Silvia Vecchiarelli

Pancreatic Ductal Adenocarcinoma Associated with Autoimmune Pancreatitis

Autoimmune pancreatitis (AIP), in contrast to other benign chronic pancreatic diseases, can be cured with immunosuppressant drugs, thus the differentiation of AIP from pancreatic cancer is of particular interest in clinical practice. There is the possibility that some patients with AIP may develop pancreatic cancer, and this possibility contributes to increasing our difficulties in differentiating AIP from pancreatic cancer. We herein report the case of a 70-year-old man in whom pancreatic adenocarcinoma and AIP were detected simultaneously. We must carefully monitor AIP patients for the simultaneous presence of pancreatic cancer, even when a diagnosis of AIP is confirmed.

State of the art biological therapies in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies with a five-year survival rate of approximately 5%. Several target agents have been tested in PDAC, but almost all have failed to demonstrate efficacy in late phase clinical trials, despite the better understanding of PDAC molecular biology generated by large cancer sequencing initiatives in the past decade. Eroltinib (a small-molecule tyrosine-kinase inhibitor of epidermal growth factor receptor) plus gemcitabine is the only schedule with a biological agent approved for advanced pancreatic cancer, but it has resulted in a very modest survival benefit in unselected patients. In our work, we report a summary of the main clinical trials (closed and ongoing) that refer to biological therapy evaluation in pancreatic cancer treatment.

Characterization of pancreatic ductal adenocarcinoma using whole transcriptome sequencing and copy number analysis by single-nucleotide polymorphism array

The aim of the current study was to implement whole transcriptome massively parallel sequencing (RNASeq) and copy number analysis to investigate the molecular biology of pancreatic ductal adenocarcinoma (PDAC). Samples from 16 patients with PDAC were collected by ultrasound‑guided biopsy or from surgical specimens for DNA and RNA extraction. All samples were analyzed by RNASeq performed at 75x2 base pairs on a HiScanSQ Illumina platform. Single‑nucleotide variants (SNVs) were detected with SNVMix and filtered on dbSNP, 1000 Genomes and Cosmic. Non‑synonymous SNVs were analyzed with SNPs&GO and PROVEAN. A total of 13 samples were analyzed by high resolution copy number analysis on an Affymetrix SNP array 6.0. RNAseq resulted in an average of 264 coding non‑synonymous novel SNVs (ranging from 146‑374) and 16 novel insertions or deletions (In/Dels) (ranging from 6‑24) for each sample, of which a mean of 11.2% were disease‑associated and somatic events, while 34.7% were frameshift somatic In/Dels. From this analysis, alterations in the known oncogenes associated with PDAC were observed, including Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations (93.7%) and inactivation of cyclin‑dependent kinase inhibitor 2A (CDKN2A) (50%), mothers against decapentaplegic homolog 4 (SMAD4) (50%), and tumor protein 53 (TP53) (56%). One case that was negative for KRAS exhibited a G13D neuroblastoma RAS viral oncogene homolog mutation. In addition, gene fusions were detected in 10 samples for a total of 23 different intra‑ or inter‑chromosomal rearrangements, however, a recurrent fusion transcript remains to be identified. SNP arrays identified macroscopic and cryptic cytogenetic alterations in 85% of patients. Gains were observed in the chromosome arms 6p, 12p, 18q and 19q which contain KRAS, GATA binding protein 6, protein kinase B and cyclin D3. Deletions were identified on chromosome arms 1p, 9p, 6p, 18q, 10q, 15q, 17p, 21q and 19q which involve TP53, CDKN2A/B, SMAD4, runt‑related transcription factor 2, AT‑rich interactive domain‑containing protein 1A, phosphatase and tensin homolog and serine/threonine kinase 11. In conclusion, genetic alterations in PDCA were observed to involve numerous pathways including cell migration, transforming growth factor‑β signaling, apoptosis, cell proliferation and DNA damage repair. However, signaling alterations were not observed in all tumors and key mutations appeared to differ between PDAC cases.

