Virus Genotype-Dependent Transcriptional Alterations in Lipid Metabolism and Inflammation Pathways in the Hepatitis C Virus-infected Liver.

Despite advances in antiviral therapy, molecular drivers of Hepatitis C Virus (HCV)-related liver disease remain poorly characterised. Chronic infection with HCV genotypes (1 and 3) differ in presentation of liver steatosis and virological response to therapies, both to interferon and direct acting antivirals. To understand what drives these clinically important differences, liver expression profiles of patients with HCV Genotype 1 or 3 infection (n = 26 and 33), alcoholic liver disease (n = 8), and no liver disease (n = 10) were analysed using transcriptome-wide microarrays. In progressive liver disease, HCV genotype was the major contributor to altered liver gene expression with 2151 genes differentially expressed >1.5-fold between HCV Genotype 1 and 3. In contrast, only 6 genes were altered between the HCV genotypes in advanced liver disease. Induction of lipogenic, lipolytic, and interferon stimulated gene pathways were enriched in Genotype 1 injury whilst a broad range of immune-associated pathways were associated with Genotype 3 injury. The results are consistent with greater lipid turnover in HCV Genotype 1 patients. Moreover, the lower activity in inflammatory pathways associated with HCV genotype 1 is consistent with relative resistance to interferon-based therapy. This data provides a molecular framework to explain the clinical manifestations of HCV-associated liver disease.

Ductal Carcinoma of the Prostate: An Uncommon Entity With Atypical Behaviour.

AIMS: Ductal adenocarcinoma is a rare variant of prostate cancer, and as such clinical outcomes and best management are not well defined. This series demonstrates the atypical presentation and unusual clinical behaviour of ductal adenocarcinoma and proposes management guidelines to assist clinicians. MATERIALS AND METHODS: A retrospective review of pure (nine patients) and mixed (18 patients) ductal adenocarcinoma of the prostate referred to the Departments of Radiation Oncology of the Sydney Cancer Centre, Royal Prince Alfred Hospital and Northern Sydney Cancer Centre, Royal North Shore Hospital, between 2000 and 2015. RESULTS: Twenty-seven patients were treated with definitive radiotherapy, nine patients (33%) with pure ductal and 18 (67%) with mixed ductal-acinar adenocarcinoma. The median follow-up was 38 months. Four patients (15%) failed locally, all of whom received less than 80 Gy, or no brachytherapy boost. Five patients (19%) failed distantly, four with biopsy-proven lung metastases. All distant failures occurred with a prostate-specific antigen (PSA) < 3 ng/ml. CONCLUSION: This series shows the atypical clinical presentation of this entity, as well as its propensity to metastasise to unusual sites. Relapse may occur at low absolute PSA values and is often asymptomatic. Ductal cancer should not simply be regarded as a high Gleason grade cancer. We propose management guidelines, including regular computed tomography examinations (rather than relying solely on PSA levels) as part of the follow-up for patients with any component of ductal adenocarcinoma.

A qualitative study of the development of a multidisciplinary case conference review methodology to reduce involved margins in pelvic exenteration surgery for recurrent rectal cancer.

AIM: Pelvic exenteration surgery remains the only curative option for recurrent rectal cancer. Microscopically involved surgical margins (R1) are associated with a higher risk of local recurrence and decreased survival. Our study aimed to develop a post hoc multidisciplinary case conference review and investigate its potential for identifying areas for improvement. METHOD: Patients who underwent pelvic exenteration surgery for recurrent rectal cancer with R1 resections at a tertiary referral centre between April 2014 and January 2016 were retrospectively reviewed from a prospectively maintained database. Patients with non-rectal cancers or who underwent palliative surgery were excluded. Cases, imaging and histopathology were evaluated by a dedicated panel including colorectal surgeons, an abdominal radiologist and a gastrointestinal pathologist. RESULTS: R1 resections were reported in 32 of 110 pelvic exenterations. Patients with other tumours were excluded and one patient had a palliative resection. Nine male patients with 11 exenterations were included with a median age of 56 years. All patients had positive soft tissue margins, and one patient also had an involved bony margin. Failures were due to (interdisciplinary) communication problems, specific management of tumour biology (multifocality, spiculated tumours), which can lead to radiological undercalling, and inadequate surgical technical planning. In hindsight, surgery would have been withheld from one patient. CONCLUSION: A retrospective multidisciplinary case evaluation of pelvic exenteration patients with involved surgical margins led to a list of recommendations which included the need to plan for wider surgical soft tissue resections and improvement in interdisciplinary communication. Lessons learned may increase clear margin rates in future resections.

