78 %, 7/9) in mucinous carcinomas, with or without associated IPMN. In contrast, GNAS mutations were rarely observed in PDAs (1 %, 1/88) and PanINs (3 %, 2/77), and not at all in mucinous cystic neoplasms (MCNs) (0/10), neuroendocrine neoplasms (0/52), acinar cell neoplasms (0/16), serous cystadenomas (0/10), and solid-pseudopapillary neoplasms (0/14). We found GNAS mutations in 55/91 IPMNs with or without associated invasive carcinoma, solely in intestinal-type (78 %, 21/27) and gastric-type (62 %, 34/55) IPMNs. Of the IPMN-associated adenocarcinomas, mucinous-subtype tumours harboured GNAS mutations more frequently (83 %, 5/6) than tubular-subtype tumours (25 %, 6/24) (p = 0.02). We separately analysed GNAS in the adenocarcinoma and the IPMN component in the IPMN-associated adenocarcinomas. In all mucinous-subtype tumours, the two components exhibited identical genotypes. In contrast, the two components in 8 of 24 tubular-subtype tumours exhibited different genotypes, indicating intratumour heterogeneity. In conclusion, mucinous carcinomas with or without associated IPMN as well as IPMNs frequently harbour a GNAS mutation, reinforcing the notion that these constitute a spectrum of pancreatic tumours. Clinically and pathologically, these tumours are associated, but GNAS mutation sheds further light on this spectrum.

A vascular model of Tsc1 deficiency accelerates renal tumor formation with accompanying hemangiosarcomas.

UNLABELLED: Tuberous sclerosis complex (TSC) is an autosomal disease caused by inactivating mutations in either of the tumor suppressor genes TSC1 or TSC2. TSC-associated tumor growth is present in multiple tissues and organs including brain, kidney, liver, heart, lungs, and skin. In the kidney, TSC angiomyolipomas have aberrant vascular structures with abnormal endothelial cells, suggesting a role for endothelial mTORC1 function. In the current report, a genetically engineered mouse model (GEMM) with a conditional knockout allele of Tsc1 with a Darpp32-Cre allele displayed accelerated formation of both kidney cystadenomas and paw hemangiosarcomas. All mutant mice developed hemangiosarcomas on multiple paws by 6 weeks of age. By 16 weeks of age, the average mutant hind paw was 4.0 mm in diameter, nearly double the size of control mice. Furthermore, the hemangiosarcomas and kidney cystadenomas were responsive to intraperitoneal rapamycin treatment. Immunoblotting and immunostaining for phospho-S6 (pS6) and phospho-CAD showed that the effect of rapamycin on tumor size was through inhibition of the mTOR signaling pathway. Finally, elevated VEGF mRNA levels were also observed in hemangiosarcoma specimens. Because paw hemangiosarcomas are easily detectable and scorable for size and growth, this novel mouse model enables accelerated in vivo drug testing for therapies of TSC-related tumors. IMPLICATIONS: These findings provide a strong rationale for simultaneous use of this conditional knockout mouse as an in vivo genetic model while seeking new cancer therapies for TSC-related tumors.

Spectrum of magnetic resonance imaging findings in pancreatic and other abdominal manifestations of Von Hippel-Lindau disease in a series of 23 patients: a pictorial review.

CONTEXT: Von Hippel Lindau disease is a rare autosomal dominantly inherited multisystem disorder characterized by development of benign and malignant tumors. The abdominal manifestation of the syndrome are protean. Magnetic resonance plays an important role in identification of abdominal abnormalities and follow-up of lesions. OBJECTIVE: To describe magnetic resonance imaging findings and patterns of pancreatic and other principal abdominal manifestations in a series of von Hippel-Lindau (VHL) disease patients and to review literature. METHODS: We retrospectively reviewed abdominal magnetic resonance studies performed in 23 patients (10 males, 13 females) diagnosed of VHL. RESULTS: In all examined patients abdominal involvement was present. The pancreatic imaging findings detected were: unilocular cystic lesions (6/23: 26.1%); serous cystadenomas (11/23: 47.8%), including diffuse lesions (8/23: 34.8%); solid neuroendocrine tumors (8/23: 34.8%); cystic neuroendocrine tumors (1/23: 4.3%). The renal findings detected were: simple renal cysts (18/23: 78.3%); complex renal cysts (13/23: 56.5%), including benign lesions (10/23: 43.5%) and malignant lesions (3/23: 13.0%); renal carcinomas (11/23: 47.8%) and 5 of these (45.5%) were multiple and bilateral. Five patients (21.7%) presented pheochromocytoma (4 of these were bilateral; 80.0%) and 1 patient (4.3%) presented cystadenoma of the epididymis. CONCLUSIONS: In VHL disease patients, magnetic resonance imaging plays an essential role in the identification of pancreatic and other abdominal lesions, in their follow-up, in the screening of asymptomatic gene carriers, and in their long-term surveillance.

