Biomarkers and biomarker validation: a pathologist's guide to getting it right.

Biomarkers are central to the delivery of personalised/precision medicine and are increasingly used across all areas of medicine to improve diagnostic accuracy, determine prognosis and predict response to treatment. Biomarkers can be used to develop assays that are then further developed into diagnostic tests, or in vitro diagnostic devices, which require an exhaustive validation and approval process. Pathologists play a critical role in the ordering and interpretation of biomarker assays. However, the evolution of a new biomarker from discovery to clinical implementation is complex, subject to various levels of scientific, clinical and regulatory scrutiny, with an approval process that varies significantly between jurisdictions. Therefore, it is important that pathologists have a solid understanding of how biomarkers are developed, the process of biomarker validation, how new biomarkers are approved for clinical use and the potential issues around implementation of biomarker testing that may lead to inaccurate results. This paper aims to provide an overview of the process of biomarker development, approval and validation, and practical tips for anatomical pathologists involved in the testing of biomarkers in routine practice.

Mesenchyme to epithelial transition protein expression, gene copy number and clinical outcome in a large non-small cell lung cancer surgical cohort.

BACKGROUND: In non-small cell lung cancer (NSCLC), mesenchyme to epithelial transition (MET) protein abundance increases with disease stage and is implicated in resistance to tyrosine kinase inhibitors. To better clarify the impact of MET overexpression on tumor behavior, we investigated a large cohort of patients who underwent curative surgical resection to determine whether MET gene amplification or protein abundance was prognostic. METHODS: Tissue microarrays (TMAs) were constructed using triplicate 1 mm cores of FFPE primary NSCLC specimens. TMAs underwent immunohistochemical (IHC) staining with the SP44 clone (Ventana) and cores were considered positive if >50% of tumor exhibited 2+ staining. The highest of triplicate values was used. MET gene amplification was detected using either SISH using Ventana's MET DNP probe or FISH using the D7S486/CEP 7 Abbott Probe. DNA was subjected to mutational profiling using Sequenom's LungCarta panel. RESULTS: Data from two institutions comprising 763 patients (516; 68%) male were generated, including 360 stage I, 226 stage II, 160 stage III and 18 resected stage IV. High MET protein expression was detected in 25% (193/763), and was significantly more common in adenocarcinomas than squamous cell carcinoma (P<0.01). MET gene copy number (GCN) correlated with high MET protein expression by IHC (P=0.01). Increased MET protein expression was associated with EGFR and KRAS mutations (P<0.01 for both). Once polysomy was excluded, true MET gene amplification was detected in only 8/763 (1%) of samples. In multivariate analysis, neither MET protein abundance nor GCN were correlated to overall patient survival. CONCLUSIONS: MET expression by IHC and GCN amplification was not prognostic in this large Caucasian surgical series. MET's primary role remains as a therapeutic target.

Determining HER2 (ERBB2) amplification status in women with breast cancer: final results from the Australian in situ hybridisation program.

Appropriate and accurate determination of HER2 status in women with breast cancer is critical for stratifying anti-HER2 therapies, and for access to subsidised treatment in the Australian setting. We conducted a regulated, nationwide program providing HER2 in situ hybridisation (ISH) testing for patients with newly diagnosed breast cancer. Cases with equivocal or non-diagnostic ISH test results at the local laboratory were sent to a high volume central testing laboratory for analysis using fluorescence ISH (FISH). We tested 78,408 early breast cancers and 3469 metastatic cancers using ISH. Of these, 12,405 early breast cancers (15.8%) and 798 metastatic cancers (23.0%) were HER2 positive. During the testing period, the proportion of core biopsy samples increased, the number of repeat tests remained stable and testing turnaround time declined. Discordant 3+ IHC, ISH negative results dropped from 20% to 13% in early breast cancers and from 35% to 8% among metastatic breast cancers. Following central laboratory FISH testing only 87 samples remained non-diagnostic (1.9% of FISH-tested samples, 0.1% of the whole cohort), most being decalcified specimens. This is a successful story of a cohesive service determining HER2 status in women with breast cancer in a 'real-world' setting.

Intraosseous lipomatous meningioma.

