Publications by authors named "Robert A Anders"

154 Publications

G-protein coupled receptor 35 (GPR35) regulates the colonic epithelial cell response to enterotoxigenic Bacteroides fragilis.

Commun Biol 2021 May 14;4(1):585. Epub 2021 May 14.

Johns Hopkins University, Department of Medicine, Division of Infectious Diseases, Baltimore, MD, USA.

G protein-coupled receptor (GPR)35 is highly expressed in the gastro-intestinal tract, predominantly in colon epithelial cells (CEC), and has been associated with inflammatory bowel diseases (IBD), suggesting a role in gastrointestinal inflammation. The enterotoxigenic Bacteroides fragilis (ETBF) toxin (BFT) is an important virulence factor causing gut inflammation in humans and animal models. We identified that BFT signals through GPR35. Blocking GPR35 function in CECs using the GPR35 antagonist ML145, in conjunction with shRNA knock-down and CRISPRcas-mediated knock-out, resulted in reduced CEC-response to BFT as measured by E-cadherin cleavage, beta-arrestin recruitment and IL-8 secretion. Importantly, GPR35 is required for the rapid onset of ETBF-induced colitis in mouse models. GPR35-deficient mice showed reduced death and disease severity compared to wild-type C57Bl6 mice. Our data support a role for GPR35 in the CEC and mucosal response to BFT and underscore the importance of this molecule for sensing ETBF in the colon.
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http://dx.doi.org/10.1038/s42003-021-02014-3DOI Listing
May 2021

Development, Practice Patterns, and Early Clinical Outcomes of a Multidisciplinary Liver Cancer Clinic.

Cancer Control 2021 Jan-Dec;28:10732748211009945

Department of Radiation Oncology and Molecular Radiation Sciences, 1500Johns Hopkins University School of Medicine, Baltimore, MD, USA.

Multidisciplinary care has been associated with improved survival in patients with primary liver cancers. We report the practice patterns and real world clinical outcomes for patients presenting to the Johns Hopkins Hospital (JHH) multidisciplinary liver clinic (MDLC). We analyzed hepatocellular carcinoma (HCC, n = 100) and biliary tract cancer (BTC, n = 76) patients evaluated at the JHH MDLC in 2019. We describe the conduct of the clinic, consensus decisions for patient management based on stage categories, and describe treatment approaches and outcomes based on these categories. We describe subclassification of BCLC stage C into 2 parts, and subclassification of cholangiocarcinoma into 4 stages. A treatment consensus was finalized on the day of MDLC for the majority of patients (89% in HCC, 87% in BTC), with high adherence to MDLC recommendations (91% in HCC, 100% in BTC). Among patients presenting for a second opinion regarding management, 28% of HCC and 31% of BTC patients were given new therapeutic recommendations. For HCC patients, at a median follow up of 11.7 months (0.7-19.4 months), median OS was not reached in BCLC A and B patients. In BTC patients, at a median follow up of 14.2 months (0.9-21.1 months) the median OS was not reached in patients with resectable or borderline resectable disease, and was 11.9 months in patients with unresectable or metastatic disease. Coordinated expert multidisciplinary care is feasible for primary liver cancers with high adherence to recommendations and a change in treatment for a sizeable minority of patients.
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http://dx.doi.org/10.1177/10732748211009945DOI Listing
April 2021

Nuclear PTEN and p53 suppress stress-induced liver cancer through distinct mechanisms.

Biochem Biophys Res Commun 2021 Apr 2;549:83-90. Epub 2021 Mar 2.

Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA. Electronic address:

PTEN and p53 are highly mutated in many cancers. These two tumor suppressors have critical functions in the nucleus, such as DNA repair, cell cycle progression, and genome maintenance. However, the in vivo functional relationship of nuclear PTEN and p53 is unknown. Here, we analyzed the liver of mice in which nuclear PTEN and p53 are individually or simultaneously depleted. We found that nuclear PTEN loss greatly upregulates p53 expression upon oxidative stress, while the loss of p53 potentiates stress-induced accumulation of PTEN in the nucleus. Next, we examined oxidative stress-induced DNA damage in hepatocytes, and found that nuclear PTEN loss aggravated the damage while p53 loss did not. Notably, mice lacking nuclear PTEN had increased hepatocellular carcinoma under oxidative stress, while mice lacking p53 in hepatocytes had accelerated hepatocellular carcinoma and intrahepatic cholangiocarcinoma. The formation of cholangiocarcinoma appears to involve the transformation of hepatocytes into cholangiocarcinoma. Simultaneous loss of nuclear PTEN and p53 exacerbated both types of liver cancers. These data suggest that nuclear PTEN and p53 suppress liver cancers through distinct mechanisms.
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http://dx.doi.org/10.1016/j.bbrc.2021.02.093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7995232PMC
April 2021

Bacterial-driven inflammation and mutant BRAF expression combine to promote murine colon tumorigenesis that is sensitive to immune checkpoint therapy.

Cancer Discov 2021 Feb 25. Epub 2021 Feb 25.

Medicine, Johns Hopkins University

Colorectal cancer (CRC) is multi-faceted with subtypes defined by genetic, histological, and immunologic features which are potentially influenced by inflammation, mutagens, and/or microbiota. CRCs with activating mutations in BRAF are associated with distinct clinical characteristics though the pathogenesis is not well understood. The Wnt-driven multiple intestinal neoplasia (MinApc[triangle]716/+) enterotoxigenic Bacteroides fragilis (ETBF) murine model is characterized by IL-17-dependent, distal colon adenomas. Herein, we report that addition of the BRAFV600E mutation to this model results in emergence of a distinct locus of mid-colon tumors. In ETBF-colonized BRAFV600ELgr5CreMin (BLM) mice, tumors have similarities to human BRAFV600E tumors, including histology, CpG island DNA hypermethylation, and immune signatures. In comparison to Min ETBF tumors, BLM ETBF tumors are infiltrated by CD8+ T cells, express interferon-gamma signatures, and are sensitive to anti-PDL1 treatment. These results provide direct evidence for critical roles of host genetic and microbiota interactions in CRC pathogenesis and sensitivity to immunotherapy.
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http://dx.doi.org/10.1158/2159-8290.CD-20-0770DOI Listing
February 2021

A feasibility study of combined epigenetic and vaccine therapy in advanced colorectal cancer with pharmacodynamic endpoint.

Clin Epigenetics 2021 Feb 2;13(1):25. Epub 2021 Feb 2.

Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, Bloomberg~Kimmel Institute for Cancer Immunotherapy, Johns Hopkins University School of Medicine, 1650 Orleans Street, Office 4M10, Baltimore, MD, 21287, USA.