Overexpressing TNF-alpha in pancreatic ductal adenocarcinoma cells and fibroblasts modifies cell survival and reduces fatty acid synthesis via downregulation of sterol regulatory element binding protein-1 and activation of acetyl CoA carboxylase.

The effect of tumor necrosis factor-alpha (TNF-α) gene delivery has been suggested as a potentially useful therapeutic approach to improve the chemotherapeutic treatment of patients with pancreatic ductal adenocarcinoma (PDA), but the exact mechanism of its action is not clearly understood. In this study, we analyzed the expression profile of TNF-α in PDA tissue and explored its potential role in fatty acid synthase (FAS) regulation in PDA cells and in fibroblasts. Quantitative real-time polymerase chain reaction was used to examine the expression of TNF-α in PDA, matching adjacent tissues, and benign lesions. Logistic regression models with robust variance were used to analyze the gene expression levels, and Kaplan-Meier survival curves were generated. In vitro, we overexpressed the TNF-α gene in PDA cells and fibroblasts and analyzed its effect on cell survival, migration, and on members of the FAS signaling pathway. We also evaluated TNF-α effects on a panel of inflammation-, angiogenesis-, and metastasis-related markers. In the tumor tissue of PDA patients, compared with their matched adjacent tissue, expression levels of TNF-α were not statistically different and did not correlate with survival or any other examined clinicopathological features. Overexpression of TNF-α significantly (p < 0.05) reduced PDA and fibroblast cell migration. In PDA cells that highly overexpress TNF-α, this was associated with a significant reduction of FAS mRNA and protein expression levels and significant (p < 0.05) reduction of SREBP-1 and ACC mRNA. Reduction of FAS by TNF-α was inhibited when either SREBP-1 or ACC was knocked down by siRNA. PDA cells and fibroblasts that overexpress TNF-α displayed differential regulation of several inflammation-related markers and reduced levels of metastasis-related genes. Our data demonstrate a previously unknown multi-targeted involvement of TNF-α in PDA lipogenesis and inflammation and metastasis and suggest that intratumoral introduction of TNF-α may have the potential as a novel therapeutic approach in human PDA.

Elafin is downregulated during breast and ovarian tumorigenesis but its residual expression predicts recurrence.

INTRODUCTION: Elafin is an endogenous serine protease inhibitor. The majority of breast cancer cell lines lack elafin expression compared to human mammary epithelial cells. In this study, we hypothesized that elafin is downregulated during breast and ovarian tumorigenesis. METHODS: We examined elafin expression by immunohistochemistry (IHC) in specimens of normal breast tissue (n = 24), ductal carcinoma in situ (DCIS) (n = 54), and invasive breast cancer (n = 793). IHC analysis of elafin expression was also performed in normal fallopian tube tissue (n = 20), ovarian cystadenomas (n = 9), borderline ovarian tumors (n = 21), and invasive ovarian carcinomas (n = 216). To understand the significance of elafin in luminal breast cancer cell lines, wild-type or M25G elafin (lacking the protease inhibitory function) were exogenously expressed in MCF-7 and T47D cells. RESULTS: Elafin expression was downregulated in 24% of DCIS and 83% of invasive breast tumors when compared to elafin expression in the normal mammary epithelium. However, the presence of elafin-positive cells in invasive breast tumors, even at low frequency, correlated with poor recurrence-free survival (RFS), reduced overall survival (OS), and clinicopathological markers of aggressive tumor behavior. Elafin-positive cells were an especially strong and independent prognostic marker of reduced RFS in IHC-defined luminal A-like tumors. Elafin was also downregulated in 33% of ovarian cystadenomas, 43% of borderline ovarian tumors, and 86% of invasive ovarian carcinomas when compared to elafin expression in the normal fallopian tube. In ovarian tumors, elafin-positive cells were correlated with reduced RFS, OS and disease-specific survival (DSS) only in stage I/II patients and not in stage III/IV patients. Notably, exogenous expression of elafin or elafin M25G in the luminal breast cancer cell lines MCF-7 and T47D significantly decreased cell proliferation in a protease inhibitory domain-independent manner. CONCLUSIONS: Elafin predicts poor outcome in breast and ovarian cancer patients and delineates a subset of endocrine receptor-positive breast cancer patients susceptible to recurrence who could benefit from more aggressive intervention. Our in vitro results suggest that elafin arrests luminal breast cancer cells, perhaps suggesting a role in tumor dormancy.

