C. Max Schmidt

Detection of Circulating Tumor DNA in Early- and Late-Stage Human Malignancies

Circulating tumor DNA can be used in a variety of clinical and investigational settings across tumor types and stages for screening, diagnosis, and identifying mutations responsible for therapeutic response and drug resistance. Circulating Tumor DNA for Early Detection and Managing Resistance Cancer evolves over time, without any warning signs. Similarly, the development of resistance to therapy generally becomes apparent only when there are obvious signs of tumor growth, at which point the patient may have lost valuable time. Although a repeat biopsy may be able to identify drug-resistant mutations before the tumor has a chance to regrow, it is usually not feasible to do many repeat biopsies. Now, two studies are demonstrating the utility of monitoring the patients’ blood for tumor DNA to detect cancer at the earliest stages of growth or resistance. In one study, Bettegowda and coauthors showed that sampling a patient’s blood may be sufficient to yield information about the tumor’s genetic makeup, even for many early-stage cancers, without a need for an invasive procedure to collect tumor tissue, such as surgery or endoscopy. The authors demonstrated the presence of circulating DNA from many types of tumors that had not yet metastasized or released detectable cells into the circulation. They could detect more than 50% of patients across 14 tumor types at the earliest stages, when these cancers may still be curable, suggesting that a blood draw could be a viable screening approach to detecting most cancers. They also showed that in patients with colorectal cancer, the information derived from circulating tumor DNA could be used to determine the optimal course of treatment and identify resistance to epidermal growth factor receptor (EGFR) blockade. Meanwhile, Misale and colleagues illustrated a way to use this information to overcome treatment resistance. These authors also found that mutations associated with EGFR inhibitor resistance could be detected in the blood of patients with colorectal cancer. In addition, they demonstrated that adding MEK inhibitors, another class of anticancer drugs, can successfully overcome resistance when given in conjunction with the EGFR inhibitors. Thus, the studies from Bettegowda and Misale and their colleagues show the effectiveness of analyzing circulating DNA from a variety of tumors and highlight the potential investigational and clinical applications of this novel technology for early detection, monitoring resistance, and devising treatment plans to overcome resistance. The development of noninvasive methods to detect and monitor tumors continues to be a major challenge in oncology. We used digital polymerase chain reaction–based technologies to evaluate the ability of circulating tumor DNA (ctDNA) to detect tumors in 640 patients with various cancer types. We found that ctDNA was detectable in >75% of patients with advanced pancreatic, ovarian, colorectal, bladder, gastroesophageal, breast, melanoma, hepatocellular, and head and neck cancers, but in less than 50% of primary brain, renal, prostate, or thyroid cancers. In patients with localized tumors, ctDNA was detected in 73, 57, 48, and 50% of patients with colorectal cancer, gastroesophageal cancer, pancreatic cancer, and breast adenocarcinoma, respectively. ctDNA was often present in patients without detectable circulating tumor cells, suggesting that these two biomarkers are distinct entities. In a separate panel of 206 patients with metastatic colorectal cancers, we showed that the sensitivity of ctDNA for detection of clinically relevant KRAS gene mutations was 87.2% and its specificity was 99.2%. Finally, we assessed whether ctDNA could provide clues into the mechanisms underlying resistance to epidermal growth factor receptor blockade in 24 patients who objectively responded to therapy but subsequently relapsed. Twenty-three (96%) of these patients developed one or more mutations in genes involved in the mitogen-activated protein kinase pathway. Together, these data suggest that ctDNA is a broadly applicable, sensitive, and specific biomarker that can be used for a variety of clinical and research purposes in patients with multiple different types of cancer.