A prospective multicentre phase III validation study of AZGP1 as a biomarker in localized prostate cancer.

BACKGROUND: Prostate cancers (PCs) with similar characteristics at the time of diagnosis can have very different disease outcomes. Conventional biomarkers of PC still lack precision in identifying individuals at high risk of PC recurrence. While many candidate biomarkers are proposed in the literature, few are in clinical practice as they lack rigorous validation. This study prospectively enrolled an independent phase III cohort to evaluate the clinical utility of zinc-alpha 2-glycoprotein (AZGP1) as a prognostic biomarker in localized PC. PATIENTS AND METHODS: In our multicentre, prospective phase III study, AZGP1 status in 347 radical prostatectomy specimens was assayed by immunohistochemistry in a NATA-accredited laboratory. The AZGP1 score was assessed in a multivariable model incorporating established prognostic factors. We also report extended outcomes from our previous phase II study. The primary endpoint was biochemical relapse-free survival (BRFS). Secondary endpoints were metastasis-free survival (MFS) and PC-specific survival (PCSS). RESULTS: In the phase II cohort, with a median follow-up of 15.8 years, low/absent AZGP1 expression was an independent predictor of poor BRFS (HR, 1.4; 95% CI, 1.1-1.9; P = 0.03), MFS (HR, 2.8; 95% CI, 1.2-6.6; P = 0.02) and PCSS (HR, 3.8; 95% CI, 1.5-9.5; P = 0.005). These results were validated in our prospective phase III cohort. Low/absent AZGP1 expression independently predicted for BRFS (HR, 1.9; 95% CI, 1.1-3.3; P = 0.02), with shorter MFS (HR, 2.0; 95% CI, 1.1-3.4; P = 0.02). AZGP1 improved the discriminatory value when incorporated into existing prognostic risk models. CONCLUSION: Our study provides prospective phase III validation that absent/low AZGP1 expression provides independent prognostic value in PC. This study provides robust evidence for the incorporation of this biomarker into clinical practice.

Meta-analysis of radical resection rates and margin assessment in pancreatic cancer.

BACKGROUND: R0 resection rates (complete tumour removal with negative resection margins) in pancreatic cancer are 70-80 per cent when a 0-mm margin is used, declining to 15-24 per cent with a 1-mm margin. This review evaluated the R0 resection rates according to different margin definitions and techniques. METHODS: Three databases (MEDLINE from 1946, PubMed from 1946 and Embase from 1949) were searched to mid-October 2014. The search terms included 'pancreatectomy OR pancreaticoduodenectomy' and 'margin'. A meta-analysis was performed with studies in three groups: group 1, axial slicing technique (minimum 1-mm margin); group 2, other slicing techniques (minimum 1-mm margin); and group 3, studies with minimum 0-mm margin. RESULTS: The R0 rates were 29 (95 per cent c.i. 26 to 32) per cent in group 1 (8 studies; 882 patients) and 49 (47 to 52) per cent in group 2 (6 studies; 1568 patients). The combined R0 rate (groups 1 and 2) was 41 (40 to 43) per cent. The R0 rate in group 3 (7 studies; 1926 patients) with a 0-mm margin was 72 (70 to 74) per cent The survival hazard ratios (R1 resection/R0 resection) revealed a reduction in the risk of death of at least 22 per cent in group 1, 12 per cent in group 2 and 23 per cent in group 3 with an R0 compared with an R1 resection. Local recurrence occurred more frequently with an R1 resection in most studies. CONCLUSION: Margin clearance definitions affect R0 resection rates in pancreatic cancer surgery. This review collates individual studies providing an estimate of achievable R0 rates, creating a benchmark for future trials.

Expression of E6AP and PML predicts for prostate cancer progression and cancer-specific death.