Acinar cell cystadenoma of the pancreas: a benign neoplasm or non-neoplastic ballooning of acinar and ductal epithelium?

Acinar cell cystadenoma (ACA) of the pancreas was initially described as a non-neoplastic cyst of the pancreas and, at that time, referred to as "acinar cystic transformation." In subsequent studies, these lesions were given the designation of "-oma," despite the relative lack of evidence supporting a neoplastic process. To characterize these lesions further, we examined the clinical, pathologic, and immunohistochemical features of 8 ACAs. The majority of patients were female (7 of 8, 88%) and ranged in age from 18 to 57 years (mean, 43 y). Grossly, the cysts involved the head (n=5), body (n=1), or the entire pancreas (n=2). ACAs were either multilocular (n=4) or unilocular (n=4) and ranged in size from 1.8 to 15 cm (mean, 6.8 cm). Histologically, multilocular ACAs were lined by patches of acinar and ductal epithelium. Immunolabeling, including double-labeling for cytokeratin 19 and chymotrypsin, highlighted the patchy pattern of the ductal and acinar cells lining the cysts. In some areas, the cysts with patches of acinar and ductal differentiation formed larger locules with incomplete septa as they appeared to fuse with other cysts. In contrast, the unilocular cases were lined by 1 to 2 cell layers of acinar cells with little intervening ductal epithelium. Nuclear atypia, mitotic figures, necrosis, infiltrative growth, and associated invasive carcinoma were absent in all cases. In addition, we assessed the clonal versus polyclonal nature of ACAs, occurring in women, using X-chromosome inactivation analysis of the human androgen receptor (AR) gene. Five of 7 cases were informative and demonstrated a random X-chromosome inactivation pattern. Clinical follow-up information was available for all patients, and follow-up ranged from 10 months to 7.8 years (mean, 3.6 y), with no evidence of recurrence or malignant transformation. We hypothesize that early lesions are marked by acinar dilatation that expands into and incorporates smaller ductules and later larger ducts. As the cysts increase in size, they fuse forming larger cysts. Later lesions demonstrate a unilocular cyst lined by predominantly acinar epithelium with scattered ductal cells. The term cystadenoma, with its neoplastic connotation, does not seem to accurately reflect the histologic, immunohistochemical, or molecular features of these lesions. We suggest readopting the term "acinar cystic transformation" until the non-neoplastic versus neoplastic origin of these lesions can be resolved.

MicroRNA from pancreatic duct aspirate differentiates cystic lesions of the pancreas.

INTRODUCTION: Prognostication for cystic neoplasms of the pancreas continues to evolve. Beyond simple size and cystic fluid CEA determination, microRNA (miRNA) detection holds great promise as molecular diagnostics for cancer risk. In this study, we sought to identify miRNAs that could predict malignant potential of pancreatic cystic lesions. METHODS: RNA was harvested from the pancreatic duct aspirate of 72 cystic neoplasms of the pancreas. Samples with adequate RNA concentration (≥ 3 ng/µL) were selected for qRTPCR profiling using assays to 379 of the most common miRNAs. miRNA profiles were correlated with histopathology from resected specimens and grouped by benign (serous cystadenomas), premalignant (intraductal papillary mucinous neoplasms and mucinous cystadenomas), or malignant lesions (adenocarcinoma). RESULTS: Adequate RNA for analysis was obtained from 42 (58.3 %) of the samples. Malignant lesions were more likely to have adequate RNA (n = 17, 81 %) than either benign (n = 6, 33 %) or premalignant lesions (n = 19, 59 %; p = 0.011). Nine miRNA were identified as differentially expressed between benign and premalignant/malignant lesions (p < 0.05). A significant correlation was found between the number of differentially expressed miRNA and the likelihood of a premalignant/malignant lesion. All premalignant or malignant lesions expressed at least one miRNA surpassing the threshold of mean miRNA expression, whereas no benign lesions had more than one miRNA surpassing the threshold. CONCLUSIONS: The presence of RNA in the duct aspirate from patients with pancreatic cystic neoplasms may be a predictor of premalignancy or malignancy. miRNA may be utilized to further differentiate between benign, premalignant, and malignant cystic lesions of the pancreas.

