Association of Mutant KRAS Alleles With Morphology and Clinical Outcomes in Pancreatic Ductal Adenocarcinoma.

CONTEXT.­: Mutant KRAS is the main oncogenic driver in pancreatic ductal adenocarcinomas (PDACs). However, the clinical and phenotypic implications of harboring different mutant KRAS alleles remain poorly understood. OBJECTIVE.­: To characterize the potential morphologic and clinical outcome differences in PDACs harboring distinct mutant KRAS alleles. DESIGN.­: Cohort 1 consisted of 127 primary conventional PDACs with no neoadjuvant therapy, excluding colloid/mucinous, adenosquamous, undifferentiated, and intraductal papillary mucinous neoplasm-associated carcinomas, for which an in-house 42-gene mutational panel had been performed. A morphologic classification system was devised wherein each tumor was assigned as conventional, papillary/large duct (P+LD, defined as neoplastic glands with papillary structure and/or with length ≥0.5 mm), or poorly differentiated (when the aforementioned component was 60% or more of the tumor). Cohort 2 was a cohort of 88 PDACs in The Cancer Genome Atlas, which were similarly analyzed. RESULTS.­: In both cohorts, there was significant enrichment of P+LD morphology in PDACs with KRAS G12V and G12R compared with G12D. In the entire combined cohort, Kaplan-Meier analyses showed longer overall survival (OS) with KRAS G12R as compared with G12D (median OS of 1255 versus 682 days, P = .03) and in patients whose PDACs displayed P+LD morphology as compared with conventional morphology (median OS of 1175 versus 684 days, P = .04). In the adjuvant-only subset, KRAS G12R had the longest OS compared with G12D, G12V, and other alleles (median OS unreached/undefined versus 1009, 1129, and 1222 days, respectively). CONCLUSIONS.­: PDACs with different mutant KRAS alleles are associated with distinct morphologies and clinical outcomes, with KRAS G12R allele associated with P+LD morphology and longer OS when compared with G12D using Kaplan-Meier studies.

Euglycemic diabetic ketoacidosis (EDKA) after pancreaticoduodenectomy: An under-recognized metabolic abnormality with outcome implications.

BACKGROUND: Euglycemic diabetic ketoacidosis is a metabolic condition characterized by relative euglycemia, ketonemia, and metabolic acidosis that occurs through mechanisms resembling starvation. Pancreaticoduodenectomy is a complex abdominal operation that subjects patients to a prolonged fasting and an inflammatory state. This study examined the incidence of euglycemic diabetic ketoacidosis and potential opportunities for early diagnosis and management in patients undergoing pancreaticoduodenectomy. METHODS: A single-institution retrospective review of 350 patients who underwent pancreaticoduodenectomy between 2017 and 2020 was performed. Primary endpoints were peak beta-hydroxybutyrate levels, peak lactate levels, lowest pH, peak base deficits, and urinary output within the first 24 hours, postoperatively. Additional endpoints included incidence of postoperative pancreatic fistula, delayed gastric emptying, total complications, postoperative hospital length of stay, readmission rates, and changes in insulin regimen at discharge. RESULTS: Of the 350 cases reviewed, 39 (11.1%) patients developed euglycemic diabetic ketoacidosis. Male sex and pancreatic cancer were associated with a risk for euglycemic diabetic ketoacidosis (P < .05). Patients with euglycemic diabetic ketoacidosis had significantly higher peak beta-hydroxybutyrate levels than patients without euglycemic diabetic ketoacidosis (mean difference = 19.8 mg/dL, 95% confidence interval = 14.7-24.9, P < .001), and were nearly four times more likely to require insulin at discharge (odds ratio 3.8, 95% confidence interval = 1.1-13.0, P < .05). CONCLUSION: This is the first large descriptive study that investigates euglycemic diabetic ketoacidosis after pancreaticoduodenectomy. Euglycemic diabetic ketoacidosis after pancreaticoduodenectomy is associated with significantly higher beta-hydroxybutyrate levels and new or increased insulin requirement at discharge. Our study demonstrates potential markers for euglycemic diabetic ketoacidosis after pancreaticoduodenectomy, offering an opportunity to identify and successfully treat this disease in a timely manner.