BACKGROUND: The promyelocytic leukemia (PML) tumor suppressor plays an important role in the response to a variety of cellular stressors and its expression is downregulated or lost in a range of human tumors. We have previously shown that the E3 ligase E6-associated protein (E6AP) is an important regulator of PML protein stability but the relationship and clinical impact of PML and E6AP expression in prostatic carcinoma is unknown. METHODS: E6AP and PML expression was assessed in tissue microarrays from a phase I discovery cohort of 170 patients treated by radical prostatectomy for localized prostate cancer (PC). Correlation analysis was carried out between PML and E6AP expression and clinicopathological variates including PSA as a surrogate of disease recurrence. The results were confirmed in a phase II validation cohort of 318 patients with associated clinical recurrence and survival data. RESULTS: Survival analysis of the phase I cohort revealed that patients whose tumors showed reduced PML and high E6AP expression had reduced time to PSA relapse (P = 0.012). This was confirmed in the phase II validation cohort where the expression profile of high E6AP/low PML was significantly associated with reduced time to PSA relapse (P < 0.001), clinical relapse (P = 0.016) and PC-specific death (P = 0.014). In multivariate analysis, this expression profile was an independent prognostic indicator of PSA relapse and clinical relapse independent of clinicopathologic factors predicting recurrence. CONCLUSION: This study identifies E6AP and PML as potential prognostic markers in localized prostate carcinoma and supports a role for E6AP in driving the downregulation or loss of PML expression in prostate carcinomas.

Adjuvant chemotherapy in elderly patients with pancreatic cancer.

BACKGROUND: Adjuvant chemotherapy improves survival for patients with resected pancreatic cancer. Elderly patients are under-represented in Phase III clinical trials, and as a consequence the efficacy of adjuvant therapy in older patients with pancreatic cancer is not clear. We aimed to assess the use and efficacy of adjuvant chemotherapy in older patients with pancreatic cancer. METHODS: We assessed a community cohort of 439 patients with a diagnosis of pancreatic ductal adenocarcinoma who underwent operative resection in centres associated with the Australian Pancreatic Cancer Genome Initiative. RESULTS: The median age of the cohort was 67 years. Overall only 47% of all patients received adjuvant therapy. Patients who received adjuvant chemotherapy were predominantly younger, had later stage disease, more lymph node involvement and more evidence of perineural invasion than the group that did not receive adjuvant treatment. Overall, adjuvant chemotherapy was associated with prolonged survival (median 22.1 vs 15.8 months; P<0.0001). Older patients (aged ≥70) were less likely to receive adjuvant chemotherapy (51.5% vs 29.8%; P<0.0001). Older patients had a particularly poor outcome when adjuvant therapy was not delivered (median survival=13.1 months; HR 1.89, 95% CI: 1.27-2.78, P=0.002). CONCLUSION: Patients aged ≥70 are less likely to receive adjuvant therapy although it is associated with improved outcome. Increased use of adjuvant therapy in older individuals is encouraged as they constitute a large proportion of patients with pancreatic cancer.

Phosphorylated Akt expression is a prognostic marker in early-stage non-small cell lung cancer.

AIMS: To determine the prognostic significance of pAkt expression in order to identify high-risk stage IB patients with non-small cell lung cancer (NSCLC) in an exploratory study. METHODS: We identified 471 consecutive patients with stage IB primary NSCLC according to the American Joint Commission on Cancer 6th edition tumour-node-metastasis (TNM) staging system, who underwent surgical resection between 1990 and 2008. Patients who received neoadjuvant or adjuvant treatments were excluded. Pathology reports were reviewed, and pathological characteristics were extracted. Expression of phosphorylated Akt (pAkt) in both cytoplasmic and nuclear locations was assessed by immunohistochemistry, and clinicopathological factors were analysed against 10-year overall survival using Kaplan-Meier and Cox proportional hazards model. RESULTS: 455 (96.6%) cancers were adequate for pAkt immunohistochemical analysis. The prevalence of pAkt expression in the cytoplasm and nucleus of the cancers was 60.7% and 43.7%, respectively. Patients whose cancers expressed higher levels of cytoplasmic pAkt had a trend towards longer overall survival than those with lower levels (p=0.06). Conversely, patients whose cancers expressed higher levels of nuclear pAkt had a poorer prognosis than those with lower levels of expression (p=0.02). Combined low cytoplasmic/high nuclear expression of pAkt was an independent predictor of overall survival (HR=2.86 (95% CI 1.35 to 6.04); p=0.006) when modelled with age (HR=1.05 (95% CI 1.03 to 1.07); p<0.001), extent of operation (HR=2.11 (95% CI 1.48 to 3.01); p<0.001), visceral pleural invasion (HR=1.63 (95% CI 1.24 to 2.15); p<0.001), gender, tumour size, histopathological type and grade (p>0.05). CONCLUSIONS: Level of expression of pAkt in the cytoplasm and nucleus is an independent prognostic factor that may help to select patients with high-risk disease.

