Outcomes of rectal cancer patients who refuse surgery after incomplete clinical response to neoadjuvant therapy.

BACKGROUND AND OBJECTIVES: Total mesorectal excision (TME) remains the standard of care for patients with rectal cancer who have an incomplete response to total neoadjuvant therapy (TNT). A minority of patients will refuse curative intent resection. The aim of this study is to examine the outcomes for these patients. METHODS: A retrospective cohort study of stage 1-3 rectal adenocarcinoma patients who underwent neoadjuvant chemoradiation therapy or TNT at a single institution. Patients either underwent TME, watch-and-wait protocol, or if they refused TME, were counseled and watched (RCW). Clinical outcomes and resource utilization were examined in each group. RESULTS: One hundred seventy-one patients (Male 59%) were included with a median surveillance of 43 months. Twenty-nine patients (17%) refused TME and had shortened overall survival (OS). Twelve patients who refused TME converted to a complete clinical response (cCR) on subsequent staging with a prolonged OS. 92% of these patients had a near cCR at initial staging endoscopy. Increased physician visits and testing was utilized in RCW and WW groups. CONCLUSION: A significant portion of patients convert to cCR and have prolonged OS. Lengthening the time to declare cCR may be considered in select patients, such as those with a near cCR at initial endoscopic staging.

Safe implementation of a minimally invasive hepatopancreatobiliary program, a narrative review and institutional experience.

Laparoscopic and robotic-assisted approaches to hepatopancreatobiliary (HPB) operations have expanded worldwide. As surgeons and medical centers contemplate initiating and expanding minimally invasive surgical (MIS) programs for complex HPB surgical operations, there are many factors to consider. This review highlights the key components of developing an MIS HPB program and shares our recent institutional experience with the adoption and expansion of an MIS approach to pancreaticoduodenectomy.

Production of an interleukin-10 blocking antibody by genetically engineered macrophages increases cancer cell death in human gastrointestinal tumor slice cultures.

Although it can promote effector T-cell function, the summative effect of interleukin-10 (IL-10) in the tumor microenvironment (TME) appears to be suppressive; therefore, blocking this critical regulatory cytokine has therapeutic potential to enhance antitumor immune function. As macrophages efficiently localize to the TME, we hypothesized that they could be used as a delivery vehicle for drugs designed to block this pathway. To test our hypothesis, we created and evaluated genetically engineered macrophages (GEMs) that produce an IL-10-blocking antibody (αIL-10). Healthy donor human peripheral blood mononuclear cells were differentiated and transduced with a novel lentivirus (LV) encoding BT-063, a humanized αIL-10 antibody. The efficacy of αIL-10 GEMs was assessed in human gastrointestinal tumor slice culture models developed from resected specimens of pancreatic ductal adenocarcinoma primary tumors and colorectal cancer liver metastases. LV transduction led to sustained production of BT-063 by αIL-10 GEMs for at least 21 days. Transduction did not alter GEM phenotype as evaluated by flow cytometry, but αIL-10 GEMs produced measurable quantities of BT-063 in the TME that was associated with an ~5-fold higher rate of tumor cell apoptosis than control.

Glypican-3 targeted positron emission tomography detects sub-centimeter tumors in a xenograft model of hepatocellular carcinoma.