Recurrent GNAS Mutations Define an Unexpected Pathway for Pancreatic Cyst Development

A mutation in the gene GNAS serves as a marker for pancreatic cysts that can progress to become invasive adenocarcinomas, guiding therapy. Staying Out of the Operating Room Like bad traffic, surgery is something to be avoided if at all possible. This was the guiding principle of Wu et al. when conducting their research on pancreatic cysts, found in more than 2% of the population. Physician and patient face a dilemma: Some pancreatic cysts will progress to form dangerous invasive carcinoma and some persist harmlessly forever. So, to err on the side of safety, many harmless cysts are removed, exposing patients to unnecessary risk. By sequencing DNA from pancreatic cysts and invasive pancreatic cancers, the authors have now determined that the presence of mutations in two oncogenes can identify the cysts that are likely to progress to carcinoma and so are good candidates for surgical removal. (Without KRAS or GNAS mutations, the cyst may be better left in place.) The authors assessed DNA from the cyst fluid of 19 intraductal papillary mucinous neoplasms and the corresponding normal tissue by massively parallel sequencing for mutations in 169 cancer genes. Two results stood out—one expected and one unexpected. Fourteen of the 19 tumors showed mutations in the known pancreatic oncogene KRAS, and 6 of the 19 carried a mutation in GNAS, a well-known oncogene in other tumor types. Of a larger set of fluid samples from these cystic neoplasms (132), 96% carried a mutation in at least one of the two oncogenes. In contrast, 44 benign cysts exhibited no GNAS or KRAS mutations. Cystic fluid that contains DNA with both the GNAS and KRAS mutations indicates, with high sensitivity and specificity, that the lesion is likely to be an intraductal papillary mucinous neoplasm (at risk for developing into an invasive carcinoma) and not a benign serous cystadenomas. These genetic markers could distinguish between benign tumors and more problematic neoplasms, enabling some patients to avoid the undesirable experience of surgery. More than 2% of the adult U.S. population harbors a pancreatic cyst. These often pose a difficult management problem because conventional criteria cannot always distinguish cysts with malignant potential from those that are innocuous. One of the most common cystic neoplasms of the pancreas, and a bona fide precursor to invasive adenocarcinoma, is called intraductal papillary mucinous neoplasm (IPMN). To help reveal the pathogenesis of these lesions, we purified the DNA from IPMN cyst fluids from 19 patients and searched for mutations in 169 genes commonly altered in human cancers. In addition to the expected KRAS mutations, we identified recurrent mutations at codon 201 of GNAS. A larger number (113) of additional IPMNs were then analyzed to determine the prevalence of KRAS and GNAS mutations. In total, we found that GNAS mutations were present in 66% of IPMNs and that either KRAS or GNAS mutations could be identified in 96%. In eight cases, we could investigate invasive adenocarcinomas that developed in association with IPMNs containing GNAS mutations. In seven of these eight cases, the GNAS mutations present in the IPMNs were also found in the invasive lesion. GNAS mutations were not found in other types of cystic neoplasms of the pancreas or in invasive adenocarcinomas not associated with IPMNs. In addition to defining a new pathway for pancreatic neoplasia, these data suggest that GNAS mutations can inform the diagnosis and management of patients with cystic pancreatic lesions.

Whole-exome sequencing of neoplastic cysts of the pancreas reveals recurrent mutations in components of ubiquitin-dependent pathways