Epigenetic therapies may modulate the tumor microenvironment. We evaluated the safety and optimal sequence of combination DNA methyltransferase inhibitor guadecitabine with a granulocyte macrophage-colony-stimulating-factor (GM-CSF) secreting colon cancer (CRC) vaccine (GVAX) using a primary endpoint of change in CD45RO + T cells. 18 patients with advanced CRC enrolled, 11 underwent paired biopsies and were evaluable for the primary endpoint. No significant increase in CD45RO + cells was noted. Grade 3-4 toxicities were expected and manageable. Guadecitabine + GVAX was tolerable but demonstrated no significant immunologic activity in CRC. We report a novel trial design to efficiently evaluate investigational therapies with a primary pharmacodynamic endpoint.Trial registry Clinicaltrials.gov: NCT01966289. Registered 21 October, 2013.
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http://dx.doi.org/10.1186/s13148-021-01014-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7856736PMC
February 2021

Vaccine-Induced Intratumoral Lymphoid Aggregates Correlate with Survival Following Treatment with a Neoadjuvant and Adjuvant Vaccine in Patients with Resectable Pancreatic Adenocarcinoma.

Clin Cancer Res 2021 Mar 4;27(5):1278-1286. Epub 2020 Dec 4.

Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland.

Purpose: Immunotherapy is currently ineffective for nearly all pancreatic ductal adenocarcinomas (PDAC), largely due to its tumor microenvironment (TME) that lacks antigen-experienced T effector cells (Teff). Vaccine-based immunotherapies are known to activate antigen-specific Teffs in the peripheral blood. To evaluate the effect of vaccine therapy on the PDAC TME, we designed a neoadjuvant and adjuvant clinical trial of an irradiated, GM-CSF-secreting, allogeneic PDAC vaccine (GVAX).

Patients And Methods: Eighty-seven eligible patients with resectable PDAC were randomly assigned (1:1:1) to receive GVAX alone or in combination with two forms of low-dose cyclophosphamide. Resected tumors following neoadjuvant immunotherapy were assessed for the formation of tertiary lymphoid aggregates (TLA) in response to treatment. The clinical endpoints are disease-free survival (DFS) and overall survival (OS).

Results: The neoadjuvant treatment with GVAX either alone or with two forms of low-dose cyclophosphamide is safe and feasible without adversely increasing the surgical complication rate. Patients in Arm A who received neoadjuvant and adjuvant GVAX alone had a trend toward longer median OS (35.0 months) than that (24.8 months) in the historical controls who received adjuvant GVAX alone. However, Arm C, who received low-dose oral cyclophosphamide in addition to GVAX, had a significantly shorter DFS than Arm A. When comparing patients with OS > 24 months to those with OS < 15 months, longer OS was found to be associated with higher density of intratumoral TLA.

Conclusions: It is safe and feasible to use a neoadjuvant immunotherapy approach for PDACs to evaluate early biologic responses. In-depth analysis of TLAs is warranted in future neoadjuvant immunotherapy clinical trials.
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http://dx.doi.org/10.1158/1078-0432.CCR-20-2974DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7925374PMC
March 2021

The Loss of Nuclear PTEN Increases Tumorigenesis in a Preclinical Mouse Model for Hepatocellular Carcinoma.

iScience 2020 Oct 10;23(10):101548. Epub 2020 Sep 10.

Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.

The gene is highly mutated in many cancers, including hepatocellular carcinoma. The PTEN protein is located at different subcellular regions-PTEN at the plasma membrane suppresses PI3-kinase signaling in cell growth, whereas PTEN in the nucleus maintains genome integrity. Here, using nuclear PTEN-deficient mice, we analyzed the role of PTEN in the nucleus in hepatocellular carcinoma that is induced by carcinogen and oxidative stress-producing hepatotoxin. Upon oxidative stress, PTEN was accumulated in the nucleus of the liver, and this accumulation promoted repair of DNA damage in wild-type mice. In contrast, nuclear PTEN-deficient mice had increased DNA damage and accelerated hepatocellular carcinoma formation. Both basal and oxidative stress-induced localization of PTEN in the nucleus require ubiquitination of lysine 13 in PTEN. Taken together, these data suggest the critical role of nuclear PTEN in the protection from DNA damage and tumorigenesis .
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http://dx.doi.org/10.1016/j.isci.2020.101548DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7516300PMC
October 2020

Bariatric Arterial Embolization with Calibrated Radiopaque Microspheres and an Antireflux Catheter Suppresses Weight Gain and Appetite-Stimulating Hormones in Swine.

J Vasc Interv Radiol 2020 Sep 14;31(9):1483-1491. Epub 2020 Aug 14.

Russell H. Morgan Department of Radiology and Radiological Science, Johns Hopkins University School of Medicine, Baltimore, Maryland.

Purpose: To examine safety and efficacy of bariatric arterial embolization (BAE) with x-ray-visible embolic microspheres (XEMs) and an antireflux catheter in swine.

Material And Methods: BAE with selective infusion of XEMs (n = 6) or saline (n = 4, control) into gastric fundal arteries was performed under x-ray guidance. Weight and plasma hormone levels were measured at baseline and weekly for 4 weeks after embolization. Cone-beam CT images were acquired immediately after embolization and weekly for 4 weeks. Hormone-expressing cells in the stomach were assessed by immunohistochemical staining.

Results: BAE pigs lost weight 1 week after embolization followed by significantly impaired weight gain relative to control animals (14.3% vs 20.9% at 4 weeks, P = .03). Plasma ghrelin levels were significantly lower in BAE pigs than in control animals (1,221.6 pg/mL vs 1,706.2 pg/mL at 4 weeks, P < .01). XEMs were visible on x-ray and cone-beam CT during embolization, and radiopacity persisted over 4 weeks (165.5 HU at week 1 vs 158.5 HU at week 4, P = .9). Superficial mucosal ulcerations were noted in 1 of 6 BAE animals. Ghrelin-expressing cell counts were significantly lower in the gastric fundus (17.7 vs 36.8, P < .00001) and antrum (24.2 vs 46.3, P < .0001) of BAE pigs compared with control animals. Gastrin-expressing cell counts were markedly reduced in BAE pigs relative to control animals (98.5 vs 127.0, P < .02). Trichrome staining demonstrated significantly more fibrosis in BAE animals compared with control animals (13.8% vs 8.7%, P < .0001).

Conclusions: XEMs enabled direct visualization of embolic material during and after embolization. BAE with XEMs and antireflux microcatheters was safe and effective.
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http://dx.doi.org/10.1016/j.jvir.2020.04.038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7483389PMC
September 2020

A Phase II Study of Allogeneic GM-CSF-Transfected Pancreatic Tumor Vaccine (GVAX) with Ipilimumab as Maintenance Treatment for Metastatic Pancreatic Cancer.

Clin Cancer Res 2020 10 26;26(19):5129-5139. Epub 2020 Jun 26.

Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, The Skip Viragh Center for Pancreas Cancer Clinical Research and Patient Care, and The Bloomberg-Kimmel Institute for Cancer Immunotherapy at Johns Hopkins University School of Medicine, Baltimore, Maryland.

Purpose: This phase II study tested granulocyte-macrophage colony-stimulating factor (GM-CSF)-allogeneic pancreatic tumor cells (GVAX) and ipilimumab in metastatic pancreatic ductal adenocarcinoma (PDA) in the maintenance setting.