A 49-year-old man with intermittent headaches and right sided parietal lump was found to have an intraosseous right parietal lesion on computed tomography (CT) and magnetic resonance imaging (MRI). A stereotactic craniectomy and excision of the lesion were performed with histopathology confirming features consistent with primary lipomatous meningioma with intraosseous extension. Lipomatous meningiomas are very uncommon subtype of meningiomas, with ongoing discussions as to their true pathogenesis. To our knowledge this case represents the first reported case of a lipomatous meningioma with predominant intraosseous extension.

Reflex ALK immunohistochemistry is feasible and highly specific for ALK gene rearrangements in lung cancer.

Fluorescence in situ hybridisation (FISH) is considered the gold standard for the detection of ALK gene rearrangements in lung adenocarcinoma. The presence of ALK gene rearrangement predicts response to specific targeted therapy, but these rearrangements are relatively rare and FISH studies are expensive, not widely available, potentially challenging to interpret and therefore difficult to undertake in all patients with non-small cell lung cancer. We developed and then deployed into the routine clinical setting a screening program for ALK gene rearrangement in all non-small cell lung cancer patients based on immunohistochemistry (IHC) with a mouse monoclonal antibody (clone 5A4).ALK IHC was strongly positive in 12 (4%) of 307 tumours from consecutive patients. Only 10 of these cancers were initially thought to be rearranged by diagnostic FISH studies. The two tumours which were IHC positive but initially interpreted as FISH negative underwent repeat FISH testing because of the discrepancy. Repeat FISH testing confirmed the presence of ALK gene rearrangement with the discrepancy being attributable to an atypical FISH pattern.Therefore, in our experienced hands, IHC for ALK performed on initial diagnosis of lung cancer is 100% specific for the presence of ALK gene rearrangement. When ALK IHC and FISH studies are discrepant, IHC may outperform FISH. Although our study was not intended to formally assess the sensitivity of ALK IHC, the 4% rate of gene rearrangements identified by this approach is consistent with the expected incidence in our population.We conclude that reflex ALK IHC followed by confirmatory FISH testing can be readily integrated into the routine clinical setting and represents a cost effective and practical approach to screening for these clinically significant gene rearrangements.

Metastatic progression of breast cancer: insights from 50 years of autopsies.

There remain no clear guidelines for the optimal management of patients with metastatic breast cancer. To better understand its natural history, we undertook a detailed examination of 197 autopsies performed on women who died of breast cancer. We reviewed clinical, treatment and pathological aspects of all cases and, additionally, pathological features and biomarker expression (ER, PgR, HER2, EGFR, p53, Ki67, c-Kit, CK AE1/AE3) were assessed in detail for the primary tumour and matched metastases for 55 of the cases. Genomes of the primary tumour and multiple metastases were analysed by array-based comparative genomic hybridization for six cases(##) . 945 metastatic deposits were identified, with a median of four/patient. The most common organs involved were lung/pleura (80%), bone (74%), liver (71%) and non-axillary lymph nodes (55%). Major findings included: (a) patients with CNS metastases were more likely to have bone metastases (p < 0.013); (b) younger age was associated with metastasis to the liver (≤ 49 years; p < 0.001) and to gynaecological organs (≤ 49 years; p = 0.001); (c) surgical excision of the primary tumour was associated with metastasis to the liver (p = 0.002); and (d) ER and PgR showed down-regulation during progression in a non-random manner, particularly in lung/pleura (ER; p < 0.001), liver and bone metastases. Genomic analysis revealed DNA copy number variation between the primary tumour and metastases (e.g. amplification of 2q11.2-q12.1 and 10q22.2-q22.3) but little variation between metastases from the same patient. In summary, the association of CNS and bone metastases, liver and gynaecological metastases in young women and the risk of liver metastases following surgery have important implications for the management of patients with breast cancer. Clonal heterogeneity of the primary tumour is important in developing metastatic propensity and the change in tumour phenotype during progression/colonization highlights the importance of sampling metastatic disease for optimal treatment strategies.

Prohibitin expression is associated with high grade breast cancer but is not a driver of amplification at 17q21.33.