BACKGROUND: Early intrahepatic recurrence is common after surgical resection of hepatocellular carcinoma (HCC) and leads to increased morbidity and mortality. Insensitive and nonspecific diagnostic imaging contributes to EIR and results in missed treatment opportunities. In addition, novel modalities are needed to identify targets amenable for targeted molecular therapy. In this study, we evaluated a zirconium-89 radiolabeled glypican-3 (GPC3) targeting antibody conjugate (89Zr-αGPC3) for use in positron emission tomography (PET) for detection of small, GPC3+ HCC in an orthotopic murine model. Athymic nu/J mice received hepG2, a GPC3+ human HCC cell line, into the hepatic subcapsular space. Tumor-bearing mice were imaged by PET/computerized tomography (CT) 4 days after tail vein injection of 89Zr-αGPC3. Livers were then excised for the tumors to be identified, measured, bisected, and then serially sectioned at 500 µm increments. Sensitivity and specificity of PET/CT for 89Zr-αGPC3-avid tumors were assessed using tumor confirmation on histologic sections as the gold standard. RESULTS: In tumor-bearing mice, 89Zr-αGPC3 avidly accumulated in the tumor within four hours of injection with ongoing accumulation over time. There was minimal off-target deposition and rapid bloodstream clearance. Thirty-eight of 43 animals had an identifiable tumor on histologic analysis. 89Zr-αGPC3 immuno-PET detected all 38 histologically confirmed tumors with a sensitivity of 100%, with the smallest tumor detected measuring 330 µm in diameter. Tumor-to-liver ratios of 89Zr-αGPC3 uptake were high, creating excellent spatial resolution for ease of tumor detection on PET/CT. Two of five tumors that were observed on PET/CT were not identified on histologic analysis, yielding a specificity of 60%. CONCLUSIONS: 89Zr-αGPC3 avidly accumulated in GPC3+ tumors with minimal off-target sequestration. 89Zr-αGPC3 immuno-PET yielded a sensitivity of 100% and detected sub-millimeter tumors. This technology may improve diagnostic sensitivity of small HCC and select GPC3+ tumors for targeted therapy. Human trials are warranted to assess its impact.

Blockade of interleukin 10 potentiates antitumour immune function in human colorectal cancer liver metastases.

OBJECTIVE: Programmed cell death protein 1 (PD-1) checkpoint inhibition and adoptive cellular therapy have had limited success in patients with microsatellite stable colorectal cancer liver metastases (CRLM). We sought to evaluate the effect of interleukin 10 (IL-10) blockade on endogenous T cell and chimeric antigen receptor T (CAR-T) cell antitumour function in CRLM slice cultures. DESIGN: We created organotypic slice cultures from human CRLM (n=38 patients' tumours) and tested the antitumour effects of a neutralising antibody against IL-10 (αIL-10) both alone as treatment and in combination with exogenously administered carcinoembryonic antigen (CEA)-specific CAR-T cells. We evaluated slice cultures with single and multiplex immunohistochemistry, in situ hybridisation, single-cell RNA sequencing, reverse-phase protein arrays and time-lapse fluorescent microscopy. RESULTS: αIL-10 generated a 1.8-fold increase in T cell-mediated carcinoma cell death in human CRLM slice cultures. αIL-10 significantly increased proportions of CD8+ T cells without exhaustion transcription changes, and increased human leukocyte antigen - DR isotype (HLA-DR) expression of macrophages. The antitumour effects of αIL-10 were reversed by major histocompatibility complex class I or II (MHC-I or MHC-II) blockade, confirming the essential role of antigen presenting cells. Interrupting IL-10 signalling also rescued murine CAR-T cell proliferation and cytotoxicity from myeloid cell-mediated immunosuppression. In human CRLM slices, αIL-10 increased CEA-specific CAR-T cell activation and CAR-T cell-mediated cytotoxicity, with nearly 70% carcinoma cell apoptosis across multiple human tumours. Pretreatment with an IL-10 receptor blocking antibody also potentiated CAR-T function. CONCLUSION: Neutralising the effects of IL-10 in human CRLM has therapeutic potential as a stand-alone treatment and to augment the function of adoptively transferred CAR-T cells.

A novel rat model for the study of postoperative pancreatic fistula.

While over the past several decades mortality after pancreatic surgery has decreased to <5%, postoperative morbidity remains remarkably high, ranging from 15% to 65%. The development of a postoperative pancreatic fistula (POPF) is a significant contributor to morbidity in patients undergoing pancreatic surgery. POPF can lead to life-threatening conditions such as intra-abdominal abscess, uncontrolled hemorrhage, sepsis, and death. Rates of POPF have not significantly changed over time, despite the introduction of multiple technical and pharmacologic interventions aimed at their treatment and prevention. Unfortunately, there are few POPF experimental models that have been described in the literature and existing models are unable to reliably reproduce the clinical sequelae of POPF, limiting the development of new methods to prevent and treat POPF. Herein, we describe a new rat experimental model that reliably creates a POPF via transection of the common pancreatic duct.

Hypoxia-inducible lentiviral gene expression in engineered human macrophages.