Preoperative Gemcitabine and Oxaliplatin in a Patient with Ovarian Metastasis from Pancreatic Cystadenocarcinoma

We describe a case of clinical benefit and partial response with gemcitabine and oxaliplatin (GEMOX) in a young patient with ovarian metastasis from cystadenocarcinoma of the pancreas. A young woman complained of abdominal pain and constipation. Computed tomography (CT) and magnetic resonance imaging scans disclosed two bilateral ovarian masses with pancreatic extension. She underwent bilateral ovarian and womb resection. During surgery peritoneal carcinosis, a pancreatic mass and multiple abdominal lesions were found. The final diagnosis was mucinous pancreatic cystadenocarcinoma with ovarian and peritoneal metastases. She started chemotherapy with GEMOX (gemcitabine 1,000 mg/m2/d1 and oxaliplatin 100 mg/m2/d2 every 2 weeks). After 12 cycles of chemotherapy a CT scan showed reduction of the pancreatic mass. She underwent distal pancreatic resection, regional lymphadenectomy and splenectomy. Pathologic examination documented prominent fibrous tissue and few neoplastic cells with mucin-filled cytoplasm. Chemotherapy was continued with gemcitabine as adjuvant treatment for another 3 cycles. There is currently no evidence of disease. As reported in the literature, GEMOX is associated with an improvement in progression-free survival and clinical benefit in patients with advanced pancreatic cancer. This is an interesting case in whom GEMOX transformed inoperable pancreatic cancer into a resectable tumor.

Comparing RECIST and Choi’s Criteria to Evaluate Radiological Response to Chemotherapy in Patients with Advanced Pancreatic Cancer

Context Assessment of response after chemotherapy (CTH) for pancreatic cancer (PC) is currently based on RECIST criteria. In 2007 Choi et al . published a new classification system. Objectives To evaluate the accuracy of the two classification systems for radiological response to CTH in patients affected by advanced PC. Methods From 2006 to 2011, 61 untreated patients affected by advanced pancreatic adenocarcinoma underwent palliative CTH. Thirty-seven (60.7 %) had a locally advanced PC and 24 (39.3%) a metastatic disease. All patients were treated with a bemcitabine-based CTH. We assessed radiological response after three months of first-line therapy applying both RECIST criteria and Choi’s criteria, which consider changes both in size and in density at CT. We evaluated the accuracy in restaging, comparing the class of response with overall survival (OS). OS was calculated with Kaplan-Meier method. The concordance with the two classification systems was evaluated with Kendall’s test. The accuracy in restaging was assessed through log rank test. Results At restaging, using RECIST criteria, we registered 6 (9.8%) patients with partial response (PR), 32 (52.5%) with stable disease (SD), and 23 (37.7%) with disease progression (PD). Instead Choi’s criteria assessed 18 PR (29.5%), 12 SD (19.7%) and 31 PD (50.8%). The concordance test showed that the two systems matched (P<0.001). Comparing each classification with OS, we observed that patients with different prognosis were better stratified with Choi’s criteria. Using RECIST criteria we did not found any significant difference in OS between patients with PR (12 months), SD (16 months) and PD (10 months). Using Choi’s criteria we found that OS in patients with PR was similar to patients with SD with 16 and 19 months (P=0.634). Patients with PR had an OS significantly higher than patients with PD (16 vs . 9 months; P=0.009; RR=2.3). Conclusions Choi’s criteria seem to better assess radiological response of CTH in PC patients than RECIST criteria. Due to the small number of patients, larger prospective studies are needed.

Copy number gain of chromosome 3q is a recurrent event in patients with intraductal papillary mucinous neoplasm (IPMN) associated with disease progression