AIMS: In a study of ductal carcinoma in situ of the breast, we identified five genes at chromosome 17q21.33 that were over-expressed in high grade cases, and showed a correlation between expression and gene copy number. The aim of this study was to investigate potential drivers of genomic amplification at 17q21.33. METHODS: Analysis of high resolution comparative genomic hybridisation and published data specified a minimum region of amplification at 17q21.33. Prohibitin (PHB) expression was examined by immunohistochemistry in 285 invasive breast cancers. Gene copy number was examined by fluorescence in situ hybridisation. RESULTS: The minimum region of amplification at 17q21.33 included ten genes with PHB selected as a candidate driver. Increased PHB expression was associated with higher grade breast cancer and poorer survival. Amplification of PHB was detected in 13 of 235 cases (5.5%) but was not associated with PHB expression. PHB amplification was most common in the ERBB2+ breast cancer subtype, although high expression was most prevalent in basal-like and luminal B cancers. CONCLUSIONS: Amplification at 17q21.33 is a recurrent feature of breast cancer that forms part of a 'firestorm' pattern of genomic aberration. PHB is not a driver of amplification, however PHB may contribute to high grade breast cancer.

Clinical and molecular characterization of HER2 amplified-pancreatic cancer.

BACKGROUND: Pancreatic cancer is one of the most lethal and molecularly diverse malignancies. Repurposing of therapeutics that target specific molecular mechanisms in different disease types offers potential for rapid improvements in outcome. Although HER2 amplification occurs in pancreatic cancer, it is inadequately characterized to exploit the potential of anti-HER2 therapies. METHODS: HER2 amplification was detected and further analyzed using multiple genomic sequencing approaches. Standardized reference laboratory assays defined HER2 amplification in a large cohort of patients (n = 469) with pancreatic ductal adenocarcinoma (PDAC). RESULTS: An amplified inversion event (1 MB) was identified at the HER2 locus in a patient with PDAC. Using standardized laboratory assays, we established diagnostic criteria for HER2 amplification in PDAC, and observed a prevalence of 2%. Clinically, HER2- amplified PDAC was characterized by a lack of liver metastases, and a preponderance of lung and brain metastases. Excluding breast and gastric cancer, the incidence of HER2-amplified cancers in the USA is >22,000 per annum. CONCLUSIONS: HER2 amplification occurs in 2% of PDAC, and has distinct features with implications for clinical practice. The molecular heterogeneity of PDAC implies that even an incidence of 2% represents an attractive target for anti-HER2 therapies, as options for PDAC are limited. Recruiting patients based on HER2 amplification, rather than organ of origin, could make trials of anti-HER2 therapies feasible in less common cancer types.

Assessing HER2 amplification in breast cancer: findings from the Australian In Situ Hybridization Program.

In August 2006, the Australian government approved subsidized trastuzumab therapy for human epidermal growth factor receptor 2 (HER2)-positive early breast cancer, and it was mandated that HER2 testing should be performed using in situ hybridization (ISH) rather than immunohistochemistry (IHC). Here we review results of the first regulated, nationwide program to provide HER2 ISH testing for all newly diagnosed breast cancer patients, with a particular emphasis on cases where IHC and ISH results were discordant. Data from all laboratories participating in the program were collated. Cases with an equivocal ISH test result [by chromogenic ISH (CISH) or silver ISH (SISH)] were tested centrally by fluorescence ISH. Most laboratories also performed HER2 IHC, and 200 cases with discordant IHC and ISH results were selected for further analysis in a central laboratory. A total of 26 laboratories were involved and 53,402 tests were reported. Over a 4-year period the HER2 positivity rate decreased for primary cancers from 23.8 to 14.6 %, but remained relatively constant for samples from metastases. Average ISH reporting times were <5 days for all yearly reporting periods. Test-repeat rates decreased for CISH (8.9-3.6 %) and SISH (13.7-8.4 %). Only 12 of 196 cases remained discordant after retesting in a central laboratory. These findings demonstrate the successful implementation of a regulated, national program that continues to collect data on HER2 status. The results also highlight the differences in IHC interpretation between local laboratories and a central, more experienced, laboratory. This model could be used to establish future biomarker-testing programs in other countries.