EGFR mutant-specific immunohistochemistry has high specificity and sensitivity for detecting targeted activating EGFR mutations in lung adenocarcinoma.

AIM: We assessed the diagnostic accuracy of epidermal growth factor receptor (EGFR) mutant-specific antibodies for detecting two common activating EGFR mutations. METHODS: Immunohistochemical expression of mutation-specific antibodies against EGFR exon 19 deletion E746-A750 ((c.2235_2249del15 or c.2236_2250del15, p. Glu746_Ala750del) and exon 21 L858R point mutation (c.2573T>G, p.Leu858Arg) were assessed in a cohort of 204 resected early stage node negative lung adenocarcinomas, and protein expression was compared with DNA analysis results from mass spectrometry analysis. RESULTS: Of seven cases with L858R point mutation, six were positive by immunohistochemistry (IHC). There were three false positive cases using L858R IHC (sensitivity 85.7%, specificity 98.5%, positive predictive value 66.7%, negative predictive value 99.5%). All seven E746-A750 exon 19 deletions identified by mutation analysis were positive by IHC. Four additional cases were positive for exon 19 IHC but negative by mutation analysis. The sensitivity of exon 19 IHC for E746-A750 was 100%, specificity 98.0%, positive predictive value 63.6% and negative predictive value 100%. CONCLUSIONS: Mutant-specific EGFR IHC has good specificity and sensitivity for identifying targeted activating EGFR mutations. Although inferior to molecular genetic analysis of the EGFR gene, IHC is highly specific and sensitive for the targeted EGFR mutations. The antibodies are likely to be of clinical value in cases where limited tumour material is available, or in situations where molecular genetic analysis is not readily available.

Biopsy-proven brain metastases from prostate cancer: a series of four cases with review of the literature.

AIMS: Prostate cancer is very common and is the second most common cause of cancer death in males in Australia; however, brain metastases are exceedingly rare. MATERIALS AND METHODS: We review four cases of biopsy-proven brain metastases from prostate cancer and review the relevant literature. RESULTS: Three of four patients had acinar adenocarcinoma of prostate with one patient having ductal adenocarcinoma variant on histopathology. Three patients had the brain as the only site of metastatic disease. All patients underwent surgery, and three of four patients underwent adjuvant palliative radiotherapy to the brain. CONCLUSION: Brain metastases from prostate cancer are rare, but brain metastases without other sites of metastatic disease are exceedingly rare and may be more common with ductal adenocarcinoma variant.

Epigenetic-induced repression of microRNA-205 is associated with MED1 activation and a poorer prognosis in localized prostate cancer.

Deregulation of microRNA (miRNA) expression can have a critical role in carcinogenesis. Here we show in prostate cancer that miRNA-205 (miR-205) transcription is commonly repressed and the MIR-205 locus is hypermethylated. LOC642587, the MIR-205 host gene of unknown function, is also concordantly inactivated. We show that miR-205 targets mediator 1 (MED1, also called TRAP220 and PPARBP) for transcriptional silencing in normal prostate cells, leading to reduction in MED1 mRNA levels, and in total and active phospho-MED1 protein. Overexpression of miR-205 in prostate cancer cells negatively affects cell viability, consistent with a tumor suppressor function. We found that hypermethylation of the MIR-205 locus was strongly related with a decrease in miR-205 expression and an increase in MED1 expression in primary tumor samples (n=14), when compared with matched normal prostate (n=7). An expanded patient cohort (tumor n=149, matched normal n=30) also showed significant MIR-205 DNA methylation in tumors compared with normal, and MIR-205 hypermethylation is significantly associated with biochemical recurrence (hazard ratio=2.005, 95% confidence interval (1.109, 3.625), P=0.02), in patients with low preoperative prostate specific antigen. In summary, these results suggest that miR-205 is an epigenetically regulated tumor suppressor that targets MED1 and may provide a potential biomarker in prostate cancer management.

The prognostic and predictive value of serum CA19.9 in pancreatic cancer.

