Raida Ahmad

MicroRNAs targeting oncogenes are down-regulated in pancreatic malignant transformation from benign tumors.

Background MicroRNA (miRNA) expression profiles have been described in pancreatic ductal adenocarcinoma (PDAC), but these have not been compared with pre-malignant pancreatic tumors. We wished to compare the miRNA expression signatures in pancreatic benign cystic tumors (BCT) of low and high malignant potential with PDAC, in order to identify miRNAs deregulated during PDAC development. The mechanistic consequences of miRNA dysregulation were further evaluated. Methods Tissue samples were obtained at a tertiary pancreatic unit from individuals with BCT and PDAC. MiRNA profiling was performed using a custom microarray and results were validated using RT-qPCR prior to evaluation of miRNA targets. Results Widespread miRNA down-regulation was observed in PDAC compared to low malignant potential BCT. We show that amongst those miRNAs down-regulated, miR-16, miR-126 and let-7d regulate known PDAC oncogenes (targeting BCL2, CRK and KRAS respectively). Notably, miR-126 also directly targets the KRAS transcript at a “seedless” binding site within its 3′UTR. In clinical specimens, miR-126 was strongly down-regulated in PDAC tissues, with an associated elevation in KRAS and CRK proteins. Furthermore, miR-21, a known oncogenic miRNA in pancreatic and other cancers, was not elevated in PDAC compared to serous microcystic adenoma (SMCA), but in both groups it was up-regulated compared to normal pancreas, implicating early up-regulation during malignant change. Conclusions Expression profiling revealed 21 miRNAs down-regulated in PDAC compared to SMCA, the most benign lesion that rarely progresses to invasive carcinoma. It appears that miR-21 up-regulation is an early event in the transformation from normal pancreatic tissue. MiRNA expression has the potential to distinguish PDAC from normal pancreas and BCT. Mechanistically the down-regulation of miR-16, miR-126 and let-7d promotes PDAC transformation by post-transcriptional up-regulation of crucial PDAC oncogenes. We show that miR-126 is able to directly target KRAS; re-expression has the potential as a therapeutic strategy against PDAC and other KRAS-driven cancers.

MicroRNAs Cooperatively Inhibit a Network of Tumor Suppressor Genes to Promote Pancreatic Tumor Growth and Progression

BACKGROUND & AIMS There has not been a broad analysis of the combined effects of altered activities of microRNAs (miRNAs) in pancreatic ductal adenocarcinoma (PDAC) cells, and it is unclear how these might affect tumor progression or patient outcomes. METHODS We combined data from miRNA and messenger RNA (mRNA) expression profiles and bioinformatic analyses to identify an miRNA-mRNA regulatory network in PDAC cell lines (PANC-1 and MIA PaCa-2) and in PDAC samples from patients. We used this information to identify miRNAs that contribute most to tumorigenesis. RESULTS We identified 3 miRNAs (MIR21, MIR23A, and MIR27A) that acted as cooperative repressors of a network of tumor suppressor genes that included PDCD4, BTG2, and NEDD4L. Inhibition of MIR21, MIR23A, and MIR27A had synergistic effects in reducing proliferation of PDAC cells in culture and growth of xenograft tumors in mice. The level of inhibition was greater than that of inhibition of MIR21 alone. In 91 PDAC samples from patients, high levels of a combination of MIR21, MIR23A, and MIR27A were associated with shorter survival times after surgical resection. CONCLUSIONS In an integrated data analysis, we identified functional miRNA-mRNA interactions that contribute to growth of PDACs. These findings indicate that miRNAs act together to promote tumor progression; therapeutic strategies might require inhibition of several miRNAs.