Patients And Methods: Patients with PDA who were treated with front-line chemotherapy consisting of 5-fluorouracil, leucovorin, irinotecan, and oxaliplatin (FOLFIRINOX) in the metastatic setting and had ongoing response or stable disease after 8-12 doses were eligible. Patients were randomized 1:1 to treatment with GVAX and ipilimumab given every 3 weeks for four doses then every 8 weeks (Arm A) or to FOLFIRINOX continuation (Arm B). The primary objective was to compare overall survival (OS) between the two arms.

Results: Eighty-two patients were included in the final analysis (Arm A: 40; Arm B: 42). The study was stopped for futility after interim analysis. Median OS was 9.38 months [95% confidence interval (CI), 5.0-12.2] for Arm A and 14.7 months (95% CI, 11.6-20.0) for Arm B (HR, 1.75; = 0.019). Using immune-related response criteria, two partial responses (5.7%) were observed in Arm A and four (13.8%) in Arm B. GVAX + ipilimumab promoted T-cell differentiation into effector memory phenotypes both in the periphery and in the tumor microenvironment and increased M1 macrophages in the tumor.

Conclusions: GVAX and ipilimumab maintenance therapy did not improve OS over continuation of chemotherapy and resulted in a numerically inferior survival in metastatic PDA. However, clinical responses and biological effects on immune cells were observed. Further study of novel combinations in the maintenance treatment of metastatic PDA is feasible.
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http://dx.doi.org/10.1158/1078-0432.CCR-20-1025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7541669PMC
October 2020

Inhibition of miR-21 Regulates Mutant KRAS Effector Pathways and Intercepts Pancreatic Ductal Adenocarcinoma Development.

Cancer Prev Res (Phila) 2020 07 14;13(7):569-582. Epub 2020 May 14.

Department of Oncology, Skip Viragh Center for Pancreas Cancer, Bloomberg Kimmel Institute for Cancer Immunotherapy, and the Sidney Kimmel Cancer Center at Johns Hopkins University School of Medicine, Baltimore, Maryland.

Almost all pancreatic ductal adenocarcinomas (PDA) develop following KRAS activation, which triggers epithelial transformation and recruitment of desmoplastic stroma through additional transcriptional and epigenetic regulation, but only a few of these regulatory mechanisms have been described. We profiled dysregulated miRNAs starting with the earliest premalignant pancreatic intraepithelial neoplasias (PanIN) in genetically engineered mutated KRAS and P53 (KPC) mice programmed to recapitulate human PDA tumorigenesis. We identified miR-21 and miR-224 as cell-specific and compartment-specific regulators in PanINs and PDA. miR-21 is overexpressed in tumor epithelial cells of premalignant ducts, while miR-224 is overexpressed in cancer-associated fibroblasts in PDA stroma. Inhibition of miR-21 reverted protumorigenic functionalities to baseline levels. Overexpression of miR-224 induced activated phenotypes in normal fibroblasts. miR-21 inhibition improved survival in established PDA. Importantly, early systemic miR-21 inhibition completely intercepted premalignant progression. Finally, an evaluation of miR-21 expression in the PDA cohort of The Cancer Genome Atlas identified a correlation between tumor epithelial cell content and miR-21 expression in human tumors providing further rationale for conducting human studies. Thus, miR-21 may be useful for early PanIN detection, and for intercepting developing premalignant pancreatic lesions and other KRAS-driven premalignancies.
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http://dx.doi.org/10.1158/1940-6207.CAPR-20-0053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7372516PMC
July 2020

The Immunosuppressive Niche of Soft-Tissue Sarcomas is Sustained by Tumor-Associated Macrophages and Characterized by Intratumoral Tertiary Lymphoid Structures.

Clin Cancer Res 2020 08 24;26(15):4018-4030. Epub 2020 Apr 24.

Departement of Oncology, Johns Hopkins University School of Medicine and Sidney Kimmel Comprehensive Cancer Center, Baltimore, MD.

Purpose: Clinical trials with immune checkpoint inhibition in sarcomas have demonstrated minimal response. Here, we interrogated the tumor microenvironment (TME) of two contrasting soft-tissue sarcomas (STS), rhabdomyosarcomas and undifferentiated pleomorphic sarcomas (UPS), with differing genetic underpinnings and responses to immune checkpoint inhibition to understand the mechanisms that lead to response.

Experimental Design: Utilizing fresh and formalin-fixed, paraffin-embedded tissue from patients diagnosed with UPS and rhabdomyosarcomas, we dissected the TME by using IHC, flow cytometry, and comparative transcriptomic studies.

Results: Our results demonstrated both STS subtypes to be dominated by tumor-associated macrophages and infiltrated with immune cells that localized near the tumor vasculature. Both subtypes had similar T-cell densities, however, their distribution diverged. UPS specimens demonstrated diffuse intratumoral infiltration of T cells, while rhabdomyosarcomas samples revealed intratumoral T cells that clustered with B cells near perivascular beds, forming tertiary lymphoid structures (TLS). T cells in UPS specimens were comprised of abundant CD8 T cells exhibiting high PD-1 expression, which might represent the tumor reactive repertoire. In rhabdomyosarcomas, T cells were limited to TLS, but expressed immune checkpoints and immunomodulatory molecules which, if appropriately targeted, could help unleash T cells into the rest of the tumor tissue.

Conclusions: Our work in STS revealed an immunosuppressive TME dominated by myeloid cells, which may be overcome with activation of T cells that traffic into the tumor. In rhabdomyosarcomas, targeting T cells found within TLS may be key to achieve antitumor response.
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http://dx.doi.org/10.1158/1078-0432.CCR-19-3416DOI Listing
August 2020

Evaluation of Cyclophosphamide/GVAX Pancreas Followed by Listeria-Mesothelin (CRS-207) with or without Nivolumab in Patients with Pancreatic Cancer.

Clin Cancer Res 2020 07 9;26(14):3578-3588. Epub 2020 Apr 9.

Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, Maryland.

Purpose: Two studies in previously treated metastatic pancreatic cancer have been completed combining GVAX pancreas vaccine (GM-CSF-secreting allogeneic pancreatic tumor cells) with cyclophosphamide (Cy) and CRS-207 (live, attenuated -expressing mesothelin). In the current study, we compared Cy/GVAX followed by CRS-207 with (Arm A) or without nivolumab (Arm B).

Patients And Methods: Patients with pancreatic adenocarcinoma who received one prior therapy for metastatic disease and RECIST measurable disease were randomized 1:1 to receive treatment on Arm A or Arm B. The primary objective was to compare overall survival (OS) between the arms. Additional objectives included assessment of progression-free survival, safety, tumor responses, CA19-9 responses, and immunologic correlates.

Results: Ninety-three patients were treated (Arm A, 51; Arm B, 42). The median OS in Arms A and B were 5.9 [95% confidence interval (CI), 4.7-8.6] and 6.1 (95% CI, 3.5-7.0) months, respectively, with an HR of 0.86 (95% CI, 0.55-1.34). Objective responses were seen in 3 patients using immune-related response criteria (4%, 2/51, Arm A; 2%, 1/42, Arm B). The grade ≥3 related adverse event rate, whereas higher in Arm A (35.3% vs. 11.9%) was manageable. Changes in the microenvironment, including increase in CD8 T cells and a decrease in CD68 myeloid cells, were observed in long-term survivors in Arm A only.

