A comprehensive preanalytical protocol for fresh solid tumor biospecimens.

Nearly seventy percent of diagnostic lab test errors occur due to variability in preanalytical factors. These are the parameters involved with all aspects of tissue processing, starting from the time tissue is collected from the patient in the operating room, until it is received and tested in the laboratory. While there are several protocols for transporting fixed tissue, organs, and liquid biopsies, such protocols are lacking for transport and handling of live solid tumor tissue specimens. There is a critical need to establish preanalytical protocols to reduce variability in biospecimen integrity and improve diagnostics for personalized medicine. Here, we provide a comprehensive protocol for the standard collection, handling, packaging, cold-chain logistics, and receipt of solid tumor tissue biospecimens to preserve tissue viability.

Detection of Residual Peritoneal Metastases Following Cytoreductive Surgery Using Pegsitacianine, a pH-Sensitive Imaging Agent: Final Results from a Phase II Study.

BACKGROUND: For patients with peritoneal carcinomatosis, extent of disease and completeness of cytoreductive surgery (CRS) are major prognostic factors for long-term survival. Assessment of these factors could be improved using imaging agents. Pegsitacianine is a pH-sensitive polymeric micelle conjugated to the fluorophore indocyanine green. The micelle disassembles in acidic microenvironments, such as tumors, resulting in localized fluorescence unmasking. We assessed the utility of pegsitacianine in detecting residual disease following CRS. PATIENTS AND METHODS: NCT04950166 was a phase II, non-randomized, open-label, multicenter US study. Patients eligible for CRS were administered an intravenous dose of pegsitacianine at 1 mg/kg 24-72 h before surgery. Following CRS, the peritoneal cavity was reexamined under near-infrared (NIR) illumination to evaluate for fluorescent tissue. Fluorescent tissue identified was excised and evaluated by histopathology. The primary outcome was the rate of clinically significant events (CSE), defined as detection of histologically confirmed residual disease excised with pegsitacianine or a revision in the assessment of completeness of CRS. Secondary outcomes included acceptable safety and pegsitacianine performance. RESULTS: A total of 53 patients were screened, 50 enrolled, and 40 were evaluable for CSE across six primary tumor types. Residual disease was detected with pegsitacianine in 20 of 40 (50%) patients. Pegsitacianine showed high sensitivity and was well tolerated with no serious adverse events (SAEs). Transient treatment-related, non-anaphylactic infusion reactions occurred in 28% of patients. CONCLUSIONS: Pegsitacianine was well tolerated and facilitated the recognition of occult residual disease following CRS. The high rate of residual disease detected suggests that the use of pegsitacianine augmented surgeon assessment and performance during CRS.

A Novel Assessment of Metabolic Pathways in Peritoneal Metastases from Low-Grade Appendiceal Mucinous Neoplasms.

BACKGROUND: There is a paucity of targeted therapies for patients with pseudomyxoma peritonei (PMP) secondary to low-grade appendiceal mucinous neoplasms (LAMNs). Dysregulated metabolism has emerged as a hallmark of cancer, and the relationship of metabolomics and cancer is an area of active scientific exploration. We sought to characterize phenotypic differences found in peritoneal metastases (PM) derived from LAMN versus adenocarcinoma. METHODS: Tumors were washed with phosphate-buffered saline (PBS), microdissected, then dissociated in ice-cold methanol dried and reconstituted in pyridine. Samples were derivatized in tert-butyldimethylsilyl (TBDMS) and subjected to gas chromatography-coupled mass spectrometry. Metabolites were assessed based on a standard library. RNA sequencing was performed, with pathway and network analyses on differentially expressed genes. RESULTS: Eight peritoneal tumor samples were obtained and analyzed: LAMNs (4), and moderate to poorly differentiated adenocarcinoma (colon [1], appendix [3]). Decreases in pyroglutamate, fumarate, and cysteine in PM from LAMNs were found compared with adenocarcinoma. Analyses showed the differential gene expression was dominated by the prevalence of metabolic pathways, particularly lipid metabolism. The gene retinol saturase (RETSAT), downregulated by LAMN, was involved in the multiple metabolic pathways that involve lipids. Using network mapping, we found IL1B signaling to be a potential top-level modulation candidate. CONCLUSIONS: Distinct metabolic signatures may exist for PM from LAMN versus adenocarcinoma. A multitude of genes are differentially regulated, many of which are involved in metabolic pathways. Additional research is needed to identify the significance and applicability of targeting metabolic pathways in the potential development of novel therapeutics for these challenging tumors.

