Christopher L. Wolfgang

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.

DAXX/ATRX, MEN1, and mTOR pathway genes are frequently altered in pancreatic neuroendocrine tumors.

A rare but deadly form of human pancreatic cancer harbors mutations in chromatin remodeling genes. Pancreatic neuroendocrine tumors (PanNETs) are a rare but clinically important form of pancreatic neoplasia. To explore the genetic basis of PanNETs, we determined the exomic sequences of 10 nonfamilial PanNETs and then screened the most commonly mutated genes in 58 additional PanNETs. The most frequently mutated genes specify proteins implicated in chromatin remodeling: 44% of the tumors had somatic inactivating mutations in MEN1, which encodes menin, a component of a histone methyltransferase complex, and 43% had mutations in genes encoding either of the two subunits of a transcription/chromatin remodeling complex consisting of DAXX (death-domain–associated protein) and ATRX (α thalassemia/mental retardation syndrome X-linked). Clinically, mutations in the MEN1 and DAXX/ATRX genes were associated with better prognosis. We also found mutations in genes in the mTOR (mammalian target of rapamycin) pathway in 14% of the tumors, a finding that could potentially be used to stratify patients for treatment with mTOR inhibitors.

Whole genomes redefine the mutational landscape of pancreatic cancer

Pancreatic cancer remains one of the most lethal of malignancies and a major health burden. We performed whole-genome sequencing and copy number variation (CNV) analysis of 100 pancreatic ductal adenocarcinomas (PDACs). Chromosomal rearrangements leading to gene disruption were prevalent, affecting genes known to be important in pancreatic cancer (TP53, SMAD4, CDKN2A, ARID1A and ROBO2) and new candidate drivers of pancreatic carcinogenesis (KDM6A and PREX2). Patterns of structural variation (variation in chromosomal structure) classified PDACs into 4 subtypes with potential clinical utility: the subtypes were termed stable, locally rearranged, scattered and unstable. A significant proportion harboured focal amplifications, many of which contained druggable oncogenes (ERBB2, MET, FGFR1, CDK6, PIK3R3 and PIK3CA), but at low individual patient prevalence. Genomic instability co-segregated with inactivation of DNA maintenance genes (BRCA1, BRCA2 or PALB2) and a mutational signature of DNA damage repair deficiency. Of 8 patients who received platinum therapy, 4 of 5 individuals with these measures of defective DNA maintenance responded.

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.

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Laparoscopic distal pancreatectomy is associated with significantly less overall morbidity compared to the open technique: a systematic review and meta-analysis

Objective:To compare laparoscopic distal pancreatectomy (LDP) versus open distal pancreatectomy (ODP) by using meta-analytical techniques. Background:LDP is increasingly performed as an alternative approach for distal pancreatectomy in selected patients. Multiple studies have tried to assess the safety and efficacy of LDP compared with ODP. Methods:A systematic review of the literature was performed to identify studies comparing LDP and ODP. Intraoperative outcomes, postoperative recovery, oncologic safety, and postoperative complications were evaluated. Meta-analysis was performed using a random-effects model. Results:Eighteen studies matched the selection criteria, including 1814 patients (43% laparoscopic, 57% open). LDP had lower blood loss by 355 mL (P < 0.001) and hospital length of stay by 4.0 days (P < 0.001). Overall complications were significantly lower in the laparoscopic group (33.9% vs 44.2%; odds ratio [OR] = 0.73, 95% confidence interval [CI] 0.57–0.95), as was surgical site infection (2.9% vs 8.1%; OR = 0.45, 95% CI 0.24–0.82). There was no difference in operative time, margin positivity, incidence of postoperative pancreatic fistula, and mortality. Conclusions:LDP has lower blood loss and reduced length of hospital stay. There was a lower risk of overall postoperative complications and wound infection, without a substantial increase in the operative time. Although a thorough evaluation of oncological outcomes was not possible, the rate of margin positivity was comparable to the open technique. The improved complication profile of LDP, taken together with the lack of compromise of margin status, suggests that this technique is a reasonable approach in selected cancer patients.

Analysis of Fluorouracil-Based Adjuvant Chemotherapy and Radiation After Pancreaticoduodenectomy for Ductal Adenocarcinoma of the Pancreas: Results of a Large, Prospectively Collected Database at the Johns Hopkins Hospital