Conclusions: Although the study did not meet its primary endpoint of improvement in OS of Arm A over Arm B, the OS was comparable with standard therapy. Objective responses and immunologic changes in the tumor microenvironment were evident.
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http://dx.doi.org/10.1158/1078-0432.CCR-19-3978DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7727397PMC
July 2020

Role of baseline volumetric functional MRI in predicting histopathologic grade and patients' survival in hepatocellular carcinoma.

Eur Radiol 2020 Jul 6;30(7):3748-3758. Epub 2020 Mar 6.

Russell H. Morgan Department of Radiology and Radiological Sciences, Johns Hopkins Hospital, Baltimore, MD, USA.

Objectives: We aimed to evaluate the role of volumetric ADC (vADC) and volumetric venous enhancement (vVE) in predicting the grade of tumor differentiation in hepatocellular carcinoma (HCC).

Methods: The study population included 136 HCC patients (188 lesions) who had baseline MR imaging and histopathological report. Measurements of vVE and vADC were performed on baseline MRI. Tumors were histologically classified into low-grade and high-grade groups. The parameters between the two groups were compared using Mann-Whitney U and chi-square tests for continuous and categorical parameters, respectively. Area under receiver operating characteristic (AUROC) was calculated to investigate the accuracy of vADC and vVE. Logistic regression and multivariable Cox regression were used to unveil the potential parameters associated with high-grade HCC and patient's survival, respectively.

Results: Lesions with higher vADC values and a higher absolute vADC skewness were more likely to be high grade on histopathology assessment (p = 0.001 and p = 0.0291, respectively). Also, vVE showed a trend to be higher in low-grade lesions (p = 0.079). Adjusted multivariable model including vADC, vVE, and vADC skewness could strongly predict HCC degree of differentiation (AUROC = 83%). Additionally, a higher Child-Pugh score (HR = 2.39 [p = 0.02] for score 2 and HR = 3.47 [p = 0.001] for score 3), vADC skewness (HR = 1.52, p = 0.02; per increments in skewness), and tumor volume (HR = 1.1, p = 0.001; per 100 cm increments) showed the highest association with patients' survival.

Conclusions: vADC and vVE have the potential to accurately predict HCC differentiation. Additionally, some imaging features in combination with patients' clinical characteristics can predict patient survival.

Key Points: • Volumetric functional MRI metrics can be considered as non-invasive measures for determining tumor histopathology in HCC. • Estimating patient survival based on clinical and imaging parameters can be used for modifying management approach and preventing unnecessary adverse events.
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http://dx.doi.org/10.1007/s00330-020-06742-8DOI Listing
July 2020

A phase 2 study of GVAX colon vaccine with cyclophosphamide and pembrolizumab in patients with mismatch repair proficient advanced colorectal cancer.

Cancer Med 2020 02 26;9(4):1485-1494. Epub 2019 Dec 26.

Bloomberg-Kimmel Institute for Cancer Immunotherapy, Johns Hopkins University School of Medicine, Baltimore, MD, USA.

Background: Mismatch repair proficient (MMRp) colorectal cancer (CRC) has been refractory to single-agent programmed cell death protein 1 (PD1) inhibitor therapy. Colon GVAX is an allogeneic, whole-cell, granulocyte-macrophage colony-stimulating factor -secreting cellular immunotherapy that induces T-cell immunity against tumor-associated antigens and has previously been studied in combination with low-dose cyclophosphamide (Cy) to inhibit regulatory T cells.

Methods: We conducted a single-arm study of GVAX/Cy in combination with the PD1 inhibitor pembrolizumab in patients with advanced MMRp CRC. Patients received pembrolizumab plus Cy on day 1, GVAX on day 2, of a 21-day cycle. The primary endpoint was the objective response rate by Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. Secondary objectives included safety, overall survival, progression-free survival, changes in carcinoembryonic antigen (CEA) levels, and immune-related correlates.

Results: Seventeen patients were enrolled. There were no objective responses, and the disease control rate was 18% by RECIST 1.1. The median progression-free survival was 82 days (95% confidence interval [CI], 48-97 days) and the median overall survival was 213 days (95% CI 179-441 days). Biochemical responses (≥30% decline in CEA) were observed in 7/17 (41%) of patients. Grade ≥ 3 treatment-related adverse events were observed in two patients (hemolytic anemia and corneal transplant rejection). Paired pre- and on-treatment biopsy specimens showed increases in programmed death-ligand 1 expression and tumor necrosis in a subset of patients.

Conclusions: GVAX/Cy plus pembrolizumab failed to meet its primary objective in MMRp CRC. Biochemical responses were observed in a subset of patients and have not previously been observed with pembrolizumab monotherapy in MMRp CRC, indicating that GVAX may modulate the antitumor immune response.
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http://dx.doi.org/10.1002/cam4.2763DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7013064PMC
February 2020

The number of microspheres in Y90 radioembolization directly affects normal tissue radiation exposure.

Eur J Nucl Med Mol Imaging 2020 04 18;47(4):816-827. Epub 2019 Nov 18.

Department of Radiology, Division of Vascular and Interventional Radiology, The Johns Hopkins Hospital, Baltimore, MD, USA.

Purpose: In Y90 radioembolization, the number of microspheres infused varies by more than a factor of 20 over the shelf-life of the glass radioembolization device. We investigated the effect of the number of Y90 microspheres on normal liver tissue.

Method: Healthy pigs received lobar radioembolization with glass Y90 microspheres at 4, 8, 12, and 16 days post-calibration, representing a > 20× range in the number of microspheres deposited per milliliter in tissue. Animals were survived for 1-month post-treatment and the livers were explanted and scanned on a micro CT system to fully characterize the microscopic distribution of individual microspheres. A complete 3D microdosimetric evaluation of each liver was performed with a spatially correlated analysis of histopathologic effect.

Results: Through whole-lobe microscopic identification of each microsphere, a consistent number of microspheres per sphere cluster was found at 4, 8, and 12 days postcalibration, despite an 8-fold increase in total microspheres infused from days 4 to 12. The additional microspheres instead resulted in more clusters formed and, therefore, a more homogeneous microscopic absorbed dose. The increased absorbed-dose homogeneity resulted in a greater volume fraction of the liver receiving a potentially toxic absorbed dose based on radiobiologic models. Histopathologic findings in the animals support a possible increase in normal liver toxicity in later treatments with more spheres (i.e., ≥ day 12) compared to early treatments with less spheres (i.e., ≤ day 8).

Conclusion: The microdosimetric evidence presented supports a recommendation of caution when treating large volumes (e.g., right lobe) using glass Y microspheres at more than 8 days post-calibration, i.e., after "2nd week" Monday. The favorable normal tissue microscopic distribution and associated low toxicity of first week therapies may encourage opportunities for dose escalation with glass microspheres and could also be considered for patients with decreased hepatic reserve.
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http://dx.doi.org/10.1007/s00259-019-04588-xDOI Listing
April 2020

Pan-Tumor Pathologic Scoring of Response to PD-(L)1 Blockade.