Disparities in Care Access to Liver-Directed Therapy Among Medicare Beneficiaries with Colorectal Cancer Liver Metastasis.

BACKGROUND: Liver-directed therapies (LDT) are important components of the multidisciplinary care of patients with colorectal cancer liver metastases (CRCLM) that contribute to improved long-term outcomes. Factors associated with receipt of LDT are poorly understood. PATIENTS AND METHODS: Patients > 65 years old diagnosed with CRCLM were identified within the Medicare Standard Analytic File (2013-2017). Patients with extrahepatic metastatic disease were excluded. Mixed-effects analyses were used to assess patient factors associated with the primary outcome of LDT, defined as hepatectomy, ablation, and/or hepatic artery infusion chemotherapy (HAIC), as well as the secondary outcome of hepatectomy. RESULTS: Among 23,484 patients with isolated CRCLM, only 2004 (8.5%) received LDT, although resectability status could not be determined for the entire cohort. Among patients who received LDT, 61.7% underwent hepatectomy alone, 28.1% received ablation alone, 8.5% underwent hepatectomy and ablation, and 1.8% received HAIC either alone (0.8%) or in combination with hepatectomy and/or ablation (0.9%). Patient factors independently associated with lower odds of LDT included older age, female sex, Black race, greater comorbidity burden, higher social vulnerability index, primary rectal cancer, synchronous liver metastasis, and further distance from a high-volume liver surgery center (p < 0.05). Results were similar for receipt of hepatectomy. CONCLUSIONS: Despite the well-accepted role of LDT for CRCLM, only a small proportion of Medicare beneficiaries with CRCLM receive LDT. Increasing access to specialized centers with expertise in LDT, particularly for Black patients, female patients, and those with higher levels of social vulnerability or long travel distances, may improve outcomes for patients with CRCLM.

Microsatellite instability should not determine candidacy for cytoreductive surgery and hyperthermic intraperitoneal chemoperfusion in patients with peritoneal metastases from colorectal cancer.

BACKGROUND: Cytoreductive surgery (CRS) plus hyperthermic intraperitoneal chemotherapy (HIPEC) is a multimodal therapeutic option for the management of peritoneal metastases (PM). Treatment outcomes for patients with colorectal cancer (CRC) PM undergoing CRS+HIPEC with microsatellite instability (MSI) remain unknown. We examined the patient characteristics and outcomes in patients with MSI CRC after CRS+HIPEC. METHODS: This was a retrospective cohort study of a prospectively maintained database of all patients with CRC PM undergoing CRS+HIPEC (2010-2020). Categorical and continuous variables were analyzed using the chi-square test and independent samples t test, respectively. Survival was evaluated with the Kaplan-Meier analysis. RESULTS: There were 324 patients diagnosed as having CRC PM undergoing CRS+HIPEC (MSI, n = 23; microsatellite stable [MSS], n = 301). There was no statistically significant difference in patient demographics, tumor characteristics, or perioperative factors between the 2 groups. There was a trend toward improved survival in the MSI group with a median overall survival (OS) of 96.7 month compared with patients with MSS disease (median OS, 51.4 months; P = .10). Patients with MSI demonstrated median progression-free survival (PFS) 8.5 months compared with 11.4 months in the MSS cohort (P = .28). CONCLUSION: Patients with CRC PM, regardless of MSI or MSS status, demonstrate similar OS and PFS after CRS+HIPEC. MSI status should not change a patient's candidacy for CRS+HIPEC.

Recurrence following Resection of Intraductal Papillary Mucinous Neoplasms: A Systematic Review to Guide Surveillance.

Patients who undergo resection for non-invasive IPMN are at risk for long-term recurrence. Further evidence is needed to identify evidence-based surveillance strategies based on the risk of recurrence. We performed a systematic review of the current literature regarding recurrence patterns following resection of non-invasive IPMN to summarize evidence-based recommendations for surveillance. Among the 61 studies reviewed, a total of 8779 patients underwent resection for non-invasive IPMN. The pooled overall median follow-up time was 49.5 months (IQR: 38.5-57.7) and ranged between 14.1 months and 114 months. The overall median recurrence rate for patients with resected non-invasive IPMN was 8.8% (IQR: 5.0, 15.6) and ranged from 0% to 27.6%. Among the 33 studies reporting the time to recurrence, the overall median time to recurrence was 24 months (IQR: 17, 46). Existing literature on recurrence rates and post-resection surveillance strategies for patients with resected non-invasive IPMN varies greatly. Patients with resected non-invasive IPMN appear to be at risk for long-term recurrence and should undergo routine surveillance.