Mammary analogue secretory carcinoma mimicking salivary adenoma.

Mammary analogue secretory carcinoma (MASC) is a recently described salivary gland tumor characterized by ETV6 translocation. It appears that prior studies have identified MASC by reviewing salivary gland carcinomas, such as acinic cell carcinoma and adenocarcinoma, not otherwise specified. To address the possibility of MASC mimicking benign salivary neoplasms we reviewed 12 salivary gland (cyst)adenomas diagnosed prior to the discovery of MASC. One encapsulated (cyst)adenoma of the parotid gland demonstrated features of MASC. The diagnosis was confirmed by fluorescence in situ hybridization with an ETV6 break-apart probe. An unusual complex pattern of ETV6 rearrangement with duplication of the telomeric/distal ETV6 probe was identified. This case illustrates that MASC may mimic salivary (cyst)adenomas. To more accurately assess true clinical and morphologic spectrum of MASC, future studies may have to include review of salivary (cyst)adenomas. The differential diagnosis of MASC may have to be expanded to include cases resembling salivary (cyst)adenomas.

A combined blood based gene expression and plasma protein abundance signature for diagnosis of epithelial ovarian cancer--a study of the OVCAD consortium.

BACKGROUND: The immune system is a key player in fighting cancer. Thus, we sought to identify a molecular 'immune response signature' indicating the presence of epithelial ovarian cancer (EOC) and to combine this with a serum protein biomarker panel to increase the specificity and sensitivity for earlier detection of EOC. METHODS: Comparing the expression of 32,000 genes in a leukocytes fraction from 44 EOC patients and 19 controls, three uncorrelated shrunken centroid models were selected, comprised of 7, 14, and 6 genes. A second selection step using RT-qPCR data and significance analysis of microarrays yielded 13 genes (AP2A1, B4GALT1, C1orf63, CCR2, CFP, DIS3, NEAT1, NOXA1, OSM, PAPOLG, PRIC285, ZNF419, and BC037918) which were finally used in 343 samples (90 healthy, six cystadenoma, eight low malignant potential tumor, 19 FIGO I/II, and 220 FIGO III/IV EOC patients). Using new 65 controls and 224 EOC patients (thereof 14 FIGO I/II) the abundances of six plasma proteins (MIF, prolactin, CA125, leptin, osteopondin, and IGF2) was determined and used in combination with the expression values from the 13 genes for diagnosis of EOC. RESULTS: Combined diagnostic models using either each five gene expression and plasma protein abundance values or 13 gene expression and six plasma protein abundance values can discriminate controls from patients with EOC with Receiver Operator Characteristics Area Under the Curve values of 0.998 and bootstrap .632+ validated classification errors of 3.1% and 2.8%, respectively. The sensitivities were 97.8% and 95.6%, respectively, at a set specificity of 99.6%. CONCLUSIONS: The combination of gene expression and plasma protein based blood derived biomarkers in one diagnostic model increases the sensitivity and the specificity significantly. Such a diagnostic test may allow earlier diagnosis of epithelial ovarian cancer.

Circadian gene expression and clinicopathologic correlates in pancreatic cancer.