Wide-field fluorescence lifetime imaging of cancer

Optical imaging of tissue autofluorescence has the potential to provide rapid label-free screening and detection of surface tumors for clinical applications, including when combined with endoscopy. Quantitative imaging of intensity-based contrast is notoriously difficult and spectrally resolved imaging does not always provide sufficient contrast. We demonstrate that fluorescence lifetime imaging (FLIM) applied to intrinsic tissue autofluorescence can directly contrast a range of surface tissue tumors, including in gastrointestinal tissues, using compact, clinically deployable instrumentation achieving wide-field fluorescence lifetime images of unprecedented clarity. Statistically significant contrast is observed between cancerous and healthy colon tissue for FLIM with excitation at 355 nm. To illustrate the clinical potential, wide-field fluorescence lifetime images of unstained ex vivo tissue have been acquired at near video rate, which is an important step towards real-time FLIM for diagnostic and interoperative imaging, including for screening and image-guided biopsy applications.

Integrated molecular analysis to investigate the role of microRNAs in pancreatic tumour growth and progression

BACKGROUND MicroRNAs (miRNAs) are small non-coding RNAs involved in the post-transcriptional regulation of mRNAs and are aberrantly expressed in cancer with important roles in tumorigenesis. A broad analysis of the combined effects of altered activities of miRNAs in pancreatic ductal adenocarcinoma (PDAC) has not been done, and how miRNAs might affect tumour progression or patient outcomes is unclear. METHODS We combined data from miRNA and mRNA expression profiles from PDAC and normal pancreas samples (each n=9) and used bioinformatic analyses to identify a miRNA-mRNA regulatory network in PDAC. We validated our findings in PDAC cell-lines (PANC-1, MIA PaCa-2, LPc006, and LPc167), subcutaneous PDAC xenografts in mice, and laser capture microdissected PDACs from patients (n=91). We used this information to identify miRNAs that contributed most to tumorigenesis. FINDINGS We identified three miRNAs (miR-21, miR-23a, and miR-27a) that acted as cooperative repressors of a network of tumour suppressor genes that included PDCD4, BTG2, and NEDD4L. Inhibition of miR-21, miR-23a, and miR-27a had synergistic effects in reducing proliferation of PDAC cells in culture and the growth of xenograft tumours. The level of inhibition was greater than that of silencing oncomiR-21 alone. In PDACs from patients, high levels of the combination of miR-21, miR-23a, and miR-27a was a strong independent predictor of short overall survival after surgical resection (hazard ratio 3·21, 95% CI 1·78-5·78). High expression of this combination was also associated with a more aggressive tumour phenotype: more microscopic tumour infiltration at resection margin and increased perineural invasion. INTERPRETATION In an integrated data analysis, we identified functional miRNA-mRNA interactions that contribute to PDAC growth. These findings indicate that miRNAs act together to promote tumour progression and that future therapeutic strategies might require inhibition of several miRNAs. Furthermore, high tumour expression of the miR-21, miR-23a, and miR-27a combination could have potential use in the future as a prognostic signature for patients with PDAC. FUNDING Peel Medical Research Trust, Alliance Family Foundation, Action Against Cancer, National Institute for Health Research, Association for International Cancer Research, Jason Boas Fellowship, Imperial Biomedical Research Centre, Rosetrees Trust, Joseph Ettedgui Charitable Foundation.

Poorly-Differentiated Signet-Ring Cell Carcinoma of the Ampulla of Vater: Report of a Rare Malignancy

CONTEXT Signet-ring cell carcinoma (SRCC) of the ampulla of Vater is a very rare clinical entity, which is infrequently reported in medical literature. CASE REPORT A 78-year-old woman was admitted with jaundice, pruritus and postprandial vomiting. Abdominal ultrasound and computed tomography scanning demonstrated gross dilatation of the common bile and pancreatic ducts with gallbladder calculi. Endoscopic retrograde cholangiopancreatography suggested a duodenal tumour at the ampulla. The patient underwent Whipples procedure with cholecystectomy. Immunohistopathological examination confirmed poorly-differentiated SRCC of the ampulla of Vater. The tumour had infiltrated the duodenal muscularis propria and pancreatic parenchyma, but local lymph nodes were clear (T3N0M0). The patient was disease-free at 6-month follow-up. CONCLUSIONS We here report a case of poorly-differentiated SRCC of the Ampulla of Vater. The majority of patients with such tumours undergo pancreaticoduodenectomy, which affords good outcomes in early disease. However, owing to the rarity of cases, the exact prognosis of ampullary SRCC remains as yet undetermined.