Radiation therapy for retroperitoneal sarcoma: practice patterns in North America.

BACKGROUND: The addition of radiation therapy (RT) to surgery in retroperitoneal sarcoma (RPS) remains controversial. We examined practice patterns in the use of RT for patients with RPS over time in a large, national cohort. METHODS: Patients in the National Cancer Database (2004-2017) who underwent resection of RPS were included. Trends over time for proportions were calculated using contingency tables with Cochran-Armitage Trend test. RESULTS: Of 7,485 patients who underwent resection, 1,821 (24.3%) received RT (adjuvant: 59.9%, neoadjuvant: 40.1%). The use of RT decreased annually by < 1% (p = 0.0178). There was an average annual increase of neoadjuvant RT by 13% compared to an average annual decrease of adjuvant RT by 6% (p < 0.0001). Treatment at high-volume centers (OR 14.795, p < 0.0001) and tumor > 10 cm (OR 2.009, p = 0.001) were associated with neoadjuvant RT. In contrast liposarcomas (OR 0.574, p = 0.001) were associated with adjuvant RT. There was no statistically significant difference in overall survival between patients treated with surgery alone versus surgery and RT (p = 0.07). CONCLUSION: In the United States, the use of RT for RPS has decreased over time, with a shift towards neoadjuvant RT. However, a large percentage of patients are still receiving adjuvant RT and this mostly occurs at low-volume hospitals.

Progression to adrenocortical tumorigenesis in mice and humans through insulin-like growth factor 2 and ß-catenin.

Dysregulation of the WNT and insulin-like growth factor 2 (IGF2) signaling pathways has been implicated in sporadic and syndromic forms of adrenocortical carcinoma (ACC). Abnormal ß-catenin staining and CTNNB1 mutations are reported to be common in both adrenocortical adenoma and ACC, whereas elevated IGF2 expression is associated primarily with ACC. To better understand the contribution of these pathways in the tumorigenesis of ACC, we examined clinicopathological and molecular data and used mouse models. Evaluation of adrenal tumors from 118 adult patients demonstrated an increase in CTNNB1 mutations and abnormal ß-catenin accumulation in both adrenocortical adenoma and ACC. In ACC, these features were adversely associated with survival. Mice with stabilized ß-catenin exhibited a temporal progression of increased adrenocortical hyperplasia, with subsequent microscopic and macroscopic adenoma formation. Elevated Igf2 expression alone did not cause hyperplasia. With the combination of stabilized ß-catenin and elevated Igf2 expression, adrenal glands were larger, displayed earlier onset of hyperplasia, and developed more frequent macroscopic adenomas (as well as one carcinoma). Our results are consistent with a model in which dysregulation of one pathway may result in adrenal hyperplasia, but accumulation of a second or multiple alterations is necessary for tumorigenesis.

Endoscopic Management and Surgical Considerations for Familial Adenomatous Polyposis.

Familial adenomatous polyposis (FAP) is the development of many adenomatous colorectal polyps. Colonoscopy is recommended to start at age 10 to 12 years at intervals of 1 to 2 years. Colectomy is clearly indicated for malignancy or significant colorectal symptoms. After colectomy, endoscopic surveillance is still critical. Duodenal and gastric polyposis is also found in almost all patients with FAP. Screening with upper endoscopy and ampullary visualization is recommended, generally determined by age and staging of duodenal polyposis, but guidelines are increasingly factoring in ampullary and gastric manifestations. Surgical management of malignancy or advanced upper tract manifestations is needed.

Disparities in access to care among patients with appendiceal or colorectal cancer and peritoneal metastases: A medicare insurance-based study in the United States.