BACKGROUND: Human immune cells, including monocyte-derived macrophages, can be engineered to deliver proinflammatory cytokines, bispecific antibodies, and chimeric antigen receptors to support immune responses in different disease settings. When gene expression is regulated by constitutively active promoters, lentiviral payload gene expression is unregulated, and can result in potentially toxic quantities of proteins. Regulated delivery of lentivirally encoded proteins may allow localized or conditional therapeutic protein expression to support safe delivery of adoptively transferred, genetically modified cells with reduced capacity for systemic toxicities. METHODS: In this study, we engineered human macrophages to express genes regulated by hypoxia responsive elements included in the lentiviral promoter region to drive conditional lentiviral gene expression only under hypoxic conditions. We tested transduced macrophages cultured in hypoxic conditions for the transient induced expression of reporter genes and the secreted cytokine, interleukin-12. Expression of hypoxia-regulated genes was investigated both transcriptionally and translationally, and in the presence of human tumor cells in a slice culture system. Finally, hypoxia-regulated gene expression was evaluated in a subcutaneous humanized-mouse cancer model. RESULTS: Engineered macrophages were shown to conditionally and tranisently express lentivirally encoded gene protein products, including IL-12 in hypoxic conditions in vitro. On return to normoxic conditions, lentiviral payload expression returned to basal levels. Reporter genes under the control of hypoxia response elements were upregulated under hypoxic conditions in the presence of human colorectal carcinoma cells and in the hypoxic xenograft model of glioblastoma, suggesting utility for systemic engineered cell delivery capable of localized gene delivery in cancer. CONCLUSIONS: Macrophages engineered to express hypoxia-regulated payloads have the potential to be administered systemically and conditionally express proteins in tissues with hypoxic conditions. In contrast to immune cells that function or survive poorly in hypoxic conditions, macrophages maintain a proinflammatory phenotype that may support continued gene and protein expression when regulated by conditional hypoxia responsive elements and naturally traffic to hypoxic microenvironments, making them ideal vehicles for therapeutic payloads to hypoxic tissues, such as solid tumors. With the ability to fine-tune delivery of potent proteins in response to endogenous microenvironments, macrophage-based cellular therapies may therefore be designed for different disease settings.

Glypican-3-Targeted 227Th α-Therapy Reduces Tumor Burden in an Orthotopic Xenograft Murine Model of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a significant cause of morbidity and mortality worldwide, with limited therapeutic options for advanced disease. Targeted α-therapy is an emerging class of targeted cancer therapy in which α-particle-emitting radionuclides, such as 227Th, are delivered specifically to cancer tissue. Glypican-3 (GPC3) is a cell surface glycoprotein highly expressed on HCC. In this study, we describe the development and in vivo efficacy of a 227Th-labeled GPC3-targeting antibody conjugate (227Th-octapa-αGPC3) for treatment of HCC in an orthotopic murine model. Methods: The chelator p-SCN-Bn-H4octapa-NCS (octapa) was conjugated to a GPC3-targeting antibody (αGPC3) for subsequent 227Th radiolabeling (octapa-αGPC3). Conditions were varied to optimize radiolabeling of 227Th. In vitro stability was evaluated by measuring the percentage of protein-bound 227Th by γ-ray spectroscopy. An orthotopic athymic Nu/J murine model using HepG2-Red-FLuc cells was developed. Biodistribution and blood clearance of 227Th-octapa-αGPC3 were evaluated in tumor-bearing mice. The efficacy of 227Th-octapa-αGPC3 was assessed in tumor-bearing animals with serial measurement of serum α-fetoprotein at 23 d after injection. Results: Octapa-conjugated αGPC3 provided up to 70% 227Th labeling yield in 2 h at room temperature. In the presence of ascorbate, at least 97.8% of 227Th was bound to αGPC3-octapa after 14 d in phosphate-buffered saline. In HepG2-Red-FLuc tumor-bearing mice, highly specific GPC3 targeting was observed, with significant 227Th-octapa-αGPC3 accumulation in the tumor over time and minimal accumulation in normal tissue. Twenty-three days after treatment, a significant reduction in tumor burden was observed in mice receiving a 500 kBq/kg dose of 227Th-octapa-αGPC3 by tail-vein injection. No acute off-target toxicity was observed, and no animals died before termination of the study. Conclusion:227Th-octapa-αGPC3 was observed to be stable in vitro; maintain high specificity for GPC3, with favorable biodistribution in vivo; and result in significant antitumor activity without significant acute off-target toxicity in an orthotopic murine model of HCC.