INTRODUCTION: The circadian rhythm is responsible for physiologic homeostasis, behavior, and components of multiple metabolic processes. Disruption of the circadian rhythm is associated with cancer development, and several circadian clock genes have been implicated in loss of cell cycle control, impaired DNA damage repair, and subsequent tumor formation. Here, we investigated the expression profiles of several circadian clock genes in pancreatic ductal adenocarcinoma (PDA). METHODS: Quantitative real-time polymerase chain reaction was used to examine the circadian clock genes (brain-muscle-like (Bmal)-ARNTL, circadian locomotor output cycles kaput (Clock), cryptochrome 1 (Cry1), cryptochrome 2 (Cry2), casein kinase 1ε (CK1ε), period 1 (Per1), period 2 (Per2), period 3 (Per3), timeless (Tim), and timeless-interacting protein (Tipin)) in PDA, as well as matching adjacent and benign tissue. Logistic regression models with robust variance were used to analyze the gene expression levels, and Kaplan-Meier survival curves were generated based on gene expression. RESULTS: In the tumor tissue of PDA patients, compared to their matched adjacent tissue, expression levels of all circadian genes were lower, with statistical significance for Per1, Per2, Per3, Cry1, Cry2, Tipin, Tim, CK1ε, Bmal-ARNTL, and Clock (p < 0.025). PDA tumors also expressed significantly lower levels of the circadian genes when compared to benign lesions for Per1, Per2, Per3, Cry2, Tipin, and CK1ε. A significant association between low levels of expression in the tumors and reduced survival was found with Per1, Per2, Per3, Cry2, Tipin, CK1ε, Clock, and Bmal-ARNTL. CONCLUSIONS: Our results reveal for the first time a dysregulated transcription of several circadian genes in PDA. Elevation of the gene levels in the benign and matched adjacent tissues may be indicative of their role during the process of tumorigenesis. The potential of using circadian genes as predictive markers of the outcomes and survival and distinguishing PDA from benign pancreas must be studied in larger populations to validate and demonstrate their eventual clinical utility.

Acinar cystadenoma of the pancreas: a clinicopathologic study of 10 cases including multilocular lesions with mural nodules.

Pancreatic acinar cystadenomas (ACAs) are rare cystic lesions showing acinar differentiation with benign outcome. Although debated, ACAs are favored to be neoplastic and potentially the benign counterpart of acinar cystadenocarcinoma. We present the largest single institution series to date comprising 10 cases. The mean age was 49 years with a female predominance (M:F=1:2.3). Abdominal/flank pain was the most common presentation (n=6). Serum amylase/lipase and cyst fluid amylase were often elevated. All lesions had a benign outcome on follow-up (5 to 67 mo). The lesions were unilocular (n=3) or multilocular (n=7) with mean size of 3.8 cm (range, 2.9 to 5.0 cm) and 5.1 cm (range, 2.0 to 7.5 cm), respectively. Eight lesions were unifocal with locations as follows: head (n=2), head/neck (n=2), body (n=1), tail (n=1), predominantly extrapancreatic with a microscopic intrapancreatic component (n=1), and unspecified location (n=1). Two lesions were multifocal, involving the head/uncinate/body and pancreatic head, respectively. Two aspects of ACAs that may represent a diagnostic pitfall include the propensity for acinar epithelium to appear as nondescript flat/cuboidal epithelium (trypsin/chymotrypsin immunopositive) and epithelial heterogeneity, with focal mucinous and squamous epithelium, the latter particularly in multilocular variants. In addition, 2 cases with intracystic nodules were observed. Array comparative genomic hybridization performed on 1 of these cases showed multiple chromosomal gains involving 1p, 3p, 5q, 6p, 7q, 8, 10q, 11, 14, 20, and X. These findings provide preliminary evidence that ACAs represent a cystic neoplastic lesion.

A study of the methylation status of opioid binding protein/cell adhesion molecule-like gene in ovarian cancer using nested methylation-specific polymerase chain reaction detection.

BACKGROUND: The goal was to improve the methylation-specific PCR (MSP) method and investigate the methylation status of the OPCML gene in carcinoma tissues from ovarian cancer patients. METHODS: MSP and nested MSP methods were used to examine the methylation status of the OPCML gene promoter in ovarian cancer, borderline tumor, and normal ovary tissues. RESULTS: Methylation of the OPCML gene was detected in 58.3% (14/24) and 83.3% (20/24) of the specimens from ovarian cancer patients using MSP and nested MSP methods, respectively. No methylation was observed in normal ovarian tissues using either method. CONCLUSIONS: The modified nested MSP method showed better sensitivity. The methylation of the OPCML gene was significantly higher in ovarian cancer than in normal tissue. The detection of OPCML gene methylation could serve as one of the molecular markers for the diagnosis of ovarian cancer.