BACKGROUND: Current staging methods for pancreatic cancer (PC) are inadequate, and biomarkers to aid clinical decision making are lacking. Despite the availability of the serum marker carbohydrate antigen 19.9 (CA19.9) for over two decades, its precise role in the management of PC is yet to be defined, and as a consequence, it is not widely used. METHODS: We assessed the relationship between perioperative serum CA19.9 levels, survival and adjuvant chemotherapeutic responsiveness in a cohort of 260 patients who underwent operative resection for PC. RESULTS: By specifically assessing the subgroup of patients with detectable CA19.9, we identified potential utility at key clinical decision points. Low postoperative CA19.9 at 3 months (median survival 25.6 vs 14.8 months, P=0.0052) and before adjuvant chemotherapy were independent prognostic factors. Patients with postoperative CA 19.9 levels>90 U/ml did not benefit from adjuvant chemotherapy (P=0.7194) compared with those with a CA19.9 of ≤90 U/ml (median 26.0 vs 16.7 months, P=0.0108). Normalization of CA19.9 within 6 months of resection was also an independent favorable prognostic factor (median 29.9 vs 14.8 months, P=0.0004) and normal perioperative CA19.9 levels identified a good prognostic group, which was associated with a 5-year survival of 42%. CONCLUSIONS: Perioperative serum CA19.9 measurements are informative in patients with detectable CA19.9 (defined by serum levels of >5 U/ml) and have potential clinical utility in predicting outcome and response to adjuvant chemotherapy. Future clinical trials should prioritize incorporation of CA19.9 measurement at key decision points to prospectively validate these findings and facilitate implementation.

A role for GATA-2 in transition to an aggressive phenotype in prostate cancer through modulation of key androgen-regulated genes.

GATA-2, a member of the GATA family of transcription factors, is involved in androgen receptor (AR) signaling, however, little is known regarding its role in prostate cancer. Here, we report that GATA-2 is expressed in a substantial proportion of prostate cancers and that high expression of GATA-2 is associated with biochemical recurrence and distant metastatic progression in a validation set of 203 cancers. In vitro data show that GATA-2 is directly recruited to the promoter region of the AR upon androgen stimulation of LNCaP prostate cancer cells with 5alpha-dihydroxytestosterone (DHT) for 24 h. Ectopic GATA-2 expression causes the induction of AR transcript levels under androgen-depleted conditions (P<0.05). The expression of the AR target gene, AZGP1, is induced upon androgen stimulation and this effect is repressed by GATA-2. In contrast, GATA-2 significantly increases transcript levels of KLK2, which increases further in a time-dependent manner on DHT treatment and in the presence of GATA-2. These results indicate that upregulation of GATA-2 may contribute to the progression to aggressive prostate cancer through modulation of expression of AR and key androgen-regulated genes, one of which, AZGP1, is associated with the progression to metastatic disease.

Expression of LMO4 and outcome in pancreatic ductal adenocarcinoma.

Identification of a biomarker of prognosis and response to therapy that can be assessed preoperatively would significantly improve overall outcomes for patients with pancreatic cancer. In this study, patients whose tumours exhibited high LMO4 expression had a significant survival advantage following operative resection, whereas the survival of those patients whose tumours had low or no LMO4 expression was not significantly different when resection was compared with operative biopsy alone.

Endoplasmic reticulum stress contributes to beta cell apoptosis in type 2 diabetes.

AIMS/HYPOTHESIS: Increased lipid supply causes beta cell death, which may contribute to reduced beta cell mass in type 2 diabetes. We investigated whether endoplasmic reticulum (ER) stress is necessary for lipid-induced apoptosis in beta cells and also whether ER stress is present in islets of an animal model of diabetes and of humans with type 2 diabetes. METHODS: Expression of genes involved in ER stress was evaluated in insulin-secreting MIN6 cells exposed to elevated lipids, in islets isolated from db/db mice and in pancreas sections of humans with type 2 diabetes. Overproduction of the ER chaperone heat shock 70 kDa protein 5 (HSPA5, previously known as immunoglobulin heavy chain binding protein [BIP]) was performed to assess whether attenuation of ER stress affected lipid-induced apoptosis. RESULTS: We demonstrated that the pro-apoptotic fatty acid palmitate triggers a comprehensive ER stress response in MIN6 cells, which was virtually absent using non-apoptotic fatty acid oleate. Time-dependent increases in mRNA levels for activating transcription factor 4 (Atf4), DNA-damage inducible transcript 3 (Ddit3, previously known as C/EBP homologous protein [Chop]) and DnaJ homologue (HSP40) C3 (Dnajc3, previously known as p58) correlated with increased apoptosis in palmitate- but not in oleate-treated MIN6 cells. Attenuation of ER stress by overproduction of HSPA5 in MIN6 cells significantly protected against lipid-induced apoptosis. In islets of db/db mice, a variety of marker genes of ER stress were also upregulated. Increased processing (activation) of X-box binding protein 1 (Xbp1) mRNA was also observed, confirming the existence of ER stress. Finally, we observed increased islet protein production of HSPA5, DDIT3, DNAJC3 and BCL2-associated X protein in human pancreas sections of type 2 diabetes subjects. CONCLUSIONS/INTERPRETATION: Our results provide evidence that ER stress occurs in type 2 diabetes and is required for aspects of the underlying beta cell failure.