Head Over Wheels: Traumatic Head and Neck Injuries Secondary to Mountain Biking.

OBJECTIVES: The popularity of mountain biking (MTB) in the United States has risen in recent years. We sought to identify the prevalence and distribution of MTB associated head and neck injuries presenting to emergency departments across the U.S. and identify risk factors for hospital admission in this patient population. METHODS: The National Electronic Injury Surveillance System (NEISS) was queried for MTB related injuries of the head and neck from 2009 to 2018, with analysis for incidence, age, gender, anatomic site, and diagnoses. RESULTS: A total of 486 cases were identified, corresponding to an estimated 18 952 head and neck MTB related ED visits. Patients were predominantly male (80.7%) and white (69.8%) with a median age of 35 years (interquartile range, 21-46 years). A majority (88.4%) of patients were released from the ED, but a significant proportion of patients were admitted (9.2%) or transferred (1.2%). The most common facial fractures were facial/not specified (35%), nasal bone (29%), mandible (15%), orbit (12%), and zygomaxillary complex (9%). The greatest predictors of hospital admission/transfer were injury to the mouth or neck and avulsion-type injury (P < .001). CONCLUSIONS: MTB results in a significant number of traumatic head and neck injuries nationwide. Patients are primarily adult, white males. The majority of injuries result in discharge from the ED, however a small amount of these patients experience significant morbidity necessitating hospital admission. Understanding the distribution of MTB head and neck injuries may aid in the clinical evaluation of these patients. LEVEL OF EVIDENCE: 4.

IWATE criteria are associated with perioperative outcomes in robotic hepatectomy: a retrospective review of 225 resections.

BACKGROUND: Robotic hepatectomy (RH) is increasingly utilized for minor and major liver resections. The IWATE criteria were developed to classify minimally invasive liver resections by difficulty. The objective of this study was to apply the IWATE criteria in RH and to describe perioperative and oncologic outcomes of RH over the last decade at our institution. METHODS: Perioperative and oncologic outcomes of patients who underwent RH between 2011 and 2019 were retrospectively collected. The difficulty level of each operation was assessed using the IWATE criteria, and outcomes were compared at each level. Univariate linear regression was performed to characterize the relationship between IWATE criteria and perioperative outcomes (OR time, EBL, and LOS), and a multivariable model was also developed to address potential confounding by patient characteristics (age, sex, BMI, prior abdominal surgery, ASA class, and simultaneous non-hepatectomy operation). RESULTS: Two hundred and twenty-five RH were performed. Median IWATE criteria for RH were 6 (IQR 5-9), with low, intermediate, advanced, and expert resections accounting for 23% (n = 51), 34% (n = 77), 32% (n = 72), and 11% (n = 25) of resections, respectively. The majority of resections were parenchymal-sparing approaches, including anatomic segmentectomies and non-anatomic partial resections. 30-day complication rate was 14%, conversion to open surgery occurred in 9 patients (4%), and there were no deaths within 30 days postoperatively. In the univariate linear regression analysis, IWATE criteria were positively associated with OR time, EBL, and LOS. In the multivariable model, IWATE criteria were independently associated with greater OR time, EBL, and LOS. Two-year overall survival for hepatocellular carcinoma and intrahepatic cholangiocarcinoma was 94% and 50%, respectively. CONCLUSION: In conclusion, the IWATE criteria are associated with surgical outcomes after RH. This series highlights the utility of RH for difficult hepatic resections, particularly parenchymal-sparing resections in the posterosuperior sector, extending the indication of minimally invasive hepatectomy in experienced hands and potentially offering select patients an alternative to open hepatectomy or other less definitive liver-directed treatment options.

Protocol for tissue slice cultures from human solid tumors to study therapeutic response.