More than 2% of adults harbor a pancreatic cyst, a subset of which progresses to invasive lesions with lethal consequences. To assess the genomic landscapes of neoplastic cysts of the pancreas, we determined the exomic sequences of DNA from the neoplastic epithelium of eight surgically resected cysts of each of the major neoplastic cyst types: serous cystadenomas (SCAs), intraductal papillary mucinous neoplasms (IPMNs), mucinous cystic neoplasms (MCNs), and solid pseudopapillary neoplasms (SPNs). SPNs are low-grade malignancies, and IPMNs and MCNs, but not SCAs, have the capacity to progress to cancer. We found that SCAs, IPMNs, MCNs, and SPNs contained 10 ± 4.6, 27 ± 12, 16 ± 7.6, and 2.9 ± 2.1 somatic mutations per tumor, respectively. Among the mutations identified, E3 ubiquitin ligase components were of particular note. Four of the eight SCAs contained mutations of the von Hippel–Lindau gene (VHL), a key component of the VHL ubiquitin ligase complex that has previously been associated with renal cell carcinomas, SCAs, and other neoplasms. Six of the eight IPMNs and three of the eight MCNs harbored mutations of RNF43, a gene coding for a protein with intrinsic E3 ubiquitin ligase activity that has not previously been found to be genetically altered in any human cancer. The preponderance of inactivating mutations in RNF43 unequivocally establish it as a suppressor of both IPMNs and MCNs. SPNs contained remarkably few genetic alterations but always contained mutations of CTNNB1, previously demonstrated to inhibit degradation of the encoded protein (β-catenin) by E3 ubiquitin ligases. These results highlight the essential role of ubiquitin ligases in these neoplasms and have important implications for the diagnosis and treatment of patients with cystic tumors.

Intraductal papillary mucinous neoplasms: Predictors of malignant and invasive pathology

Objective:Determine whether size and other preoperative parameters predict malignant or invasive intraductal papillary mucinous neoplasia (IPMN). Summary Background Data:From 1991 to 2006, 150 patients underwent 156 operations for IPMN. Methods:Prospectively collected, retrospective review of a single academic institutions experience. All preoperative parameters including a detailed radiologic-based classification of IPMN type, location, distribution, size, number, cytology, and mural nodularity were correlated with IPMN pathology. Results:Malignant IPMN was present in 32% of cases, whereas 19% of cases were invasive. IPMN type and main pancreatic duct diameter were significant predictors of malignant IPMN (P < 0.001). Side-branch lesion number was negatively associated with invasive IPMN (P = 0.03). Side-branch size, location, and distribution did not predict IPMN pathology. The presence of mural nodules was associated with malignant and invasive IPMN (P < 0.001; P < 0.02). Atypical cytopathology was significantly associated with malignant and invasive IPMN (P < 0.001; P < 0.001). Multivariate analysis demonstrated mural nodularity and atypical cytopathology were predictive of malignancy and/or invasion in branch-type IPMN. Conclusions:To lower the rate of invasive pathology, surgery should be recommended for fit patients with main-duct IPMN and for branch-duct IPMN with mural nodularity or positive cytology irrespective of location, distribution, or size.

A Multicenter Analysis of Distal Pancreatectomy for Adenocarcinoma: Is Laparoscopic Resection Appropriate?

BACKGROUND As compared with open distal pancreatectomy (ODP), laparoscopic distal pancreatectomy (LDP) affords improved perioperative outcomes. The role of LDP for patients with pancreatic ductal adenocarcinoma (PDAC) is not defined. STUDY DESIGN Records from patients undergoing distal pancreatectomy (DP) for PDAC from 2000 to 2008 from 9 academic medical centers were reviewed. Short-term (node harvest and margin status) and long-term (survival) cancer outcomes were assessed. A 3:1 matched analysis was performed for ODP and LDP cases using age, American Society of Anesthesiologists (ASA) class, and tumor size. RESULTS There were 212 patients who underwent DP for PDAC; 23 (11%) of these were approached laparoscopically. For all 212 patients, 56 (26%) had positive margins. The mean number of nodes (+/- SD) examined was 12.6 +/-8.4 and 114 patients (54%) had at least 1 positive node. Median overall survival was 16 months. In the matched analysis there were no significant differences in positive margin rates, number of nodes examined, number of patients with at least 1 positive node, or overall survival. Logistic regression for all 212 patients demonstrated that advanced age, larger tumors, positive margins, and node positive disease were independently associated with worse survival; however, method of resection (ODP vs. LDP) was not. Hospital stay was 2 days shorter in the matched comparison, which approached significance (LDP, 7.4 days vs. ODP, 9.4 days, p = 0.06). CONCLUSIONS LDP provides similar short- and long-term oncologic outcomes as compared with OD, with potentially shorter hospital stay. These results suggest that LDP is an acceptable approach for resection of PDAC of the left pancreas in selected patients.