Recruitment and activation of pancreatic stellate cells from the bone marrow in pancreatic cancer: a model of tumor-host interaction.

BACKGROUND AND AIMS: Chronic pancreatitis and pancreatic cancer are characterised by extensive stellate cell mediated fibrosis, and current therapeutic development includes targeting pancreatic cancer stroma and tumor-host interactions. Recent evidence has suggested that circulating bone marrow derived stem cells (BMDC) contribute to solid organs. We aimed to define the role of circulating haematopoietic cells in the normal and diseased pancreas. METHODS: Whole bone marrow was harvested from male ß-actin-EGFP donor mice and transplanted into irradiated female recipient C57/BL6 mice. Chronic pancreatitis was induced with repeat injections of caerulein, while carcinogenesis was induced with an intrapancreatic injection of dimethylbenzanthracene (DMBA). Phenotype of engrafted donor-derived cells within the pancreas was assessed by immunohistochemistry, immunofluorescence and in situ hybridisation. RESULTS: GFP positive cells were visible in the exocrine pancreatic epithelia from 3 months post transplantation. These exhibited acinar morphology and were positive for amylase and peanut agglutinin. Mice administered caerulein developed chronic pancreatitis while DMBA mice exhibited precursor lesions and pancreatic cancer. No acinar cells were identified to be donor-derived upon cessation of cerulein treatment, however rare occurrences of bone marrow-derived acinar cells were observed during pancreatic regeneration. Increased recruitment of BMDC was observed within the desmoplastic stroma, contributing to the activated pancreatic stellate cell (PaSC) population in both diseases. Expression of stellate cell markers CELSR3, PBX1 and GFAP was observed in BMD cancer-associated PaSCs, however cancer-associated, but not pancreatitis-associated BMD PaSCs, expressed the cancer PaSC specific marker CELSR3. CONCLUSIONS: This study demonstrates that BMDC can incorporate into the pancreas and adopt the differentiated state of the exocrine compartment. BMDC that contribute to the activated PaSC population in chronic pancreatitis and pancreatic cancer have different phenotypes, and may play important roles in these diseases. Further, bone marrow transplantation may provide a useful model for the study of tumor-host interactions in cancer and pancreatitis.

High Notch1 protein expression is an early event in breast cancer development and is associated with the HER-2 molecular subtype.

AIMS: Activation of Notch signalling results in hyperplasia and tumorigenesis in murine mammary epithelium. However, there is little information regarding the expression of Notch1 in premalignant lesions and early breast cancer. We investigated expression of Notch1 in breast cancer development and its association with molecular subtypes. METHODS AND RESULTS: Immunohistochemical expression of Notch1 was determined in a murine model of mammary carcinogenesis and in breast tissue from two cohorts of breast cancer patients, the first (n=222) comprising a histological progression series and the second an outcome series of 228 patients with operable invasive ductal carcinoma. Enhanced expression of Notch1 protein was an early event in both murine and human breast cancer development with progressive increases in expression with the development of hyperplasia and malignancy. High Notch1 was not prognostic in the outcome cohort. There was, however, a highly significant association of high Notch1 protein with the HER-2 molecular subtype of breast cancer (P=0.008). CONCLUSIONS: These data demonstrate that aberrant Notch regulation is an early event in mammary carcinogenesis and is associated with the HER-2 molecular subtype of breast cancer, and suggest the Notch signalling pathway may be a potential therapeutic target worthy of further investigation.

Loss of STARD10 expression identifies a group of poor prognosis breast cancers independent of HER2/Neu and triple negative status.