The impact of systemic therapy on the tumor microenvironment has been difficult to study in human solid tumors. Our protocol describes steps for establishing slice cultures to investigate response to chemotherapies, immunotherapies, or adoptive cell therapies. Endpoints include changes in viability, histology, live-cell imaging, and multi-omics analyses. The protocol has been applied to a broad array of gastrointestinal malignancies. Culture conditions and treatment parameters can be modified for specific experiments. The platform is highly flexible and easy to manipulate. For complete details on the use and execution of this protocol, please refer to Kenerson et al. (2020), Jabbari et al. (2020), Brempelis et al. (2020), and Jiang et al. (2017).

Glypican-3 targeted delivery of 89Zr and 90Y as a theranostic radionuclide platform for hepatocellular carcinoma.

Glypican-3 (GPC3) is a tumor associated antigen expressed by hepatocellular carcinoma (HCC) cells. This preclinical study evaluated the efficacy of a theranostic platform using a GPC3-targeting antibody αGPC3 conjugated to zirconium-89 (89Zr) and yttrium-90 (90Y) to identify, treat, and assess treatment response in a murine model of HCC. A murine orthotopic xenograft model of HCC was generated. Animals were injected with 89Zr-labeled αGPC3 and imaged with a small-animal positron emission/computerized tomography (PET/CT) imaging system (immuno-PET) before and 30 days after radioimmunotherapy (RIT) with 90Y-labeled αGPC3. Serum alpha fetoprotein (AFP), a marker of tumor burden, was measured. Gross tumor volume (GTV) and SUVmax by immuno-PET was measured using fixed intensity threshold and manual segmentation methods. Immuno-PET GTV measurements reliably quantified tumor burden prior to RIT, strongly correlating with serum AFP (R2 = 0.90). Serum AFP was significantly lower 30 days after RIT in 90Y-αGPC3 treated animals compared to those untreated (p = 0.01) or treated with non-radiolabeled αGPC3 (p = 0.02). Immuno-PET GTV measurements strongly correlated with tumor burden after RIT (R2 = 0.87), and GTV of animals treated with 90Y-αGPC3 was lower than in animals who did not receive treatment or were treated with non-radiolabeled αGPC3, although this only trended toward statistical significance. A theranostic platform utilizing GPC3 targeted 89Zr and 90Y effectively imaged, treated, and assessed response after radioimmunotherapy in a GPC3-expressing HCC xenograft model.

Genetically engineered macrophages persist in solid tumors and locally deliver therapeutic proteins to activate immune responses.

BACKGROUND: Though currently approved immunotherapies, including chimeric antigen receptor T cells and checkpoint blockade antibodies, have been successfully used to treat hematological and some solid tumor cancers, many solid tumors remain resistant to these modes of treatment. In solid tumors, the development of effective antitumor immune responses is hampered by restricted immune cell infiltration and an immunosuppressive tumor microenvironment (TME). An immunotherapy that infiltrates and persists in the solid TME, while providing local, stable levels of therapeutic to activate or reinvigorate antitumor immunity could overcome these challenges faced by current immunotherapies. METHODS: Using lentivirus-driven engineering, we programmed human and murine macrophages to express therapeutic payloads, including Interleukin (IL)-12. In vitro coculture studies were used to evaluate the effect of genetically engineered macrophages (GEMs) secreting IL-12 on T cells and on the GEMs themselves. The effects of IL-12 GEMs on gene expression profiles within the TME and tumor burden were evaluated in syngeneic mouse models of glioblastoma and melanoma and in human tumor slices isolated from patients with advanced gastrointestinal malignancies. RESULTS: Here, we present a cellular immunotherapy platform using lentivirus-driven genetic engineering of human and mouse macrophages to constitutively express proteins, including secreted cytokines and full-length checkpoint antibodies, as well as cytoplasmic and surface proteins that overcomes these barriers. GEMs traffic to, persist in, and express lentiviral payloads in xenograft mouse models of glioblastoma, and express a non-signaling truncated CD19 surface protein for elimination. IL-12-secreting GEMs activated T cells and induced interferon-gamma (IFNγ) in vitro and slowed tumor growth resulting in extended survival in vivo. In a syngeneic glioblastoma model, IFNγ signaling cascades were also observed in mice treated with mouse bone-marrow-derived GEMs secreting murine IL-12. These findings were reproduced in ex vivo tumor slices comprised of intact MEs. In this setting, IL-12 GEMs induced tumor cell death, chemokines and IFNγ-stimulated genes and proteins. CONCLUSIONS: Our data demonstrate that GEMs can precisely deliver titratable doses of therapeutic proteins to the TME to improve safety, tissue penetrance, targeted delivery and pharmacokinetics.