Margin-Positive Pancreatic Ductal Adenocarcinoma during Pancreaticoduodenectomy: Additional Resection Does Not Improve Survival.

BACKGROUND: The impact of resecting positive margins during pancreaticoduodenectomy (PD) for pancreatic ductal adenocarcinoma (PDA) remains debated. Additionally, the survival benefit of resecting multiple positive margins is unknown. METHODS: We identified patients with PDA who underwent PD from 2006 to 2015. Pancreatic neck, bile duct, and uncinate frozen section margins were assessed before and after resection of positive margins. Survival curves were compared with log-rank tests. Multivariable Cox regression assessed the effect of margin status on overall survival. RESULTS: Of 501 patients identified, 17.3%, 5.3%, and 19.7% had an initially positive uncinate, bile duct, or neck margin, respectively. Among initially positive bile duct and neck margins, 77.8% and 67.0% were resected, respectively. Although median survival was decreased among patients with any positive margins (15.6 vs. 20.9 months; p = 0.006), it was similar among patients with positive bile duct or neck margins with or without R1 to R0 resection (17.0 vs. 15.6 months; p = 0.20). Median survival with and without positive uncinate margins was 13.8 vs. 19.7 months (p = 0.04). Uncinate margins were never resected. Resection of additional margins when the uncinate was concurrently positive was not associated with improved survival (p = 0.37). Patients with positive margins who received adjuvant therapy had improved survival, regardless of margin resection (p = 0.03). Adjuvant therapy was independently protective against death (hazard ratio 0.6, 95% CI 0.5-0.7). CONCLUSIONS: Positive PD margins at any position are associated with reduced overall survival; however, resection of additional margins may not improve survival, particularly with concurrently positive uncinate margins. Adjuvant chemotherapy improves survival with positive margins, regardless of resection.

Staging of Biliary and Primary Liver Tumors: Current Recommendations and Workup.

Hepatobiliary malignancies are a diverse group of neoplasms involving the liver, gallbladder, and bile ducts. Although intrahepatic cholangiocarcinoma, perihilar cholangiocarcinoma, hepatocellular carcinoma, and gallbladder adenocarcinoma share many biological and anatomic features, they have distinct clinical presentations and natural histories that require individual consideration. Here, we discuss the incidence, outcomes, patient presentation, initial workup, pathologic diagnoses, staging classification, imaging and surgical staging, and determinants of resectability for each malignancy.

Surgical adhesions in mice are derived from mesothelial cells and can be targeted by antibodies against mesothelial markers.

Peritoneal adhesions are fibrous tissues that tether organs to one another or to the peritoneal wall and are a major cause of postsurgical and infectious morbidity. The primary molecular chain of events leading to the initiation of adhesions has been elusive, chiefly due to the lack of an identifiable cell of origin. Using clonal analysis and lineage tracing, we have identified injured surface mesothelium expressing podoplanin (PDPN) and mesothelin (MSLN) as a primary instigator of peritoneal adhesions after surgery in mice. We demonstrate that an anti-MSLN antibody diminished adhesion formation in a mouse model where adhesions were induced by surgical ligation to form ischemic buttons and subsequent surgical abrasion of the peritoneum. RNA sequencing and bioinformatics analyses of mouse mesothelial cells from injured mesothelium revealed aspects of the pathological mechanism of adhesion development and yielded several potential regulators of this process. Specifically, we show that PDPN+MSLN+ mesothelium responded to hypoxia by early up-regulation of hypoxia-inducible factor 1 alpha (HIF1α) that preceded adhesion development. Inhibition of HIF1α with small molecules ameliorated the injury program in damaged mesothelium and was sufficient to diminish adhesion severity in a mouse model. Analyses of human adhesion tissue suggested that similar surface markers and signaling pathways may contribute to surgical adhesions in human patients.

Prospective Evaluation of Results of Reoperation in Zollinger-Ellison Syndrome.