Clin Cancer Res 2020 02 31;26(3):545-551. Epub 2019 Oct 31.

Department of Dermatology at Johns Hopkins University School of Medicine, Baltimore, Maryland.

Purpose: Pathologic response assessment of tumor specimens from patients receiving systemic treatment provides an early indication of therapeutic efficacy and predicts long-term survival. Grading systems for pathologic response were first developed for chemotherapy in select tumor types. Immunotherapeutic agents have a mechanism of action distinct from chemotherapy and are being used across a broad array of tumor types. A standardized, universal scoring system for pathologic response that encompasses features characteristic for immunotherapy and spans tumor types is needed.

Experimental Design: Hematoxylin and eosin-stained slides from neoadjuvant surgical resections and on-treatment biopsies were assessed for features of immune-related pathologic response (irPR). A total of 258 specimens from patients with 11 tumor types as part of ongoing clinical trials for anti-PD-(L)1 were evaluated. An additional 98 specimens from patients receiving anti-PD-(L)1 in combination with other treatments were also reviewed, including those from three additional tumor types.

Results: Common irPR features (immune activation, cell death, tissue repair, and regression bed) were present in all tumor types reviewed, including melanoma, non-small cell lung, head and neck squamous cell, Merkel cell, and renal cell carcinoma, among others. Features were consistent across primary tumors, lymph nodes, and distant metastases. Specimens from patients treated with anti-PD-(L)1 in combination with another agent also exhibited irPR features.

Conclusions: irPR features are consistent across tumor types and treatment settings. Standardized, pan-tumor irPR criteria (irPRC) are defined and associated specimen-handling considerations are described. Future, prospective studies are merited to validate irPRC in larger datasets and to associate pathologic features with long-term patient outcomes.
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http://dx.doi.org/10.1158/1078-0432.CCR-19-2379DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7002263PMC
February 2020

Noninvasive Detection of Microsatellite Instability and High Tumor Mutation Burden in Cancer Patients Treated with PD-1 Blockade.

Clin Cancer Res 2019 12 10;25(23):7024-7034. Epub 2019 Sep 10.

Personal Genome Diagnostics, Baltimore, Maryland.

Purpose: Microsatellite instability (MSI) and high tumor mutation burden (TMB-High) are promising pan-tumor biomarkers used to select patients for treatment with immune checkpoint blockade; however, real-time sequencing of unresectable or metastatic solid tumors is often challenging. We report a noninvasive approach for detection of MSI and TMB-High in the circulation of patients.

Experimental Design: We developed an approach that utilized a hybrid-capture-based 98-kb pan-cancer gene panel, including targeted microsatellite regions. A multifactorial error correction method and a novel peak-finding algorithm were established to identify rare MSI frameshift alleles in cell-free DNA (cfDNA).

Results: Through analysis of cfDNA derived from a combination of healthy donors and patients with metastatic cancer, the error correction and peak-finding approaches produced a specificity of >99% ( = 163) and sensitivities of 78% ( = 23) and 67% ( = 15), respectively, for MSI and TMB-High. For patients treated with PD-1 blockade, we demonstrated that MSI and TMB-High in pretreatment plasma predicted progression-free survival (hazard ratios: 0.21 and 0.23, = 0.001 and 0.003, respectively). In addition, we analyzed cfDNA from longitudinally collected plasma samples obtained during therapy to identify patients who achieved durable response to PD-1 blockade.

Conclusions: These analyses demonstrate the feasibility of noninvasive pan-cancer screening and monitoring of patients who exhibit MSI or TMB-High and have a high likelihood of responding to immune checkpoint blockade..
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http://dx.doi.org/10.1158/1078-0432.CCR-19-1372DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6892397PMC
December 2019

Immunopathologic Stratification of Colorectal Cancer for Checkpoint Blockade Immunotherapy.

Cancer Immunol Res 2019 10 22;7(10):1574-1579. Epub 2019 Aug 22.

Bloomberg-Kimmel Institute for Cancer Immunotherapy, Johns Hopkins University, Baltimore, Maryland.

Mismatch-repair deficiency in solid tumors predicts their response to PD-1 blockade. Based on this principle, pembrolizumab is approved as standard of care for patients with unresectable or metastatic microsatellite instability-high (MSI-H) cancer. Despite this success, a large majority of metastatic colorectal cancer patients are not MSI-H and do not benefit from checkpoint blockade treatment. Predictive biomarkers to develop personalized medicines and guide clinical trials are needed for these patients. We, therefore, asked whether immunohistologic stratification of metastatic colorectal cancer based on primary tumor PD-L1 expression associated with the presence or absence of extracellular mucin defines a subset of metastatic colorectal cancer patients who exhibit a preexisting antitumor immune response and who could potentially benefit from the checkpoint blockade. To address this, we studied 26 advanced metastatic colorectal cancer patients treated with pembrolizumab (NCT01876511). To stratify patients, incorporation of histopathologic characteristics (percentage of extracellular mucin) and PD-L1 expression at the invasive front were used to generate a composite score, the CPM (composite PD-L1 and mucin) score, which discriminated patients who exhibited clinical benefit (complete, partial, or stable disease) from those patients with progressive disease. When validated in larger cohorts, the CPM score in combination with MSI testing may guide immunotherapy interventions for colorectal cancer patient treatment.
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http://dx.doi.org/10.1158/2326-6066.CIR-18-0927DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6774859PMC
October 2019

Abundant Expression of Lysyl Oxidase-like 2 Protein in Intrahepatic Bile Ducts of Infants With Biliary Atresia.

J Pediatr Gastroenterol Nutr 2019 09;69(3):344-350

Department of Pediatrics.

Introduction: Biliary atresia (BA) is characterized by rapidly progressive inflammation and fibrosis of the biliary tract, which usually progresses despite surgical intervention (Kasai hepatoportoenterostomy). Lysyl oxidase-like (LOXL2) is an extracellular matrix enzyme that catalyzes the cross-linking of fibrillar collagen and elastin and is thought to play a crucial role in tissue fibrosis; anti-LOXL2 drugs have been shown to be antifibrotic in animals.

Objective: The aim of the study was to investigate the presence of LOXL2 in BA livers and hepatic and extrahepatic control tissues.

Methods: Liver wedge biopsies from infants with BA (n = 20) were obtained at Kasai, and were compared with non-BA livers (n = 20). Liver fibrosis was scored using the Ishak scale, and immunohistochemistry was performed using a commercially available polyclonal anti-LOXL2 antibody. The expression of LOXL2 was scored for intensity and for distribution of bile duct staining by a pathologist blinded to the diagnosis. Staining of LOXL2 in pediatric control tissue, muscle (n = 5), heart (n = 5), and bone (n = 10) was performed.

Results: Tissue from patients with BA abundantly expressed LOXL2 (intensity score 2.0 vs 1.4 [P ≤ 0.001]) for non-BA and distribution of bile duct-staining score of 3.0 versus 2.8 (P = 0.001) for non-BA. Fibrosis score of all BA samples was 4.2 versus 3.1 for non-BA. Nonhepatic pediatric tissue displayed minimal to no LOXL2 staining.