Yttrium-90-Labeled Anti-Glypican 3 Radioimmunotherapy Halts Tumor Growth in an Orthotopic Xenograft Model of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the second most lethal malignancy globally and is increasing in incidence in the United States. Unfortunately, there are few effective systemic treatment options, particularly for disseminated disease. Glypican-3 (GPC3) is a proteoglycan cell surface receptor overexpressed in most HCCs and provides a unique target for molecular therapies. We have previously demonstrated that PET imaging using a 89Zr-conjugated monoclonal anti-GPC3 antibody (αGPC3) can bind to minute tumors and allow imaging with high sensitivity and specificity in an orthotopic xenograft mouse model of HCC and that serum alpha-fetoprotein (AFP) levels are highly correlated with tumor size in this model. In the present study, we conjugated 90Y, a high-energy beta-particle-emitting radionuclide, to our αGPC3 antibody to develop a novel antibody-directed radiotherapeutic approach for HCC. Luciferase-expressing HepG2 human hepatoblastoma cells were orthotopically implanted in the livers of athymic nude mice, and tumor establishment was verified at 6 weeks after implantation by bioluminescent imaging and serum AFP concentration. Tumor burden by bioluminescence and serum AFP concentration was highly correlated in our model. Yttrium-90 was conjugated to αGPC3 using the chelating agent 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and injected via the tail vein into the experimental mice at a dose of 200 µCi/mouse or 300 µCi/mouse. Control mice received DOTA-αGPC3 without radionuclide. At 30 days after a single dose of the radioimmunotherapy agent, mean serum AFP levels in control animals increased dramatically, while animals treated with 200 µCi only experienced a minor increase, indicating cessation of tumor growth, and animals treated with 300 µCi experienced a reduction in serum AFP concentration, indicating tumor shrinkage. Mean tumor-bearing liver weight in control animals was also significantly greater than that in animals that received either dose of 90Y-αGPC3. These results were achieved without significant toxicity as measured by body condition scoring and body weight. The results of this preclinical pilot demonstrate that GPC3 can be used as a target for radioimmunotherapy in an orthotopic mouse model of HCC and may be a target of clinical significance, particularly for disseminated HCC.

Multidisciplinary approach for multifocal, bilobar hepatocellular carcinoma: A case report and literature review.

BACKGROUND: Hepatocellular carcinoma (HCC) is the second most lethal malignancy worldwide. There has been virtually no change in the survivability of HCC in spite of improvement in therapies. Surgery is considered the ideal first, curative intervention, however most patients present in advanced stages with unresectable disease. Therefore, systemic and liver-directed non-operative therapies are initially offered to downstage the disease. To ensure optimal management, a multidisciplinary team approach is often warranted. Our case highlights the benefits of a multidisciplinary approach in a young woman with multifocal, bilobar HCC. CASE SUMMARY: A 36-year-old Chinese woman with untreated hepatitis B was found to have large bilobar HCC during work up for abdominal pain. Her initial serum alpha-fetoprotein was significantly elevated to 311136 ng/mL. Computed tomography scan demonstrated bulky bilobar liver masses, consistent with intermediate stage HCC, Barcelona Clinic Liver Cancer Stage B. Her case was discussed and a personalized care plan was developed at the Multidisciplinary Center for Advanced Minimally Invasive Liver Oncologic Therapies at the University of Washington. She initially underwent bilobar transarterial chemoembolization with partial response of the left lobar tumor. Salvage hypofractionated proton beam radiation therapy was delivered to the right lobe followed by two additional transarterial chemoembolizations to the left lobe with good response. Finally, to remove left lobar residual disease, she was taken to the operating room for a left hepatectomy eleven months after her initial presentation. She continues to be without evidence of disease. CONCLUSION: Coordinating the multiple HCC treatment modalities is complex and our case highlights the benefits of a multidisciplinary approach.