Is there a ‘margin’ for error in pancreatic cancer surgery?

Evaluation of: Gnerlich JL, Luka SR, Deshpande AD et al. Microscopic margins and patterns of treatment failure in resected pancreatic adenocarcinoma. Arch. Surg. 147(8), 753-760 (2012). Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with one of the worst 5-year survival rates of any malignancy. Even after potentially curative surgical resection, disease may progress rapidly. It is therefore important to identify clinicopathologic factors that influence survival and may be modified to improve outcomes. The evaluated article presents data from a retrospective review of patients who underwent surgical resection for PDAC. Local recurrence (LR), distant recurrence and survival were compared between patients with a negative resection margin (R0) and those with a positive resection margin (R1). Patients with R1 posterior margins, in particular, were more likely to have LR and worse LR-free survival. In addition, this was more pronounced if patients had lymph-node involvement. Similar results have been reported in other studies and this study illustrates that standardized pathological reporting of PDAC specimens may allow further investigation of factors affecting R1 patients.

Correlation of multiparameter flow cytometry and bone marrow trephine immunohistochemistry in the identification and characterization of neoplastic plasma cells.

Identification and quantification of neoplastic plasma cells (PC) and differentiating them from normal/reactive PC is critical for the work-up, diagnosis and classification of suspected PC neoplasms. Identification of light chain restriction and/or aberrant phenotype using either multiparameter flow cytometry (MFC) or bone marrow trephine biopsy (BMTB) immunohistochemistry (IHC) is needed as a surrogate for PC clonality. There are a few studies in the literature comparing MFC on bone marrow aspirate (BMA) and BMTB in patients with PC disorders (Paiva et al, 2009; Johnsen et al, 2010); yet no comparison between immunophenotypic results obtained through MFC and BMTB-IHC has been undertaken on a larger scale. We aimed to compare these two techniques in their ability to quantify PC, and to demonstrate light chain restriction and an aberrant phenotype. We retrospectively identified 89 presentation samples from PC myeloma (PCM) patients from our archive for which both BMTB-IHC and MFC results were available. PC quantification on BMTB was based on CD138 (clone MI15, Dako, Glostrup, Denmark) immunostain. BMTB were also immunostained for CD3 (clone LN10; Dako), CD20 (clone L26; Dako), CD79a (clone JCB117; Dako), CD56 (clone 1B6; Leica, Nussloch, Germany), cyclin D1 (CCND1; clone SP4, Thermoshandon, Billerica, MA, USA) and CD117 (Dako, polyclonal) expression. Light chain analysis was performed by IHC (Dako, polyclonal) in all cases and additionally by mRNA in situ hybridization (ISH) (Leica) in 13 cases. The criteria for a diagnosis of PC neoplasm by BMTB-IHC were the presence of PC with abnormal morphology, light chain restriction and/or aberrant PC phenotype. MFC was carried out on BMA samples using a 3-laser, 8-colour BD FACSCANTO II (Becton Dickinson BD, Franklin Lakes, NJ, USA) with the following fluorochrome-antibody combination: FITC-CD38 (clone HB7, BD), PE/CD56 (clone MY31, BD), PerCP-Cy5.5-CD138 (clone MI15; Dako), PE-cy7-CD19 (clone SJ25C1, BD), APC-CyKappa (Dako, polyclonal), APCH7-CyLambda (clone 1-155-2, BD), V450-CD20 (clone L27, BD), V500-CD45 (clone H30, BD). Data interpretation was performed using FACS Diva software (BD) and/or Infinicyt (Cytognos, Salamanca, Spain). Gating and analysis strategies used in this study have been previously described (Rawstron et al, 2008). The MFC criteria for involvement by neoplastic PCs were the presence of PCs (minimum 100 events) with cytoplasmic light chain restriction and/or abnormal PC phenotype. Data was analysed using Microsoft Excel 2010 (Microsoft, Redmond, WA, USA). Statistical analysis was performed using IBM SPSS version 22 (IBM, Armonk, NY, USA). The percentage of PCs (PC%) among all nucleated cells on BMTB-IHC was significantly higher than that by MFC (median 50% vs. 6%; P < 0 001; Table I). There was a positive correlation between the PC% by BMTB-IHC and MFC (Spearman correlation coefficient of R = 0 443, P < 0 001)