A margin-negative R0 resection accomplished with minimal postoperative complications is the surgeon’s contribution to long-term survival in pancreatic cancer

Pancreatic cancer has a poor prognosis with complete surgical resection being the only therapy to offer a realistic chance for long-term survival. The aim of this study is to identify surgery-related variables that influence long-term survival. Between 1990 and 2002, 226 consecutive patients (mean age of 64 ± 11 years) had resection for pancreatic adenocarcinoma. Prognostic variables in these patients were analyzed using univariate and multivariate analysis. Two hundred four patients (90%) had pancreaticoduodenectomy, 13 patients (6%) had distal pancreatectomy, and 9 patients (4%) had a TP. Stage I disease was present in 50 (22%), stage II disease in 170 (75%), and stage III disease in 6 (3%). R0 resections were achieved in 70%. Operative morbidity was 36% and 30-day mortality was 6%. Actual 1-year, 3-year, and 5-year survival rates were 49% (n=111), 14% (n=31), and 4% (n=9). Using multivariate analysis: tumor size, tumor differentiation, obtaining an R0 resection, and lack of postoperative complications were variables associated with long-term survival. Long-term survival in patients with pancreatic cancer after resection remains poor. Achieving a margin negative resection (R0) with no postoperative complications are prognostic variables that can be affected by the surgeon.

Effect of Hospital Volume, Surgeon Experience, and Surgeon Volume on Patient Outcomes After Pancreaticoduodenectomy: A Single-Institution Experience

OBJECTIVE To determine the importance of hospital volume, surgeon experience, and surgeon volume in performing pancreaticoduodenectomy (PD). DESIGN, SETTING, AND PATIENTS From 1980 through 2007, 1003 patients underwent PD by 19 surgeons at a university hospital. MAIN OUTCOME MEASURES Patient morbidity and mortality, quality of resection, and learning curve were examined according to hospital volume (period 1: 1980-2003 vs period 2: 2004-2007), surgeon experience (total number of PDs), and surgeon volume (number of PDs per year). RESULTS Perioperative morbidity and mortality for all 1003 PDs were 41% and 3%, respectively. Differences existed between period 1 and period 2 in percentage of PDs performed in elderly patients (7% vs 17%), mortality (4% vs 2%), estimated blood loss (1817 mL vs 780 mL), length of stay (18 days vs 12 days), and proportion of International Study Group on Pancreatic Fistula grade C pancreatic fistulae (29% vs 12%). Surgeons with less experience (<50 PDs) performed PD with higher morbidity (53% vs 39%), pancreatic fistula rate (20% vs 10%), estimated blood loss (1918 mL vs 1101 mL), and operative time (458 minutes vs 335 minutes) compared with surgeons with more experience (> or =50 PDs). Experienced surgeons had comparable outcomes irrespective of annual volume. Mortality, margins, and number of lymph nodes resected were not affected by surgeon experience or surgeon volume. Learning curves projected that less experienced surgeons would achieve morbidity and mortality rates equivalent to those of experienced surgeons when they reached 20 and 60 PDs, respectively. CONCLUSIONS Improvement in PD outcomes, including mortality, occurred with increased PD volume at a pancreatic center. Surgeon experience remained an important determinant of overall morbidity. Experienced surgeons, however, had comparable outcomes irrespective of annual volume.