OBJECTIVE: To determine the role of reoperation in patients with persistent or recurrent Zollinger-Ellison Syndrome (ZES). BACKGROUND: Approximately, 0% to 60% of ZES patients are disease-free (DF) after an initial operation, but the tumor may recur. METHODS: A prospective database was queried. RESULTS: A total of 223 patients had an initial operation for possible cure of ZES and then were subsequently evaluated serially with cross sectional imaging-computed tomography, magnetic resonance imaging, ultrasound, more recently octreoscan-and functional studies for ZES activity. The mean age at first surgery was 49 years and with an 11-year mean follow-up 52 patients (23%) underwent reoperation when ZES recurred with imageable disease. Results in this group are analyzed in the current report. Reoperation occurred on a mean of 6 years after the initial surgery with a mean number of reoperations of 1 (range 1-5). After reoperation 18/52 patients were initially DF (35%); and after a mean follow-up of 8 years, 13/52 remained DF (25%). During follow-up, 9/52 reoperated patients (17%) died, of whom 7 patients died a disease-related death (13%). The overall survival from first surgery was 84% at 20 years and 68% at 30 years. Multiple endocrine neoplasia type 1 status did not affect survival, but DF interval and liver metastases did. CONCLUSIONS: These results demonstrate that a significant proportion of patients with ZES will develop resectable persistent or recurrent disease after an initial operation. These patients generally have prolonged survival after reoperation and 25% can be cured with repeat surgery, suggesting all ZES patients postresection should have systematic imaging, and if tumor recurs, advise repeat operation.

Identification of tumorigenic cells and therapeutic targets in pancreatic neuroendocrine tumors.

Pancreatic neuroendocrine tumors (PanNETs) are a type of pancreatic cancer with limited therapeutic options. Consequently, most patients with advanced disease die from tumor progression. Current evidence indicates that a subset of cancer cells is responsible for tumor development, metastasis, and recurrence, and targeting these tumor-initiating cells is necessary to eradicate tumors. However, tumor-initiating cells and the biological processes that promote pathogenesis remain largely uncharacterized in PanNETs. Here we profile primary and metastatic tumors from an index patient and demonstrate that MET proto-oncogene activation is important for tumor growth in PanNET xenograft models. We identify a highly tumorigenic cell population within several independent surgically acquired PanNETs characterized by increased cell-surface protein CD90 expression and aldehyde dehydrogenase A1 (ALDHA1) activity, and provide in vitro and in vivo evidence for their stem-like properties. We performed proteomic profiling of 332 antigens in two cell lines and four primary tumors, and showed that CD47, a cell-surface protein that acts as a "don't eat me" signal co-opted by cancers to evade innate immune surveillance, is ubiquitously expressed. Moreover, CD47 coexpresses with MET and is enriched in CD90(hi)cells. Furthermore, blocking CD47 signaling promotes engulfment of tumor cells by macrophages in vitro and inhibits xenograft tumor growth, prevents metastases, and prolongs survival in vivo.

Multiple Endocrine Neoplasia: Genetics and Clinical Management.

Early diagnosis of multiple endocrine neoplasia (MEN) syndromes is critical for optimal clinical outcomes; before the MEN syndromes can be diagnosed, they must be suspected. Genetic testing for germline alterations in both the MEN type 1 (MEN1) gene and RET proto-oncogene is crucial to identifying those at risk in affected kindreds and directing timely surveillance and surgical therapy to those at greatest risk of potentially life-threatening neoplasia. Pancreatic, thymic, and bronchial neuroendocrine tumors are the leading cause of death in patients with MEN1 and should be aggressively considered by at least biannual computed tomography imaging.

Skin fibrosis. Identification and isolation of a dermal lineage with intrinsic fibrogenic potential.

Dermal fibroblasts represent a heterogeneous population of cells with diverse features that remain largely undefined. We reveal the presence of at least two fibroblast lineages in murine dorsal skin. Lineage tracing and transplantation assays demonstrate that a single fibroblast lineage is responsible for the bulk of connective tissue deposition during embryonic development, cutaneous wound healing, radiation fibrosis, and cancer stroma formation. Lineage-specific cell ablation leads to diminished connective tissue deposition in wounds and reduces melanoma growth. Using flow cytometry, we identify CD26/DPP4 as a surface marker that allows isolation of this lineage. Small molecule-based inhibition of CD26/DPP4 enzymatic activity during wound healing results in diminished cutaneous scarring. Identification and isolation of these lineages hold promise for translational medicine aimed at in vivo modulation of fibrogenic behavior.