Background: Prior studies attempting to identify disparities in the care of patients with appendiceal (AC) or colorectal cancer (CRC) with peritoneal metastasis (PM) are limited to single-institution, highly selected patient populations. This observational cohort study sought to identify factors associated with specialty care for Medicare beneficiaries with AC/CRC-PM. Materials and methods: Patients >65 years old in the United States diagnosed with AC/CRC and isolated PM were identified within the Medicare Standard Analytic File (2013-2017). Mixed-effects analyses assessed patient factors associated with cytoreductive surgery and hyperthermic intraperitoneal chemotherapy (CRS/HIPEC) and outpatient consultation with a peritoneal surface malignancy (PSM) surgeon, and Cox proportional-hazards analysis compared 3-year overall survival (OS) between patients receiving CRS/HIPEC versus systemic therapy alone. Results: Among 7,653 patients, only 250 (3.3%) underwent CRS/HIPEC. Among those individuals who did not undergo CRS/HIPEC (N=7,403), only 475 (6.4%) had outpatient consultation with a PSM surgeon. Patient factors independently associated with lower odds of CRS/HIPEC and PSM surgery consultation included older age, greater comorbidity burden, higher social vulnerability index, and further distance from a PSM center (p<0.05). CRS/HIPEC was independently associated with better 3-year OS compared with systemic therapy alone (HR=0.29, 95%CI=0.21-0.38). Conclusion: An exceedingly small proportion of Medicare beneficiaries with AC/CRC-PM undergo CRS/HIPEC or even have an outpatient consultation with a PSM surgeon. Significant disparities in treatment and access to care exist for patients with higher levels of social vulnerability and those that live further away from a PSM center. Future research and interventions should focus on improving access to care for these at-risk patient populations.

Do Lymph Node Metastases Matter in Appendiceal Cancer with Peritoneal Carcinomatosis? A US HIPEC Collaborative Study.

BACKGROUND: Whether formal regional lymph node (LN) evaluation is necessary for patients with appendiceal adenocarcinoma (AA) who have peritoneal metastases is unclear. The aim of this study was to evaluate the prognostic value of LN metastases on survival in patients treated with cytoreductive surgery and hyperthermic intraperitoneal chemotherapy (CRS-HIPEC). METHODS: A retrospective analysis of the US HIPEC collaborative, a multi-institutional consortium comprising 12 high-volume centers, was performed to identify patients with AA who underwent CRS-HIPEC with adequate LN sampling (≥ 12 LNs). RESULTS: Two hundred-fifty patients with AA who underwent CRS-HIPEC were included. Outcomes were compared between LN - and LN + disease. Baseline patient characteristics between groups were similar, with most patients undergoing complete cytoreduction (0/1: 86.0% vs. 76.8%, p = 0.08), respectively. More adverse tumor factors were found in patients with LN + disease, including poor differentiation, signet ring cells, and lymphovascular invasion. Multivariate analysis of overall survival (OS) found LN + disease was independently associated with worse OS (HR: 2.82 95%CI: 1.25-6.34, p = 0.01), even after correction for receipt of systemic therapy. On Kaplan-Meier analysis, median OS was lower in patients with LN + disease (25.9 months vs. 91.4 months, p < 0.01). LN + disease remained associated with poor OS following propensity score matching (HR: 4.98 95%CI: 1.72-14.40, p < 0.01) and in patients with PCI ≥ 20 (HR: 3.68 95%CI: 1.54-8.80, p < 0.01). CONCLUSIONS: In this large multi-institutional study of patients with AA undergoing CRS-HIPEC, LN status remained associated with worse OS even in the setting of advanced peritoneal carcinomatosis. Formal LN evaluation should be performed for most patients with AA undergoing CRS-HIPEC.

Neutrophils Extracellular Traps Inhibition Improves PD-1 Blockade Immunotherapy in Colorectal Cancer.

Immune checkpoint inhibitors can improve the prognosis of patients with advanced malignancy; however, only a small subset of advanced colorectal cancer patients in microsatellite-instability-high or mismatch-repair-deficient colorectal cancer can benefit from immunotherapy. Unfortunately, the mechanism behind this ineffectiveness is unclear. The tumor microenvironment plays a critical role in cancer immunity, and may contribute to the inhibition of immune checkpoint inhibitors and other novel immunotherapies in patients with advanced cancer. Herein, we demonstrate that the DNase I enzyme plays a pivotal role in the degradation of NETs, significantly dampening the resistance to anti-PD-1 blockade in a mouse colorectal cancer model by attenuating tumor growth. Remarkably, DNase I decreases tumor-associated neutrophils and the formation of MC38 tumor cell-induced neutrophil extracellular trap formation in vivo. Mechanistically, the inhibition of neutrophil extracellular traps with DNase I results in the reversal of anti-PD-1 blockade resistance through increasing CD8+ T cell infiltration and cytotoxicity. These findings signify a novel approach to targeting the tumor microenvironment using DNase I alone or in combination with immune checkpoint inhibitors.

Preclinical targeting of the type I insulin-like growth factor receptor in adrenocortical carcinoma.