Fatty pancreas: A factor in postoperative pancreatic fistula

Objective:To determine whether patients who develop a pancreatic fistula after pancreatoduodenectomy are more likely to have pancreatic fat than matched controls. Background:Pancreatic fistula continues to be a major cause of postoperative morbidity and increased length of stay after pancreatoduodenectomy. Factors associated with postoperative pancreatic fistula include a soft pancreas, a small pancreatic duct, the underlying pancreatic pathology, the regional blood supply, and surgeons experience. Fatty pancreas previously has not been considered as a contributing factor in the development of postoperative pancreatic fistula. Methods:Forty patients with and without a pancreatic fistula were identified from an Indiana University database of over 1000 patients undergoing pancreatoduodenectomy and matched for multiple parameters including age, gender, pancreatic pathology, surgeon, and type of operation. Surgical pathology specimens from the pancreatic neck were reviewed blindly for fat, fibrosis, vessel density, and inflammation. These parameters were scored (0–4+). Results:The pancreatic fistula patients were less likely (P < 0.05) to have diabetes but had significantly more intralobular (P < 0.001), interlobular (P < 0.05), and total pancreatic fat (P < 0.001). Fistula patients were more likely to have high pancreatic fat scores (50% vs. 13%, P < 0.001). Pancreatic fibrosis, vessel density, and duct size were lower (P < 0.001) in the fistula patients and negative correlations (P < 0.001) existed between fat and fibrosis (R = −0.40) and blood vessel density (R = −0.15). Conclusions:These data suggest that patients with postoperative pancreatic fistula have (1) increased pancreatic fat and (2) decreased pancreatic fibrosis, blood vessel density, and duct size. Therefore, we conclude that fatty pancreas is a risk factor for postoperative pancreatic fistula.

Neuroendocrine Tumors of the Pancreas

This literature review briefly summarizes the epidemiology, pathophysiology, clinical management, and outcomes of patients with pancreatic neuroendocrine tumors (PNETs) and highlights recent advances in PNET research. PNETs are rare neoplasms, compared with carcinomas arising from pancreatic exocrine tissue. They, like other neuroendocrine tumor types, display variable malignant potential, hormone-related syndromes (functionality), localization, and genetic background. Although tumor origin and molecular pathogenesis remain poorly understood, recently established grading and staging systems facilitate patient risk stratification, and thereby directly impact clinical decision making. Although the optimal clinical management of PNETs involves a multidisciplinary approach, surgery remains the only curative treatment for early-stage disease. Surgery may also have a role in patients with advanced-stage disease, including those with hepatic metastases. Alternative therapeutic approaches applied to PNETs, including chemotherapy, radiofrequency ablation, transarterial chemoembolization, biotherapy, polypeptide radionuclide receptor therapy, antiangiogenic therapy, and selective internal radiotherapy, have failed to demonstrate a long-term survival benefit. Surgery remains the primary therapeutic option for patients with PNETs. Research on PNETs is desperately needed to improve the therapeutic options for patients with this disease.

CT vs MRCP: optimal classification of IPMN type and extent.

IntroductionIntraductal papillary mucinous neoplasms (IPMNs) of the pancreas are being diagnosed with increased frequency. CT scanning commonly serves as the primary imaging modality before surgery. We hypothesized MRCP provides better characterization of IPMN type/extent, which more closely matches actual pathology.MethodsOf 214 patients treated with IPMN (1991–2006), 30 had both preoperative CT and MRCP. Of these, 18 met imaging study criteria. Independent readers performed retrospective, blinded analyses using standardized criteria for IPMN type and extent.ResultsA ductal connection was detected on 73% of MRCP scans and only 18% of CT. IPMN type was classified differently in seven (39%); four (22%) of which were read on CT as having main duct involvement where this was not appreciated on MRCP or found on surgical pathology. MRCP showed multifocal disease in 13(72%) versus only 9(50%) on CT. A different disease distribution was seen in 9(50%). Finally, 101 branch lesions were identified on MRCP compared to 46 on CT.ConclusionsCT falls short of MRCP in detecting a ductal connection, estimating main duct involvement, and identification of small branch duct cysts. These factors influence diagnostic accuracy, cancer risk stratification and operative strategy. MRCP should be employed for optimal management of patients with IPMN.