Sensitive Positron Emission Tomography Imaging of PD-L1 Expression in Human Breast and Lung Carcinoma Xenografts Using the Radiometalated Peptide Ga-68-TRAP-WL12

Noninvasive imaging of the immune checkpoint protein programmed death ligand 1 (PD-L1; synonyms: CD274, B7–H1) holds great promise to improve patient selection and, thus, response rates for immune checkpoint therapy (ICT) with monoclonal antibodies targeting the PD1/PD-L1 axis. The PD-L1 specific peptide WL12 (cyclo­(AcY-(NMe)­A-N-P-H-L-Hyp-W-S-W­(Me)-(NMe)­Nle-(NMe)­Nle-O-C)-G-NH2) was functionalized with the Gallium-68 chelator TRAP by means of click chemistry (CuAAC). The resulting conjugate TRAP-WL12 was labeled with Gallium-68 within 16 min, with approximately 90% radiochemical yield and 99% radiochemical purity, affording Ga-68-TRAP-WL12 with molar activities typically exceeding 100 MBq/nmol. This radiotracer was characterized by positron emission tomography (PET) imaging and ex vivo biodistribution in murine xenografts of nontransfected PD-L1 expressing tumor cell lines, MDA-MB-231 (human breast carcinoma), and H2009 (human lung adenocarcinoma). It showed a favorable biodistribution profile with rapid renal clearance and low background (tumor-to-blood ratio = 26.6, 3 h p.i.). Conjugation of the Ga-68-TRAP moiety to WL12 successfully mitigated the nonspecific uptake of this peptide in organs, particularly the liver. This was demonstrated by comparing Ga-68-TRAP-WL12 with the archetypical Ga-68-DOTA-WL12, for which tumor-to-liver ratios of 1.4 and 0.5, respectively, were found. Although immunohistochemistry (IHC) revealed a low PD-L1 expression in MDA-MB-213 and H2009 xenografts that corresponds well to the clinical situation, PET showed high tumor uptakes (6.6 and 7.3% injected activity per gram of tissue (iA/g), respectively) for Ga-68-TRAP-WL12. Thus, this tracer has the potential for routine clinical PD-L1 PET imaging because it detects even very low PD-L1 expression densities with high sensitivity and may open an avenue to replace PD-L1 IHC of biopsies as the standard means to select potential responders for ICT

Loss of Ifnar1 in Pancreatic Acinar Cells Ameliorates the Disease Course of Acute Pancreatitis

<div><p>Type I interferon constitutes an essential component of the combinational therapy against viral disease. Acute pancreatitis is one side effect of type I interferon-based therapy, implying that activation of type I interferon signaling affects the homeostasis and integrity of pancreatic acinar cells. Here, we investigated the role of type I interferon signaling in pancreatic acinar cells using a caerulein-induced murine model of acute pancreatitis. Pancreas-specific ablation of interferon (alpha and beta) receptor 1 (Ifnar1) partially protected animals from caerulein-induced pancreatitis, as demonstrated by reduced tissue damage. Profiling of infiltrating immune cells revealed that this dampened tissue damage response correlated with the number of macrophages in the pancreas. Pharmacologic depletion of macrophages reversed the protective effect of Ifnar1 deficiency. Furthermore, expression of chemokine (C-C motif) ligand 2 (Ccl2), a potent factor for macrophage recruitment, was significantly increased in the Ifnar1-deficient pancreas. Thus, type I interferon signaling in pancreatic acinar cells controls pancreatic homeostasis by affecting the macrophage-mediated inflammatory response in the pancreas.</p></div

Depletion of macrophages rescues the focally restricted inflammation phenotype in Ifnar^del mice.

<p>(A) Representative H&E staining of the pancreas from WT and Ifnar^del mice 48 hours after treatment. Mice were either treated with caerulein (regeneration model, left), caerulein plus clodronate filled liposomes (macrophage depletion, middle) or caerulein plus PBS filled liposomes (PBS control group, right) as described in the methods section (n = 3; original magnification, 100x). (B-D) Combined and individual scoring of pancreatic histological parameters from WT and Ifnar^del mice 24 and 48 hours, and 5 days following caerulein-induced injury (plain bars) or caerulein-induced injury combined with macrophage depletion (striped bars) (n = 3–9 per group. Bars indicate mean +/- SD. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, Mann-Whitney-test).</p

Release of the chemoattractant Ccl2/MCP1 in untreated Ifnar^del mice.

<p>(A) qRT PCR analysis of mRNA levels of the chemokines <i>Cxcl9</i>, <i>Cxcl10</i>, <i>Cxcl11</i>, <i>Ccl2</i>, <i>Ccl4</i>, <i>Ccl5</i> and <i>Ccl25</i> from whole pancreatic tissue of WT and Ifnar^del mice harvested 4 hours following caerulein-induced injury (n = 3 per group. Bars indicate mean +/- SD. Normalized on the mRNA of the housekeeping gene <i>Ppib</i>). (B) qRT PCR analysis of mRNA levels of the chemokines <i>Cxcl9</i>, <i>Cxcl10</i>, <i>Cxcl11</i>, <i>Ccl2</i>, <i>Ccl4</i>, <i>Ccl5</i> and <i>Ccl25</i> from whole pancreatic tissue of untreated WT and Ifnar^del mice (n = 7 per group. Bars indicate mean +/- SD. Normalized on the mRNA of the housekeeping gene <i>Ppib</i>. *P<0.05, unpaired Student´s t-test). (C) Immunohistochemical staining for MCP1-positive centro-acinar cells in the pancreas from WT and Ifnar^del mice 24 hours following caerulein-induced injury or untreated and counting of the absolute number of positive cells on five separate high power fields for each section of untreated WT and Ifnar^del mice and after 24 hours following caerulein-induced injury (n = 3–7 per group. Bars indicate mean +/- SD. ****P<0.0001, Mann-Whitney-test. Original magnification, 200x and 1000x).</p

Proinflammatory cytokine expression in WT and Ifnar^del mice.