PURPOSE To examine the efficacy of adjuvant chemoradiotherapy after pancreaticoduodenectomy (PD) for pancreatic adenocarcinoma (PC) in patients undergoing resection at Johns Hopkins Hospital (JHH; Baltimore, MD). PATIENTS AND METHODS Between August 30, 1993, and February 28, 2005, a total of 908 patients underwent PD for PC at JHH. A prospective database was reviewed to determine which patients received fluorouracil (FU) -based CRT. Excluded patients had metastatic disease, died 60 or fewer days after PD, received preoperative therapy, an experimental vaccine, adjuvant chemotherapy or radiation alone. The final cohort includes 616 patients. RESULTS The median follow-up was 17.8 months (interquartile range, 9.7 to 33.5 months). Overall median survival was 17.9 months (95% CI, 16.3 to 19.5 months). Groups were similar with respect to tumor size, nodal status, and margin status, but the CRT group was younger (P < .001), and less likely to present with a severe comorbid disease (P = .001). Patients with carcinomas larger than 3 cm (P = .001), grade 3 and 4 (P < .001), margin-positive resection (P = .001), and complications after surgery (P = .017) had poor long-term survival. Patients receiving CRT experienced an improved median (21.2 v 14.4 months; P < .001), 2-year (43.9% v 31.9%), and 5-year (20.1% v 15.4%) survival compared with no CRT. After controlling for high-risk features, CRT was still associated with improved survival (relative risk = 0.74; 95% CI, 0.62 to 0.89). CONCLUSION These data suggest that adjuvant concurrent FU-based CRT significantly improves survival after PD for PC when compared with patients not receiving CRT. These data support the use of combined adjuvant CRT for PC.

Does pancreatic duct stenting decrease the rate of pancreatic fistula following pancreaticoduodenectomy? Results of a prospective randomized trial.

Pancreatic duct stenting remains an attractive strategy to reduce the incidence of pancreatic fistulas following pancreaticoduodenectomy (PD) with encouraging results in both retrospective and prospective studies. We performed a prospective randomized trial to test the hypothesis that internal pancreatic duct stenting reduces the development of pancreatic fistulas following PD. Two hundred thirty-eight patients were randomized to either receive a pancreatic stent (S) or no stent (NS), and stratified according to the texture of the pancreatic remnant (soft/normal versus hard). Four patients were excluded from the study; in three instances due to a pancreatic duct that was too small to cannulate and in the other instance because a total pancreatectomy was performed. Patients who randomized to the S group had a 6-cm-long segment of a plastic pediatric feeding tube used to stent the pancreaticojejunostomy anastomosis. In patients with a soft pancreas, 57 randomized to the S group and 56 randomized to the NS group. In patients with a hard pancreas, 58 randomized to the S group and 63 randomized to the NS group. The S and NS groups for the entire study population, as well as for the subgroup of high-risk patients with soft pancreata, were similar as regard to demographics, past medical history, preoperative symptoms, preoperative procedures, and intraoperative data. The pancreatic fistula rate for the entire study population was 9.4%. The fistula rates in the S and NS subgroups with hard pancreata were similar, at 1.7% and 4.8% (P=0.4), respectively. The fistula rates in the S and NS subgroups with soft pancreata were also similar, at 21.1% and 10.7% (P=0.1), respectively. A nonstatistically significant increase in the pancreatic fistula rate in the S group persisted after adjusting for the operating surgeon and technical details of the operation (e.g., anastomotic technique, anastomotic orientation, pancreatic duct size, and number of intra-abdominal drains placed). In patients with soft pancreata, 63% percent of the pancreatic fistulas in stented patients required adjustment to the clinical pathway (including two deaths), compared to 47% of the pancreatic fistulas in patients in the NS group (P=0.3). Internal pancreatic duct stenting does not decrease the frequency or the severity of postoperative pancreatic fistulas.

SMAD4 Gene Mutations Are Associated with Poor Prognosis in Pancreatic Cancer

Purpose: Recently, the majority of protein coding genes were sequenced in a collection of pancreatic cancers, providing an unprecedented opportunity to identify genetic markers of prognosis for patients with adenocarcinoma of the pancreas. Experimental Design: We previously sequenced more than 750 million base pairs of DNA from 23,219 transcripts in a series of 24 adenocarcinomas of the pancreas. In addition, 39 genes that were mutated in more than one of these 24 cancers were sequenced in a separate panel of 90 well-characterized adenocarcinomas of the pancreas. Of these 114 patients, 89 underwent pancreaticoduodenectomy, and the somatic mutations in these cancers were correlated with patient outcome. Results: When adjusted for age, lymph node status, margin status, and tumor size, SMAD4 gene inactivation was significantly associated with shorter overall survival (hazard ratio, 1.92; 95% confidence interval, 1.20-3.05; P = 0.006). Patients with SMAD4 gene inactivation survived a median of 11.5 months, compared with 14.2 months for patients without SMAD4 inactivation. By contrast, mutations in CDKN2A or TP53 or the presence of multiple (≥4) mutations or homozygous deletions among the 39 most frequently mutated genes were not associated with survival. Conclusions:SMAD4 gene inactivation is associated with poorer prognosis in patients with surgically resected adenocarcinoma of the pancreas.