CONTEXT: Drug therapy for adrenocortical carcinoma (ACC), a rare and lethal malignancy, is largely empirical and ineffective. New treatments directed at molecular targets critical to the pathophysiology of ACC may prove more efficacious. OBJECTIVE: The objective of the study was to profile human adrenal tumors and ACC cell lines to assess activated IGF signaling and determine the efficacy of two IGF receptor (IGF-1R) antagonists alone and in combination with mitotane. EXPERIMENTAL DESIGN: ACC cell lines that display or lack activated IGF signaling are used to assess the effects of two IGF-1R antagonists in cultured cells and ACC xenograft tumors. RESULTS: Transcriptional profiling data derived from DNA microarray analysis of human adrenal tumors implicate IGF2 as the single highest up-regulated transcript in the vast majority of carcinomas. We show that the majority of ACC cell lines tested display constitutive IGF ligand production and activation of downstream effector pathways. Both IGF-1R antagonists cause significant dose-dependent growth inhibition in ACC cell lines. Furthermore, we observe that mitotane, the first-line adrenolytic drug used in patients with ACC, results in enhanced growth inhibition when used in combination with the IGF-1R antagonists. We next examined the activity of IGF-1R antagonists against ACC xenografts in athymic nude mice. IGF inhibition markedly reduced tumor growth greater than that observed with mitotane treatment, and combination therapy with mitotane significantly enhanced tumor growth suppression. CONCLUSION: These findings establish a critical role of IGF signaling in ACC pathophysiology and provide rationale for use of targeted IGF-1R antagonists to treat adrenocortical carcinoma in future clinical trials.

Technical Detail for Robot Assisted Pancreaticoduodenectomy.

Since its first report in 2003, robotic pancreaticoduodenectomy (RPD) has gained popularity among pancreatic surgeons. Inherent advantages of the robotic platform, including three-dimensional vision, wristed instruments, and improved ergonomics, allow the surgeon to recapitulate the principles of open pancreatoduodenectomy allowing safe oncologic dissection, hemostasis, and meticulous reconstruction. Over the course of the past decade, significant strides have been achieved in outlining the safety, feasibility, and learning curve of the robotic Whipple. When performed by high volume pancreatic surgeons experienced in RPD, recent comparative effectiveness studies show potential advantages compared to the open technique, including reductions in hospital stay and morbidity. National data also show reductions in conversion rates compared to its laparoscopic counterpart. Although long-term oncologic data are still needed, short-term oncologic surrogates of margin resection and lymph node harvest suggest no compromise in oncologic outcomes. As pancreatic surgeons increasingly integrate robotics into their practice, proficiency-based training and credentialing will be necessary for the safe application and dissemination of RPD. Here, we provide the detailed steps of a robotic pancreaticoduodenectomy performed at the University of Pittsburgh Medical Center.

Inhibition of protein kinase Cbeta by enzastaurin enhances radiation cytotoxicity in pancreatic cancer.

PURPOSE: Aberrant activation of protein kinase Cbeta (PKCbeta) by pancreatic cancer cells facilitates angiogenesis and tumor cell survival. Targeting PKCbeta with enzastaurin, a well-tolerated drug in clinical trials, would be expected to radiosensitize pancreatic tumors through direct antitumor and antivascular effects. EXPERIMENTAL DESIGN: We tested the hypothesis that enzastaurin radiosensitizes pancreatic cancer cells in culture and in vivo through inhibition of PKCbeta. We analyzed pancreatic cancer xenografts for growth delay and microvessel density after treatment with enzastaurin, radiation, or both. We determined the effect of radiation and enzastaurin on glycogen synthase kinase 3beta, a mediator of cell death in culture and in vivo. RESULTS: At concentrations attained in patients, enzastaurin reduced levels of active PKCbeta measured by phosphorylation at Thr(500) in culture and in xenografts. Enzastaurin alone did not affect pancreatic cancer cell survival, proliferation, or xenograft growth. However, enzastaurin radiosensitized pancreatic cancer cells in culture by colony formation assay. Enzastaurin alone decreased microvessel density of pancreatic cancer xenografts without appreciable effects on tumor size. When combined with radiation, enzastaurin increased radiation-induced tumor growth delay with a corresponding decrease in microvessel density. Enzastaurin inhibited radiation-induced phosphorylation of glycogen synthase kinase 3beta at Ser(9) in pancreatic cancer cells in culture and in tumor xenografts, suggesting a possible mechanism for the observed radiosensitization. CONCLUSIONS: Enzastaurin inhibits PKCbeta in pancreatic cancer cells in culture, enhancing radiation cytotoxicity. Additional antivascular effects of enzastaurin were observed in vivo, resulting in greater radiosensitization. These results provide the rationale for a clinical trial in locally advanced pancreatic cancer combining enzastaurin with radiation.