Loss of mammary epithelial prolactin receptor delays tumor formation by reducing cell proliferation in low-grade preinvasive lesions.

Top quartile serum prolactin levels confer a twofold increase in the relative risk of developing breast cancer. Prolactin exerts this effect at an ill defined point in the carcinogenic process, via mechanisms involving direct action via prolactin receptors within mammary epithelium and/or indirect action through regulation of other hormones such as estrogen and progesterone. We have addressed these questions by examining mammary carcinogenesis in transplants of mouse mammary epithelium expressing the SV40T oncogene, with or without the prolactin receptor, using host animals with a normal endocrine system. In prolactin receptor knockout transplants the area of neoplasia was significantly smaller (7 versus 17%; P < 0.001 at 22 weeks and 7 versus 14%; P = 0.009 at 32 weeks). Low-grade neoplastic lesions displayed reduced BrdU incorporation rate (11.3 versus 17% P = 0.003) but no change in apoptosis rate. Tumor latency increased (289 days versus 236 days, P < 0.001). Tumor frequency, growth rate, morphology, cell proliferation and apoptosis were not altered. Thus, prolactin acts directly on the mammary epithelial cells to increase cell proliferation in preinvasive lesions, resulting in more neoplasia and acceleration of the transition to invasive carcinoma. Targeting of mammary prolactin signaling thus provides a strategy to prevent the early progression of neoplasia to invasive carcinoma.

Aberrant p16(INK4A) and DPC4/Smad4 expression in intraductal papillary mucinous tumours of the pancreas is associated with invasive ductal adenocarcinoma.

BACKGROUND AND AIMS: Intraductal papillary mucinous tumours (IPMT) of the pancreas constitute a unique pathological entity with an overall incidence of associated invasive malignancy of 20%. The malignant potential of an individual IPMT cannot be accurately predicted. Preoperative estimation of the risk of associated invasive malignancy with IPMT would be of significant clinical benefit. As aberrations in cell cycle regulatory genes are associated with the progression of precursor pancreatic ductal lesions to invasive adenocarcinoma, we examined expression of key cell cycle regulatory genes in the cyclin D1/retinoblastoma pathway and the transforming growth factor beta/Smad4 signalling pathway in a cohort of patients with surgically resected IPMT. METHODS: Sections of formalin fixed paraffin embedded pancreatic tissue from a cohort of 18 patients with IPMT were examined using immunohistochemistry for protein expression of cell cycle regulatory genes p16(INK4A), p21(CIP1), p27(KIP1), cyclin D1, pRb, and p53, as well as the cell signalling molecule Smad4. A comparison of expression levels was made between adenoma/borderline IPMT (10 patients) and intraductal papillary mucinous carcinoma (IPMC) (eight patients, four of whom harboured invasive carcinoma). Statistical analysis was performed using the chi(2) and Fisher's exact tests. RESULTS: Aberrant expression of the proteins examined increased in frequency from adenoma/borderline IPMT to IPMC. Specifically, there was a significantly greater incidence of loss of p16(INK4A) expression in IPMC: 8/8 lesions (100%) compared with 1/10 (10%) adenoma/borderline IPMT (p<0.001). Similarly, loss of Smad4 expression was associated with IPMC: 3/8 (38%) versus adenoma/borderline IPMT 0/10 (p<0.03). Loss of Smad4 expression within the IPMT was the best marker for the presence of invasive carcinoma (p<0.001). CONCLUSIONS: These data indicate that loss of p16(INK4A) and Smad4 expression occur more frequently in IPMC alone, or with associated invasive carcinoma, compared with adenoma/borderline IPMT. Aberrant protein expression of these cell cycle regulatory genes in IPMT and pancreatic intraepithelial neoplasia in the current model of pancreatic cancer progression suggest similarities in their development and may also represent the subsequent risk of invasive carcinoma.