Conclusions: There is significant overexpression of LOXL2 in BA hepatic tissue with minimal expression in extrahepatic tissue. The over expression noted in human hepatic tissue at Kasai suggests the rationale for further investigation of anti-LOXL2 therapeutics in BA.
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http://dx.doi.org/10.1097/MPG.0000000000002414DOI Listing
September 2019

Targeting Mechanoresponsive Proteins in Pancreatic Cancer: 4-Hydroxyacetophenone Blocks Dissemination and Invasion by Activating MYH14.

Cancer Res 2019 09 29;79(18):4665-4678. Epub 2019 Jul 29.

Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, Maryland.

Metastasis is complex, involving multiple genetic, epigenetic, biochemical, and physical changes in the cancer cell and its microenvironment. Cells with metastatic potential are often characterized by altered cellular contractility and deformability, lending them the flexibility to disseminate and navigate through different microenvironments. We demonstrate that mechanoresponsiveness is a hallmark of pancreatic cancer cells. Key mechanoresponsive proteins, those that accumulate in response to mechanical stress, specifically nonmuscle myosin IIA (MYH9) and IIC (MYH14), α-actinin 4, and filamin B, were highly expressed in pancreatic cancer as compared with healthy ductal epithelia. Their less responsive sister paralogs-myosin IIB (MYH10), α-actinin 1, and filamin A-had lower expression differential or disappeared with cancer progression. We demonstrate that proteins whose cellular contributions are often overlooked because of their low abundance can have profound impact on cell architecture, behavior, and mechanics. Here, the low abundant protein MYH14 promoted metastatic behavior and could be exploited with 4-hydroxyacetophenone (4-HAP), which increased MYH14 assembly, stiffening cells. As a result, 4-HAP decreased dissemination, induced cortical actin belts in spheroids, and slowed retrograde actin flow. 4-HAP also reduced liver metastases in human pancreatic cancer-bearing nude mice. Thus, increasing MYH14 assembly overwhelms the ability of cells to polarize and invade, suggesting targeting the mechanoresponsive proteins of the actin cytoskeleton as a new strategy to improve the survival of patients with pancreatic cancer. SIGNIFICANCE: This study demonstrates that mechanoresponsive proteins become upregulated with pancreatic cancer progression and that this system of proteins can be pharmacologically targeted to inhibit the metastatic potential of pancreatic cancer cells.
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http://dx.doi.org/10.1158/0008-5472.CAN-18-3131DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6744980PMC
September 2019

Comparison of Biomarker Modalities for Predicting Response to PD-1/PD-L1 Checkpoint Blockade: A Systematic Review and Meta-analysis.

JAMA Oncol 2019 Aug;5(8):1195-1204

Department of Dermatology, Johns Hopkins Medical Institutions, Baltimore, Maryland.

Importance: PD-L1 (programmed cell death ligand 1) immunohistochemistry (IHC), tumor mutational burden (TMB), gene expression profiling (GEP), and multiplex immunohistochemistry/immunofluorescence (mIHC/IF) assays have been used to assess pretreatment tumor tissue to predict response to anti-PD-1/PD-L1 therapies. However, the relative diagnostic performance of these modalities has yet to be established.

Objective: To compare studies that assessed the diagnostic accuracy of PD-L1 IHC, TMB, GEP, and mIHC/IF in predicting response to anti-PD-1/PD-L1 therapy.

Evidence Review: A search of PubMed (from inception to June 2018) and 2013 to 2018 annual meeting abstracts from the American Association for Cancer Research, American Society of Clinical Oncology, European Society for Medical Oncology, and Society for Immunotherapy of Cancer was conducted to identify studies that examined the use of PD-L1 IHC, TMB, GEP, and mIHC/IF assays to determine objective response to anti-PD-1/PD-L1 therapy. For PD-L1 IHC, only clinical trials that resulted in US Food and Drug Administration approval of indications for anti-PD-1/PD-L1 were included. Studies combining more than 1 modality were also included. Preferred Reporting Items for Systematic Reviews and Meta-analysis guidelines were followed. Two reviewers independently extracted the clinical outcomes and test results for each individual study.

Main Outcomes And Measures: Summary receiver operating characteristic (sROC) curves; their associated area under the curve (AUC); and pooled sensitivity, specificity, positive and negative predictive values (PPV, NPV), and positive and negative likelihood ratios (LR+ and LR-) for each assay modality.

Results: Tumor specimens representing over 10 different solid tumor types in 8135 patients were assayed, and the results were correlated with anti-PD-1/PD-L1 response. When each modality was evaluated with sROC curves, mIHC/IF had a significantly higher AUC (0.79) compared with PD-L1 IHC (AUC, 0.65, P < .001), GEP (AUC, 0.65, P = .003), and TMB (AUC, 0.69, P = .049). When multiple different modalities were combined such as PD-L1 IHC and/or GEP + TMB, the AUC drew nearer to that of mIHC/IF (0.74). All modalities demonstrated comparable NPV and LR-, whereas mIHC/IF demonstrated higher PPV (0.63) and LR+ (2.86) than the other approaches.

Conclusions And Relevance: In this meta-analysis, tumor mutational burden, PD-L1 IHC, and GEP demonstrated comparable AUCs in predicting response to anti-PD-1/PD-L1 treatment. Multiplex immunohistochemistry/IF and multimodality biomarker strategies appear to be associated with improved performance over PD-L1 IHC, TMB, or GEP alone. Further studies with mIHC/IF and composite approaches with a larger number of patients will be required to confirm these findings. Additional study is also required to determine the most predictive analyte combinations and to determine whether biomarker modality performance varies by tumor type.
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http://dx.doi.org/10.1001/jamaoncol.2019.1549DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6646995PMC
August 2019

Axon Guidance Molecules Promote Perineural Invasion and Metastasis of Orthotopic Pancreatic Tumors in Mice.

Gastroenterology 2019 09 1;157(3):838-850.e6. Epub 2019 Jun 1.

Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, Maryland; Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland; Pancreatic Cancer Precision Medicine Program, Johns Hopkins University School of Medicine, Baltimore, Maryland; Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland; Skip Viragh Center for Pancreatic Cancer, Johns Hopkins University School of Medicine, Baltimore, Maryland. Electronic address:

Background & Aims: Little is known about mechanisms of perineural invasion (PNI) by pancreatic ductal adenocarcinomas (PDAs) or other tumors. Annexin A2 (ANXA2) regulates secretion of SEMA3D, an axon guidance molecule, which binds and activates the receptor PLXND1 to promote PDA invasion and metastasis. We investigated whether axon guidance molecules promote PNI and metastasis by PDA cells in mice.