Background Intraductal papillary mucinous neoplasm (IPMN) is the most common cystic preneoplastic lesion of pancreatic cancer. We used an approach coupling high resolution cytogenetic analysis (Affymetrix Oncoscan FFPE Array) with clinically-oriented bioinformatic interpretation of data to understand the most relevant alterations of precursor lesions at different stages to identify new diagnostic markers. Results We identified multiple copy number alterations, particularly in lesions with severe dysplasia, with 7 IPMN with low-intermediate dysplasia carrying a nearly normal karyotype and 13 IPMN with complex Karyotype (> 4 alterations), showing high grade dysplasia. A specific gain of chromosome arm 3q was found in IPMN with complex Karyotype (92%). This gain of 3q is particularly interesting for the presence of oncogenes such as PIK3CA, GATA2 and TERC that are part of pathways that deregulate cell growth and promote disease progression. Quantitative PCR and FISH analysis confirmed the data. Further demonstration of the overexpression of the PIK3CA gene supports the identification of this alteration as a possible biomarker in the early identification of patients with IPMN at higher risk for disease progression. Materials and methods High resolution cytogenetic analysis was performed in 20 formalin fixed paraffin embedded samples of IPMN by Oncoscan FFPE assay. Results were validated by qPCR and FISH analysis. Conclusions The identification of these markers at an early stage of disease onset could help to identify patients at risk for cancer progression and new candidates for a more specific targeted therapy.

SNP-Array High Resolution Cytogenetic Analysis of Resectable and Advanced Pancreatic Cancer

Context Pancreatic cancer (PC) is the fourth leading cause of cancer deaths. The molecular mechanisms involved in the high tumorigenicity of PC are not yet well-known. Methods Pancreatic tumor samples from 14 patients were collected by ultrasound-guided biopsy and used for DNA extraction. High resolution copy number analysis was performed on Affymetrix SNP array 6.0 and analyzed with segmentation algorithm against a reference of 270 Ceu HapMap individuals (Partek Genomic Suite). Results Nine out of 14 patients exhibited both macroscopic and cryptic cytogenetic alterations, with a mean of 10 copy number alterations (CNA) per patient, while 5 patients did not show any copy number gain or loss. Deletions outnumbered amplifications by more than 2 folds. The chromosomes showing more copy number gains were chromosomes 12, 18, 19, while chromosomes 6, 9, 17 and 18 were most frequently deleted. In particular, deletions on 9p21 encompassed CDKN2A and 2B tumor suppressor genes, that on chromosome 18q21 overlapped with SMAD4, the one on chromosome 6p21 included RUNX2, while TP53 and MAP2K4 were the target genes deleted on chromosome 17p13. Amplified regions on chromosome 12p12 encompassed KRAS and ETV6 genes, the one on chromosome 18q11 overlapped with GATA6, while that on 19q13 included AKT2. We observed that the number of alterations correlates with the clinical course, and in particular that patients with none to few alterations (≤6) showed a median time to disease progression and a median overall survival significantly longer than those having a high number of CNA (>6), with a time to disease progression of 13.7 vs . 4.1 months (P=0.015) and an overall survival of 14.6 vs . 4.8 months (P=0.035). Conclusions High resolution cytogenetic analysis by SNP-array has the potential to uncover the genetic alterations carried by pancreatic tumors, and find new markers related to patient prognosis.

Whole Transcriptome Sequencing Reveals Somatic HMGCR Mutation in a Case of Pancreatic Adenocarcinoma with Long-Term Therapy Response

Context We merged clinical history of a locally advanced pancreatic cancer (LAPC) patient with data obtained from a whole transcriptome massively parallel sequencing (RNASeq). Case report A 56-year-old man with histological diagnosis of LAPC. After obtained informed consent, we collected a fragment of pancreatic lesion. Patient received 6 induction cycles treatment with gemcitabine and oxaliplatin (GEMOX) from November 2011, followed by chemoradiotherapy with bi-weekly gemcitabine 50 mg/m 2 for 6 weeks. CT-scan demonstrated partial response, so patient received additional 12 cycles of GEMOX, with further response, which currently persist since 17 months (to 3.8 cm vs . 2.5 cm). At the same time, the RNASeq was performed at 75x2 bp on a HiScanSQ (Illumina Inc., San Diego, CA, USA) platform. Single nucleotide variants (SNVs) were detected with SNVMix2 and filtered on dbSNP, 1000 Genomes Project, and Cosmic databases. Non-synonymous SNVs were analyzed with SNPsG ATXN10-TMEM49; chromosome 22 and 17) and 2 out-of-frame fusions [ t (15;3) and t (19;22)] leading to SMAD3-KIAA1143 and LTBP4-SPATS2L, both disrupting genes of the TGFbeta pathway. Conclusion We found a novel somatic alteration involving HMGCR in LAPC. Due to the key role of HMGCR in cellular transformation, we hypothesize a strong potential in the development and outcome of LAPC, whose the optimal treatment remains to be elucidated. Trials that integrate RNASeq data with clinical options are needed.