The phospholipid transfer protein STARD10 cooperates with c-erbB signaling and is overexpressed in Neu/ErbB2 breast cancers. We investigated if STARD10 expression provides additional prognostic information to HER2/neu status in primary breast cancer. A published gene expression dataset was used to determine relationships between STARD10 and HER2 mRNA levels and patient outcome. The central findings were independently validated by immunohistochemistry in a retrospective cohort of 222 patients with breast cancer with a median follow-up of 64 months. Kaplan-Meier and Cox proportional hazards analyses were used for univariate and multivariate analyses. Patients with low STARD10 or high HER2 tumor mRNA levels formed discrete groups each associated with a poor disease-specific survival (p = 0.0001 and p = 0.0058, respectively). In the immunohistochemical study low/absent STARD10 expression i.e. < or = 10% positive cells was observed in 24 of 222 (11%) tumors. In a univariate model, low/absent STARD10 expression was significantly associated with decreased patient survival (p = 0.0008). In multivariate analyses incorporating tumor size, tumor grade, lymph node status, ER, PR and HER2 status, low STARD10 expression was an independent predictor of death from breast cancer (HR: 2.56 (95% CI: 1.27-5.18), p = 0.0086). Furthermore, low/absent STARD10 expression, HER2 amplification and triple negative status were independent prognostic variables. Loss of STARD10 expression may provide an additional marker of poor outcome in breast cancer identifying a subgroup of patients with a particularly adverse prognosis, which is independent of HER2 amplification and the triple negative phenotype.

Prediction of local recurrence, distant metastases, and death after breast-conserving therapy in early-stage invasive breast cancer using a five-biomarker panel.

PURPOSE: To determine the clinical utility of intrinsic molecular phenotype after breast-conserving therapy (BCT) with lumpectomy and whole-breast irradiation with or without a cavity boost. PATIENTS AND METHODS: Four hundred ninety-eight patients with invasive breast cancer were enrolled into a randomized trial of BCT with or without a tumor bed radiation boost. Tumors were classified by intrinsic molecular phenotype as luminal A or B, HER-2, basal-like, or unclassified using a five-biomarker panel: estrogen receptor, progesterone receptor, HER-2, CK5/6, and epidermal growth factor receptor. Kaplan-Meier and Cox proportional hazards methodology were used to ascertain relationships to ipsilateral breast tumor recurrence (IBTR), locoregional recurrence (LRR), distant disease-free survival (DDFS), and death from breast cancer. RESULTS: Median follow-up was 84 months. Three hundred ninety-four patients were classified as luminal A, 23 were luminal B, 52 were basal, 13 were HER-2, and 16 were unclassified. There were 24 IBTR (4.8%), 35 LRR (7%), 47 distant metastases (9.4%), and 37 breast cancer deaths (7.4%). The overall 5-year disease-free rates for the whole cohort were: IBTR 97.4%, LRR 95.6%, DDFS 92.9%, and breast cancer-specific death 96.3%. A significant difference was observed for survival between subtypes for LRR (P = .012), DDFS (P = .0035), and breast cancer-specific death (P = .0482), but not for IBTR (P = .346). CONCLUSION: The 5-year and 10-year survival rates varied according to molecular subtype. Although this approach provides additional information to predict time to IBTR, LRR, DDFS, and death from breast cancer, its predictive power is less than that of traditional pathologic indices. This information may be useful in discussing outcomes and planning management with patients after BCT.

Diagnosis of cutaneous melanocytic tumours by four-colour fluorescence in situ hybridisation.

BACKGROUND: Accurate classification of primary melanocytic tumours as benign or malignant is crucial for prognostic prediction and appropriate patient management. Several chromosomal aberrations have been frequently identified in melanomas, but are absent in melanocytic naevi. We performed four-colour fluorescence in situ hybridisation (FISH) analysis of melanocytic tumours to determine the accuracy of the technique in classifying melanocytic tumours as benign or malignant. METHODS: FISH was performed on paraffin-embedded tissue from 40 histologically unequivocal melanocytic tumours (10 metastatic melanomas, 10 primary melanomas and 20 benign melanocytic naevi) using the product Vysis LSI RREB1/LSI MYB/LSI CCND1/CEP 6 probes (Abbott Molecular Laboratories, USA), which is designed to detect the copy number of the RREB1 (6p25), MYB (6q23), and CCND1 (11q13) genes and FISH positivity is defined by means of a scoring algorithm. RESULTS: FISH distinguished the melanomas and the naevi with a sensitivity of 90% (10/10 primary melanoma cases and 8/10 metastatic melanoma cases, respectively), and a specificity of 95%. The most common abnormalities in the melanomas were increased copies of 11q (70%) and 6p (70%), followed by 6q loss relative to cep6 (50%). Fifteen of the 18 positive melanomas were positive by more than one criterion. CONCLUSIONS: The results of this study show that FISH, using a panel of four probes, is a sensitive and specific method of classifying benign and malignant melanocytic tumours. The four-colour FISH technique has the potential to assist in the stratification of the subgroup of melanocytic tumours which are difficult to classify using conventional histology.