Diagnostic Utility of Lymphoid Enhancer Binding Factor 1 Immunohistochemistry in Small B-Cell Lymphomas

Objectives Recent studies have shown that lymphoid enhancer binding factor 1 (LEF1) is a useful marker for chronic lymphocytic B-cell leukemia (CLL)/small lymphocytic lymphoma. Yet, it is not still being widely used in a diagnostic setting. In this study, we document the experience with LEF1 immunohistochemistry during routine diagnostics. Methods In total, 191 B-cell lymphoma cases from Hammersmith Hospital, Imperial College NHS Healthcare Trust (London, UK) were investigated by immunohistochemistry for LEF1 during routine diagnostic workup. These cases included both bone marrow trephines and lymph node biopsy specimens. The monoclonal antibody clone EPR2029Y was used. Results LEF1 expression was strong and diffuse (>70% of cells) in most cases. Few CLL cases showed a staining in proliferation centers only. Seventy-seven of 80 CLL cases expressed LEF1. Other entities expressing LEF1 included one of 38 follicular lymphomas, two of 33 marginal zone lymphomas, and one diffuse large B-cell lymphoma with a background of follicular lymphoma grade 3B. Sensitivity for LEF1 for the diagnosis of CLL was 0.96, and specificity was 0.93. Conclusions In this study, we could demonstrate the diagnostic utility of LEF1. LEF1 is a sensitive and specific marker for CLL and is helpful in the diagnosis of diagnostically challenging small B-cell lymphomas.

Systemic EBV-positive lymphoproliferative disease of childhood

A 21=2-year-old Caucasian girl presented critically ill with fever, shock, and a recent hematemesis. She had a generalized purpuric rash, bruising, and scattered petechiae. Her liver was palpable 4 cm below the right costal margin but there was no lymphadenopathy or palpable splenomegaly. Blood tests showed: white blood cell count 0.9 3 10/l, neutrophils 0.4 3 10/l, lymphocytes 0.7 3 10/l, hemoglobin concentration 79 g/l, and platelet count 7 3 10/l. Coagulation tests showed disseminated intravascular coagulation. Following resuscitation, bone marrow aspiration and trephine biopsy were performed. The bone marrow aspirate showed prominent hemophagocytosis (left and center). Normal hemopoietic cells were greatly reduced. There was an infiltrate of medium sized atypical lymphoid cells (center and right); these had a high nucleocytoplasmic ratio, slight to moderate chromatin condensation, and moderately basophilic cytoplasm, often extending into blebs. Flow cytometric immunophenotyping of the aspirate identified a small population of abnormal cytotoxic CD8-positive T cells, which showed complete loss of CD5 and CD7, co-expressed CD38, and showed partial expression of CD56. On trephine biopsy sections, the cells of the lymphoid infiltrate expressed CD2, CD3, CD8, HLADR, granzyme B, weak CD7, and EBER (Epstein–Barr virus-encoded RNA). They did not express CD4, CD5, CD56, CD57, ALK1, or terminal deoxynucleotidyl transferase. Molecular analysis showed a clonal T cell expansion on a polyclonal background. The titer of Epstein-Barr virus (EBV) on PCR of whole blood was subsequently found to be very high with 20,000,000 copies of the virus per ml. A diagnosis of systemic EBV-positive lymphoproliferative disease of childhood was made [1]. This condition is more common in Asia than in European countries and North America but some cases in Caucasian patients have been described previously [2]. Hemophagocytosis is a common feature.