Methods: We performed studies in a dorsal root ganglion (DRG) invasion system, wild-type C57BL/6 mice (controls), mice with peripheral sensory neuron-specific disruption of PlxnD1 (PLAC mice), LSL-KRAS;LSL-TP53;PDX-1-CRE (KPC) mice, and KPC mice crossed with ANXA2-knockout mice (KPCA mice). PDA cells were isolated from KPC mice and DRG cells were isolated from control mice. Levels of SEMA3D or ANXA2 were knocked down in PDA cells with small hairpin and interfering RNAs and cells were analyzed by immunoblots in migration assays, with DRGs and with or without antibodies against PLXND1. PDA cells were injected into the pancreas of control and PLAC mice, growth of tumors was assessed, and tumor samples were analyzed by histology. DRG cells were incubated with SEMA3D and analyzed by live imaging. We measured levels of SEMA3D and PLXND1 in PDA specimens from patients with PNI and calculated distances between tumor cells and nerves.

Results: DRG cells increase the migration of PDC cells in invasion assays; knockdown of SEMA3D in PDA cells or antibody blockade of PLXND1 on DRG cells reduced this invasive activity. In mice, orthotopic tumors grown from PDA cells with knockdown of SEMA3D, and in PLAC mice, orthotopic tumors grown from PDA cells, had reduced innervation and formed fewer metastases than orthotopic tumors grown from PDA cells in control mice. Increased levels of SEMA3D and PLXND1 in human PDA specimens associated with PNI.

Conclusions: DRG cells increase the migratory and invasive activities of pancreatic cancer cells, via secretion of SEMA3D by pancreatic cells and activation of PLXND1 on DRGs. Knockdown of SEMA3D and loss of neural PLXND1 reduces innervation of orthotopic PDAs and metastasis in mice. Increased levels of SEMA3D and PLXND1 in human PDA specimens associated with PNI. Strategies to disrupt the axon guidance pathway mediated by SEMA3D and PLXND1 might be developed to slow progression of PDA.
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http://dx.doi.org/10.1053/j.gastro.2019.05.065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6707836PMC
September 2019

Intratumoral Adaptive Immunosuppression and Type 17 Immunity in Mismatch Repair Proficient Colorectal Tumors.

Clin Cancer Res 2019 Sep 6;25(17):5250-5259. Epub 2019 May 6.

Bloomberg-Kimmel Institute for Cancer Immunotherapy, Johns Hopkins University, Baltimore, Maryland.

Purpose: Approximately 10% of patients with mismatch repair-proficient (MMRp) colorectal cancer showed clinical benefit to anti-PD-1 monotherapy (NCT01876511). We sought to identify biomarkers that delineate patients with immunoreactive colorectal cancer and to explore new combinatorial immunotherapy strategies that can impact MMRp colorectal cancer.

Experimental Design: We compared the expression of 44 selected immune-related genes in the primary colon tumor of 19 patients with metastatic colorectal cancer (mCRC) who responded ( = 13) versus those who did not ( = 6) to anti-PD-1 therapy (NCT01876511). We define a 10 gene-based immune signature that could distinguish responder from nonresponder. Resected colon specimens ( = 14) were used to validate the association of the predicted status (responder and nonresponder) with the immune-related gene expression, the phenotype, and the function of tumor-infiltrating lymphocytes freshly isolated from the same tumors.

Results: Although both IL17 and IL17 immunoreactive MMRp colorectal cancers are associated with intratumor correlates of adaptive immunosuppression (CD8/IFNγ and PD-L1/IDO1 colocalization), only IL17 MMRp tumors (3/14) have a tumor immune microenvironment (TME) that resembles the TME in primary colon tumors of patients with mCRC responsive to anti-PD-1 treatment.

Conclusions: The detection of a preexisting antitumor immune response in MMRp colorectal cancer (immunoreactive MMRp colorectal cancer) is not sufficient to predict a clinical benefit to T-cell checkpoint inhibitors. Intratumoral IL17-mediated signaling may preclude responses to immunotherapy. Drugs targeting the IL17 signaling pathway are available in clinic, and their combination with T-cell checkpoint inhibitors could improve colorectal cancer immunotherapy..
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http://dx.doi.org/10.1158/1078-0432.CCR-19-0114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6726531PMC
September 2019

Multiple Immune-Suppressive Mechanisms in Fibrolamellar Carcinoma.

Cancer Immunol Res 2019 05 22;7(5):805-812. Epub 2019 Mar 22.

Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, Maryland.

Fibrolamellar carcinoma (FLC) is a rare type of liver cancer that affects adolescents and young adults. The most effective treatment for FLC is surgical resection, but no standardized systemic therapy exists for patients with recurrent or unresectable FLC. As a first step to understand the immune microenvironment of FLC, we investigated targetable immune-checkpoint pathways, PD-1, PD-L1, B7-H3, IDO-1, and LAG3, in relation to CD8 cytotoxic T-lymphocyte density. Thirty-two FLC tumor specimens were analyzed using IHC staining for PD-L1, CD8, PD-1, IDO, LAG3, and B7-H3. Sixty-three percent of FLC cases demonstrated membranous PD-L1 expression on tumor cells, and almost 70% of cases demonstrated PD-L1 tumor-infiltrating lymphocytes and tumor-associated macrophages (TIL/TAM). Myeloid-derived cells appeared to be a major component of PD-L1 tumor-infiltrating immune cells. Forty percent of the cases showed B7-H3 expression in the tumor zone, with 91% cases showing B7-H3 expression in TILs and TAMs. IDO and PD-1 expression was highest in the tumor interface zone. B7-H3 or IDO expression on tumor cells significantly correlated with higher CD8 T-cell density. In conclusion, a high proportion of FLC cases showed robust expression of PD-1, PD-L1, B7-H3, and IDO in an adaptive immune-resistance pattern. Our findings provide further basis for targeting these different immune-checkpoint axes in FLC.
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http://dx.doi.org/10.1158/2326-6066.CIR-18-0499DOI Listing
May 2019

Human colon mucosal biofilms from healthy or colon cancer hosts are carcinogenic.

J Clin Invest 2019 03 11;129(4):1699-1712. Epub 2019 Mar 11.

Bloomberg-Kimmel Institute for Immunotherapy, Departments of Oncology and Medicine and the Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins School of Medicine, Johns Hopkins Medical Institutions, Baltimore, Maryland, USA.

Mucus-invasive bacterial biofilms are identified on the colon mucosa of approximately 50% of colorectal cancer (CRC) patients and approximately 13% of healthy subjects. Here, we test the hypothesis that human colon biofilms comprise microbial communities that are carcinogenic in CRC mouse models. Homogenates of human biofilm-positive colon mucosa were prepared from tumor patients (tumor and paired normal tissues from surgical resections) or biofilm-positive biopsies from healthy individuals undergoing screening colonoscopy; homogenates of biofilm-negative colon biopsies from healthy individuals undergoing screening colonoscopy served as controls. After 12 weeks, biofilm-positive, but not biofilm-negative, human colon mucosal homogenates induced colon tumor formation in 3 mouse colon tumor models (germ-free ApcMinΔ850/+;Il10-/- or ApcMinΔ850/+ and specific pathogen-free ApcMinΔ716/+ mice). Remarkably, biofilm-positive communities from healthy colonoscopy biopsies induced colon inflammation and tumors similarly to biofilm-positive tumor tissues. By 1 week, biofilm-positive human tumor homogenates, but not healthy biopsies, displayed consistent bacterial mucus invasion and biofilm formation in mouse colons. 16S rRNA gene sequencing and RNA-Seq analyses identified compositional and functional microbiota differences between mice colonized with biofilm-positive and biofilm-negative communities. These results suggest human colon mucosal biofilms, whether from tumor hosts or healthy individuals undergoing screening colonoscopy, are carcinogenic in murine models of CRC.
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http://dx.doi.org/10.1172/JCI124196DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6436866PMC
March 2019

Persistent mutant oncogene specific T cells in two patients benefitting from anti-PD-1.