Expression of S100A2 calcium-binding protein predicts response to pancreatectomy for pancreatic cancer.

BACKGROUND & AIMS: Current methods of preoperative staging and predicting outcome following pancreatectomy for pancreatic cancer (PC) are inadequate. We evaluated the utility of multiple biomarkers from distinct biologic pathways as potential predictive markers of response to pancreatectomy and patient survival. METHODS: We assessed the relationship of candidate biomarkers known, or suspected, to be aberrantly expressed in PC, with disease-specific survival and response to therapy in a cohort of 601 patients. RESULTS: Of the 17 candidate biomarkers examined, only elevated expression of S100A2 was an independent predictor of survival in both the training (n = 162) and validation sets (n = 439; hazard ratio [HR], 2.19; 95% confidence interval [CI]: 1.48-3.25; P < .0001) when assessed in a multivariate model with clinical variables. Patients with high S100A2 expressing tumors had no survival benefit with pancreatectomy compared with those with locally advanced disease, whereas those without high S100A2 expression had a survival advantage of 10.6 months (19.4 vs 8.8 months, respectively) and a HR of 3.23 (95% CI: 2.39-4.33; P < .0001). Of significance, patients with S100A2-negative tumors had a significant survival benefit from pancreatectomy even in the presence of involved surgical margins (median, 15.7 months; P = .0007) or lymph node metastases (median, 17.4 months; P = .0002). CONCLUSIONS: S100A2 expression is a good predictor of response to pancreatectomy for PC and suggests that high S100A2 expression may be a marker of a metastatic phenotype. Prospective measurement of S100A2 expression in diagnostic biopsy samples has potential clinical utility as a predictive marker of response to pancreatectomy and other therapies that target locoregional disease.

Poor-prognosis estrogen receptor-positive breast cancer identified by histopathologic subclassification.

PURPOSE: Identification of biologically and clinically distinct breast cancer subtypes could improve prognostic assessment of primary tumors. The characteristics of "molecular" breast cancer subtypes suggest that routinely assessed histopathologic features in combination with limited biomarkers may provide an informative classification for routine use. EXPERIMENTAL DESIGN: Hierarchical cluster analysis based on components of histopathologic grade (tubule formation, nuclear pleomorphism, and mitotic score), expression of ER, cytokeratin 5/6, and HER2 amplification identified four breast cancer subgroups in a cohort of 270 cases. Cluster subgroup membership was compared with observed and Adjuvant! Online predicted 10-year survival. Survival characteristics were confirmed in an independent cohort of 300 cases assigned to cluster subgroups using a decision tree model. RESULTS: Four distinct breast cancer cluster subgroups (A-D) were identified that were analogous to molecular tumor types and showed a significant association with survival in both the original and validation cohorts (P < 0.001). There was a striking difference between survival for patients in cluster subgroups A and B with ER(+) breast cancer (P < 0.001). Outcome for all tumor types was well estimated by Adjuvant! Online, with the exception of cluster B ER(+) cancers where Adjuvant! Online was too optimistic. CONCLUSIONS: Breast cancer subclassification based on readily accessible pathologic features could improve prognostic assessment of ER(+) breast cancer.

Pancreatic intraepithelial neoplasia in association with intraductal papillary mucinous neoplasms of the pancreas: implications for disease progression and recurrence.