Locally Advanced Pancreatic Cancer: Is It Possible Pancreatic Resection? A Case Report

Context Patients with locally advanced pancreatic cancer are usually treated with chemoradiotherapy and rarely they became resectable. Herein, we present the case of a patient with locally advanced pancreatic cancer. Case report A 56-year-old man was observed in October 2011 because of high blood levels of CA 19.9 (>230 U/mL) and the presence of a pancreatic mass of the uncinate process (diameter 3.8x3.5 cm) revealed by US and CT scan. An US-guided biopsy allowed the diagnosis of well differentiated pancreatic adenocarcinoma, biliopancreatic type. CT scan showed a vascular involvement of both superior mesenteric vein and artery. The disease was defined as locally advanced unresectable pancreatic cancer. The patient started chemotherapy with gemcitabine and oxaliplatin. Five months later, CT scan re-evaluation of the disease showed a stable disease. Thus, a protocol of radio-chemotherapy was suggested. Eight months later from diagnosis, the mass was still unresectable. Other eight cycles of gemcitabine and oxaliplatin were performed. In February 2013 a further CT scan evaluation demonstrated a smaller lesion (3.5x2.2 cm) and also the vascular involvement was decreased, still without a normal fat plane between the tumor and the vessels. Another cycle of gemcitabine and oxaliplatin was completed. At the end of May 2013, the 18 FDG-PET was negative; CT scan demonstrated a further decreased of the mass (maximum diameter: 2.5 cm) while the mesenteric vessels involvement still remained. Moreover, the genomic characteristics of the patient DNA were different from other the pancreatic cancer. Because of the long-term survival of the young patient, the partial regression of the disease and the genomic characteristics of the tumor, a surgical approach was indicated. The patient underwent to a total pancreatectomy with splenectomy plus segmental resection of portal mesenteric trunk. Pathological diagnosis confirmed a well-differentiated ductal pancreatic carcinoma, biliopancreatic type (T4), with R0 resection. Conclusion Our case suggests that there are locally advanced pancreatic cancers in which chemoradiotherapy can allow surgical pancreatic resection probably because they have particular genomic characteristics.

Whole Transcriptome Sequencing Reveals Molecular Prognostic Markers in Pancreatic Adenocarcinoma

Context Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy with few effective therapies. Although next-generation sequencing (NGS) further clarified the genomic complexity of PDAC, genes or pathways that specifically drive tumour progression or metastasis are not well understood. Objective Our challenge is to implement a whole transcriptome massively parallel sequencing (RNASeq) study to better understand the PDAC molecular biology for diagnosis and prognosis of PDAC. Methods We collected a total of 17 PDAC samples by ultrasound-guided biopsy or by surgical specimen for RNA extraction. Fourteen samples were analyzed by RNASeq, performed at 75x2 bp on a HiScanSQ Illumina platform. To study gene expression profiling related to poor outcome, we first studied differentially expressed genes between “poor” (overall survival 36 months; n=3) prognosis in a total of 6 PDAC sample. Results The relative presence of tumor cells in the sample was evaluated based on the presence of KRAS mutation. A total of 211 genes were differentially expressed. Genes involved in the p53 signalling pathway (CSNK1G1, TGFA), the Wnt/β-catenin (DKK1, WNT7B, WNT10A), insulin-like growth factor system (IGF2) and EGF receptor signalling pathway (EGF) were highly upregulated in “poor” PDAC samples. Interestingly, we found a strong overexpression of CA125/MUC16, recently demonstrated to be involved in PDCA and ovarian cancer invasion by stimulating matrix metalloproteinases 7. Conclusion Components of p53 signalling pathway, Wnt/β-catenin pathways, insulin-like growth factor system and MUC16 might be useful molecular markers in PDCA as their overexpression seems to be related with cancer growth, invasion and prognosis. Further validation of the role of these genes is necessary for translation in clinical practice.