RET gene mutations (genotype and phenotype) of multiple endocrine neoplasia type 2 and familial medullary thyroid carcinoma.

The rapid technical advances in molecular biology and accelerating improvements in genomic and proteomic diagnostics have led to increasingly personalized strategies for cancer therapy. Such an approach integrates the genomic, proteomic, and molecular information unique to the individual to provide an accurate genetic diagnosis, molecular risk assessment, informed family counseling, therapeutic profiling, and early preventative management that best fits the particular needs of each patient. The discovery of mutations in the RET proto-oncogene resulting in variable onset and severity of multiple endocrine neoplasia type 2 (MEN2) was the first step in developing direct genetic testing for at-risk individuals. Patients with germline RET mutations may undergo risk assessment and appropriate intervention based on specific mutations. Moreover, family members of affected individuals receive counseling based on understanding of the genetic transmission of the disease. Increasingly, clinicians are able to make therapeutic choices guided by an informative biomarker code. Improvements in detection and management of patients with MEN2 resulting from understanding of the RET proto-oncogene are evidence of the benefits of personalized cancer medicine. This review describes the discovery of the RET proto-oncogene, the association between genotype and phenotype, and the role of mutation analysis on diagnosis and treatment of MEN2.

Pancreatic neuroendocrine tumours: hypoenhancement on arterial phase computed tomography predicts biological aggressiveness.

BACKGROUND: Contrary to pancreatic adenocarcinoma, pancreatic neuroendocrine tumours (PNET) are commonly hyperenhancing on arterial phase computed tomography (APCT). However, a subset of these tumours can be hypoenhancing. The prognostic significance of the CT appearance of these tumors remains unclear. METHODS: From 2001 to 2012, 146 patients with well-differentiated PNET underwent surgical resection. The degree of tumour enhancement on APCT was recorded and correlated with clinicopathological variables and overall survival. RESULTS: APCT images were available for re-review in 118 patients (81%). The majority had hyperenhancing tumours (n = 80, 68%), 12 (10%) were isoenhancing (including cases where no mass was visualized) and 26 (22%) were hypoenhancing. Hypoenhancing PNET were larger, more commonly intermediate grade, and had higher rates of lymph node and synchronous liver metastases. Hypoenhancing PNET were also associated with significantly worse overall survival after a resection as opposed to isoenhancing and hyperenhancing tumours (5-year, 54% versus 89% versus 93%). On multivariate analysis of factors available pre-operatively, only hypoenhancement (HR 2.32, P = 0.02) was independently associated with survival. DISCUSSION: Hypoenhancement on APCT was noted in 22% of well-differentiated PNET and was an independent predictor of poor outcome. This information can inform pre-operative decisions in the multidisciplinary treatment of these neoplasms.

Current management of the Zollinger-Ellison syndrome.

In summary, ZES is a syndrome caused by gastrinoma, usually located within the gastrinoma triangle and associated with symptoms of peptic ulcer disease, GERD, and diarrhea. The diagnosis of ZES is made by measuring fasting levels of serum gastrin, BAO, and the secretin stimulation test. Because of the high association of ZES and MEN1, HPT must be excluded by obtaining a serum calcium and parathyroid hormone level. Treatment of ZES consists of medical control of symptoms with PPIs and evaluation for potentially curative surgical intervention. Noninvasive imaging studies including SRS, CT, and MRI should be performed initially to evaluate for metastases and identify resectable disease. Invasive imaging modalities such as EUS may be performed to further evaluate primary tumors. IOUS, palpation, and duodenotomy are used for intraoperative localization of gastrinomas. In patients with MEN1, surgical resection should be pursued only if there is an identifiable tumor larger than 2 cm and after surgery for the primary hyperparathyroidism (3 1/2-gland parathyroidectomy). All patients with resectable localized sporadic gastrinoma should undergo surgical exploration, even those with biochemical evidence but negative imaging studies. Tumor is most commonly found in the duodenum, and the cure rate is high. In patients with liver metastases, surgery should be considered if all identifiable tumor can be safely removed. A multidisciplinary approach including surgical and nonsurgical therapies should be taken in patients with advanced disease.