The development of pancreatic cancer (PC) several years after curative resection for noninvasive intraductal papillary mucinous neoplasm (IPMN) and the presence of PC distant from IPMN suggest that PC may develop independently of the IPMN. Here, we identified pancreatic intraepithelial neoplasia (PanIN) lesions, the putative precursors of PC, in the ducts of pancreata resected for IPMN and assessed the frequency of molecular aberrations common to PanIN and PC, within these lesions. The protein expression of p53, p21(WAF1/CIP1), cyclin D1, p16(INK4A) and DPC4/Smad4 were examined by immunohistochemistry in 267 PanIN lesions from a cohort of 23 patients with IPMN. Overexpression of p21(WAF1/CIP1) was present in PanIN-1A and -1B lesions and increased in frequency in PanIN-2 and PanIN-3. Overexpression of p53 and cyclin D1, and loss of p16(INK4A) expression were detected in PanIN-2 and PanIN-3 lesions. Loss of DPC4/Smad4 expression occurred only in the PanIN-3 lesions. PanIN lesions that were more dysplastic than the coincident IPMN were identified in 5 of 12 patients, and 2 of these contained a greater number of aberrations in protein expression than the IPMN. PanIN lesions seen in association with IPMN demonstrate molecular and histologic changes identical to PanIN lesions found in association with PC and, in some cases, are more advanced than the associated IPMN. These data suggest that PanIN lesions found in the ducts of a pancreas with IPMN may be relevant to the development of PC either coincident with IPMN or in the remnant pancreas after curative resection of IPMN.

Xanthomatous pituitary lesions: a report of two cases and review of the literature.

We describe two young men with cystic pituitary enlargement on magnetic resonance imaging (MRI) causing hypopituitarism. The first patient presented acutely unwell with headache and vomiting associated with anterior and posterior pituitary dysfunction. The second patient presented with hypopituitarism after a long history of hypogonadism. In both cases yellow/brown fluid was found at surgery and histological examination revealed inflammatory infiltrate with foamy histiocytes, lymphocytes and multinucleated giant cells containing cholesterol clefts. Full recovery of pituitary function occurred after surgery in the first but not the second patient. The first case is the first documented case of xanthomatous hypophysitis with recovery of pituitary function following surgery. The cases differed in duration of disease, as indicated by the long history of symptoms, the histological finding of marked fibrosis and the lack of recovery of pituitary function in the second. Xanthomatous pituitary lesions categorized in the literature as xanthomatous hypophysitis, xanthogranulomatous hypophysitis and xanthogranuloma of the sellar region have overlapping histological features. Our two cases revealed histological features that do not fit completely into any of the categories but share features of all three. These findings suggest that the various xanthomatous lesions of the sellar region may be a spectrum of a common inflammatory process rather than distinct pathological entities.

DPC4/Smad4 expression and outcome in pancreatic ductal adenocarcinoma.

PURPOSE: Prognostic indicators in pancreatic cancer (PC) are poorly defined and difficult to quantify preoperatively, hence they may lead to inappropriate patient selection for treatment. We examined the protein expression of key cell-cycle regulatory and cell-signaling molecules that occur at high frequency in PC and assessed their relationship to clinicopathologic parameters, response to operative resection, and outcome. PATIENTS AND METHODS: We identified 348 patients with pancreatic ductal adenocarcinoma and assessed the influence of reported clinicopathologic prognostic factors and the expression of the cell-cycle regulatory genes p21(WAF1/CIP1) (CDKN1A), cyclin D1 (CCND1), p53, and p16(INK4A) (CDKN2) and the cell-signaling molecule DPC4/Smad4 (MADH4) using immunohistochemistry in a subgroup of 129 patients. RESULTS: Independent prognostic factors in resected patients were tumor size greater than 45 mm (P =.0015), involvement of surgical margins (P <.0001), and perineural invasion (P =.014). Loss of DPC4/Smad4 expression cosegregated with resectability (P <.0001) and was associated with improved survival after resection (P <.0001), whereas resection did not improve survival in patients whose tumor expressed DPC4/Smad4 (P =.5). Aberrant expression of p21(WAF1/CIP1), cyclin D1, p53, or p16(INK4A) was not associated with a difference in survival. CONCLUSION: Tumor size (> 45 mm), resection margin involvement, and perineural invasion were independent prognostic factors. Preoperative assessment of DPC4/Smad4 expression has potential as a prognostic indicator in patients with PC since resection did not benefit those patients whose cancers expressed DPC4/Smad4 and accurate assessment of DPC4/Smad4 expression, unlike tumor size, margin status, and perineural invasion, does not require resection.