J Immunother Cancer 2019 02 11;7(1):40. Epub 2019 Feb 11.

Bloomberg-Kimmel Institute for Cancer Immunotherapy, Johns Hopkins University, Baltimore, MD, USA.

Background: Several predictive biomarkers are currently approved or are under investigation for the selection of patients for checkpoint blockade. Tumor PD-L1 expression is used for stratification of non-small cell lung (NSCLC) patients, with tumor mutational burden (TMB) also being explored with promising results, and mismatch-repair deficiency is approved for tumor site-agnostic disease. While tumors with high PD-L1 expression, high TMB, or mismatch repair deficiency respond well to checkpoint blockade, tumors with lower PD-L1 expression, lower mutational burdens, or mismatch repair proficiency respond much less frequently.

Case Presentation: We studied two patients with unexpected responses to checkpoint blockade monotherapy: a patient with PD-L1-negative and low mutational burden NSCLC and one with mismatch repair proficient colorectal cancer (CRC), both of whom lack the biomarkers associated with response to checkpoint blockade, yet achieved durable clinical benefit. Both maintained T-cell responses in peripheral blood to oncogenic driver mutations - BRAF-N581I in the NSCLC and AKT1-E17K in the CRC - years after treatment initiation. Mutation-specific T cells were also found in the primary tumor and underwent dynamic perturbations in the periphery upon treatment.

Conclusions: These findings suggest that T cell responses to oncogenic driver mutations may be more prevalent than previously appreciated and could be harnessed in immunotherapeutic treatment, particularly for patients who lack the traditional biomarkers associated with response. Comprehensive studies are warranted to further delineate additional predictive biomarkers and populations of patients who may benefit from checkpoint blockade.
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http://dx.doi.org/10.1186/s40425-018-0492-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6371497PMC
February 2019

Quantitative Characterization of CD8+ T Cell Clustering and Spatial Heterogeneity in Solid Tumors.

Front Oncol 2018 7;8:649. Epub 2019 Jan 7.

Department of Biomedical Engineering, School of Medicine, Johns Hopkins University, Baltimore, MD, United States.

Quantitative characterization of the tumor microenvironment, including its immuno-architecture, is important for developing quantitative diagnostic and predictive biomarkers, matching patients to the most appropriate treatments for precision medicine, and for providing quantitative data for building systems biology computational models able to predict tumor dynamics in the context of immune checkpoint blockade therapies. The intra- and inter-tumoral spatial heterogeneities are potentially key to the understanding of the dose-response relationships, but they also bring challenges to properly parameterizing and validating such models. In this study, we developed a workflow to detect CD8+ T cells from whole slide imaging data, and quantify the spatial heterogeneity using multiple metrics by applying spatial point pattern analysis and morphometric analysis. The results indicate a higher intra-tumoral heterogeneity compared with the heterogeneity across patients. By comparing the baseline metrics with PD-1 blockade treatment outcome, our results indicate that the number of high-density T cell clusters of both circular and elongated shapes are higher in patients who responded to the treatment. This methodology can be applied to quantitatively characterize the tumor microenvironment, including immuno-architecture, and its heterogeneity for different cancer types.
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http://dx.doi.org/10.3389/fonc.2018.00649DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6330341PMC
January 2019

Evaluation of Peritumoral Fibrosis in Metastatic Colorectal Adenocarcinoma to the Liver Using Digital Image Analysis.

Am J Clin Pathol 2019 01;151(2):226-230

Department of Pathology, The Johns Hopkins Hospital, Baltimore, MD.

Objectives: It is challenging to separate peritumoral fibrosis from fibrosis due to chronic liver disease in mass-directed liver biopsies. We evaluated the distance that peritumoral fibrosis extends from metastatic colorectal adenocarcinoma in liver.

Methods: Peritumoral and distant uninvolved liver trichrome stains from 25 cases were analyzed using digital image analysis. Fibrosis was quantitated at concentric intervals from each tumor and in uninvolved liver.

Results: There was a 3.9 fold (range 0.9-18.6) median increase in fibrosis in the first 0.5 mm of peritumoral liver compared to distant liver. Fibrosis levels returned to baseline at median 2.5 mm (interquartile range 1.5-5.0 mm) from tumor.

Conclusions: Fibrosis is markedly increased in peritumoral liver. Fibrosis levels returned to baseline by 5 mm from tumor in approximately 75% of cases. Pathologists should be cautious of fibrosis in mass-directed liver biopsies without at least 5 mm of liver tissue distal to the mass.
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http://dx.doi.org/10.1093/ajcp/aqy134DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6306046PMC
January 2019

Multidimensional, quantitative assessment of PD-1/PD-L1 expression in patients with Merkel cell carcinoma and association with response to pembrolizumab.

J Immunother Cancer 2018 10 1;6(1):99. Epub 2018 Oct 1.

Department of Dermatology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.

Background: We recently reported a 56% objective response rate in patients with advanced Merkel cell carcinoma (MCC) receiving pembrolizumab. However, a biomarker predicting clinical response was not identified.

Methods: Pretreatment FFPE tumor specimens (n = 26) were stained for CD8, PD-L1, and PD-1 by immunohistochemistry/immunofluorescence (IHC/IF), and the density and distribution of positive cells was quantified to determine the associations with anti-PD-1 response. Multiplex IF was used to test a separate cohort of MCC archival specimens (n = 16), to identify cell types expressing PD-1.

Results: Tumors from patients who responded to anti-PD-1 showed higher densities of PD-1+ and PD-L1+ cells when compared to non-responders (median cells/mm, 70.7 vs. 6.7, p = 0.03; and 855.4 vs. 245.0, p = 0.02, respectively). There was no significant association of CD8+ cell density with clinical response. Quantification of PD-1+ cells located within 20 μm of a PD-L1+ cell showed that PD-1/PD-L1 proximity was associated with clinical response (p = 0.03), but CD8/PD-L1 proximity was not. CD4+ and CD8+ cells in the TME expressed similar amounts of PD-1.

Conclusions: While the binomial presence or absence of PD-L1 expression in the TME was not sufficient to predict response to anti-PD-1 in patients with MCC, we show that quantitative assessments of PD-1+ and PD-L1+ cell densities as well as the geographic interactions between these two cell populations correlate with clinical response. Cell types expressing PD-1 in the TME include CD8+ T-cells, CD4+ T-cells, T, and CD20+ B-cells, supporting the notion that multiple cell types may potentiate tumor regression following PD-1 blockade.
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http://dx.doi.org/10.1186/s40425-018-0404-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6167897PMC
October 2018