Lymph nodes and survival in pancreatic neuroendocrine tumors.

HYPOTHESIS: Lymph node metastases decrease survival in patients with pancreatic neuroendocrine tumors (pNETs). DESIGN: Prospective database searches. SETTING: National Institutes of Health (NIH) and Stanford University Hospital (SUH). PATIENTS: A total of 326 patients underwent surgical exploration for pNETs at the NIH (n = 216) and SUH (n = 110). MAIN OUTCOME MEASURES: Overall survival, disease-related survival, and time to development of liver metastases. RESULTS: Forty patients (12.3%) underwent enucleation and 305 (93.6%) underwent resection. Of the patients who underwent resection, 117 (35.9%) had partial pancreatectomy and 30 (9.2%) had a Whipple procedure. Forty-one patients also had liver resections, 21 had wedge resections, and 20 had lobectomies. Mean follow-up was 8.1 years (range, 0.3-28.6 years). The 10-year overall survival for patients with no metastases or lymph node metastases only was similar at 80%. As expected, patients with liver metastases had a significantly decreased 10-year survival of 30% (P < .001). The time to development of liver metastases was significantly reduced for patients with lymph node metastases alone compared with those with none (P < .001). For the NIH cohort with longer follow-up, disease-related survival was significantly different for those patients with no metastases, lymph node metastases alone, and liver metastases (P < .001). Extent of lymph node involvement in this subgroup showed that disease-related survival decreased as a function of the number of lymph nodes involved (P = .004). CONCLUSIONS: As expected, liver metastases decrease survival of patients with pNETs. Patients with lymph node metastases alone have a shorter time to the development of liver metastases that is dependent on the number of lymph nodes involved. With sufficient long-term follow-up, lymph node metastases decrease disease-related survival. Careful evaluation of number and extent of lymph node involvement is warranted in all surgical procedures for pNETs.

Hypoxia-inducible factor-dependent histone deacetylase activity determines stem cell fate in the placenta.

Hypoxia-inducible factor (HIF) is a heterodimeric transcription factor composed of HIFalpha and the arylhydrocarbon receptor nuclear translocator (ARNT/HIF1beta). Previously, we have reported that ARNT function is required for murine placental development. Here, we used cultured trophoblast stem (TS) cells to investigate the molecular basis of this requirement. In vitro, wild-type TS cell differentiation is largely restricted to spongiotrophoblasts and giant cells. Interestingly, Arnt-null TS cells differentiated into chorionic trophoblasts and syncytiotrophoblasts, as demonstrated by their expression of Tfeb, glial cells missing 1 (Gcm1) and the HIV receptor CXCR4. During this process, a region of the differentiating Arnt-null TS cells underwent granzyme B-mediated apoptosis, suggesting a role for this pathway in murine syncytiotrophoblast turnover. Surprisingly, HIF1alpha and HIF2alpha were induced during TS cell differentiation in 20% O2; additionally, pVHL levels were modulated during the same time period. These results suggest that oxygen-independent HIF functions are crucial to this differentiation process. As histone deacetylase (HDAC) activity has been linked to HIF-dependent gene expression, we investigated whether ARNT deficiency affects this epigenetic regulator. Interestingly, Arnt-null TS cells had reduced HDAC activity, increased global histone acetylation, and altered class II HDAC subcellular localization. In wild-type TS cells, inhibition of HDAC activity recapitulated the Arnt-null phenotype, suggesting that crosstalk between the HIFs and the HDACs is required for normal trophoblast differentiation. Thus, the HIFs play important roles in modulating the developmental plasticity of stem cells by integrating physiological, transcriptional and epigenetic inputs.