<p>(A-B) qRT PCR analysis of mRNA levels of the proinflammatory cytokines <i>Ifnγ</i>, <i>Tnfα</i>, <i>IL-6</i>, <i>Lbp</i>, <i>G-Csf</i>, <i>Ifnα</i>, <i>IL-1α</i> and <i>IL-1β</i> (A) and the anti-inflammatory <i>cytokines IL-10</i>, <i>IL-13</i>, <i>Tgfβ1–3</i> and <i>Csf</i> (B) from whole pancreatic tissue of WT and Ifnar^del mice harvested 4 hours following caerulein-induced injury (n = 3 per group. Bars indicate mean +/- SD. Normalized on the mRNA of the housekeeping gene <i>Ppib</i>). (C-D) qRT PCR analysis of mRNA levels of the proinflammatory cytokines <i>Ifnγ</i>, <i>Tnfα</i>, <i>IL-6</i>, <i>Lbp</i>, <i>G-Csf</i>, <i>Ifnα</i>, <i>IL-1α</i> and <i>IL-1β</i> (C) and the anti- inflammatory cytokines <i>IL-10</i>, <i>IL-13</i>, <i>Tgfβ1–3</i> and <i>Csf</i> (D) from whole pancreatic tissue of untreated WT and Ifnar^del mice (n = 7 per group. Bars indicate mean +/- SD. Normalized on the mRNA of the housekeeping gene <i>Ppib</i>. *P<0.05, unpaired Student´s t-test).</p

Macrophages are the predominant immune cells infiltrating the pancreas following caerulein-induced injury in Ifnar^del mice.

<p>(A) The absolute number of CD45-positive immune cells, B220-positive B-lymphocytes, CD3-positive T-lymphocytes and MPO-positive neutrophils was counted in five separate high power fields for each section of untreated WT and Ifnar^del mice and 24, 48 hours, 3, 5, 7 and 14 days following caerulein-induced injury (n = 3–9 per group. Bars indicate mean +/- SD. *P<0.05, **P<0.01, ****P<0.0001, Student´s t-test). (B) Immunohistochemical staining for F4/80-positive macrophages in the pancreas from WT and Ifnar^del mice 24h following caerulein-induced injury and counting of the absolute number of positive cells in five separate high power fields for each section of untreated WT and Ifnar^del mice and 24, 48 hours, 3, 5, 7 and 14 days following caerulein-induced injury (n = 3–9 per group. Bars indicate mean +/- SD. *P<0.05, ****P<0.0001, Student´s t-test. Original magnification, 200x and 650x).</p

Characterization of untreated Ifnar^del mice reveals no differences to wild type mice.

<p>(A) qRT PCR analysis of mRNA levels of Ifnar1 from whole pancreatic tissue of untreated WT and Ifnar^del mice. (n = 3 per group. Bars indicate mean +/- SD. Normalized on the mRNA of the housekeeping gene <i>Ppib</i>. **P<0.01, Mann-Whitney-test). (B) Representative H&E staining of the pancreas from 8 week old WT and Ifnar^del mice without treatment (original magnification, 100x). (C) Pancreas weight/body weight ratio of 8 week old WT and Ifnar^del mice without treatment (n = 7 per group. Bars indicate mean +/- SD). (D) Amylase, lipase and LDH (lactat-dehydrogenase) levels in the serum from of 8 week old WT and Ifnar^del mice without treatment (n = 7 per group. Bars indicate mean +/- SD).</p

Pancreatic inflammation after caerulein- induced injury is limited in Ifnar^del mice.

<p>(A) Representative H&E staining of the pancreas from WT and Ifnar^del mice, 24 and 48 hours following caerulein-induced injury (original magnification, 100x). (B) Representative H&E staining of the pancreas from WT and Ifnar^del mice, 3, 7 and 14 days following caerulein-induced injury (original magnification, 100x). (C-E) Combined and individual scoring of pancreatic histological parameters from WT and Ifnar^del mice, 24, 48 hours, 3, 5, 7 and 14 days following caerulein-induced injury (n = 3–9 per group. Bars indicate mean +/- SD. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, Mann-Whitney-test).</p

Additional file 1 of PSMA-ligand uptake can serve as a novel biomarker in primary prostate cancer to predict outcome after radical prostatectomy

Additional file 1. Supplementary files. Supplementary table 1. Distribution of pT and miT. Supplementary table 2. Distribution of pN and miN. Supplementary table 3. Univariate analysis for the association of 68Ga-PSMA-11 PET findings with surgical margin status. Supplementary Fig. 1. Flowchart of inclusion and exclusion steps. Supplementary Fig. 2. Longer biochemical recurrence-free survival was associated with (A) pT = 2, (B) pN=0, (C) Gleason Score < 8 and (D) negative surgical margin

A machine learning algorithm predicts molecular subtypes in pancreatic ductal adenocarcinoma with differential response to gemcitabine-based versus FOLFIRINOX chemotherapy - Fig 1

Histopathological samples of two patients showing comparable tissue morphology in H&E staining (A,C) but a KRT81+ subtype (B) in one patient and KRT81- subtype (D) in the other patient.</p