Publications by authors named "M Widmaier"

9 Publications

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Comparison of continuous measures across diagnostic PD-L1 assays in non-small cell lung cancer using automated image analysis.

Mod Pathol 2020 03 16;33(3):380-390. Epub 2019 Sep 16.

MedImmune, Gaithersburg, MD, USA.

Tumor programmed cell death ligand-1 (PD-L1) expression is a key biomarker to identify patients with non-small cell lung cancer who may have an enhanced response to anti-programmed cell death-1 (PD-1)/PD-L1 treatment. Such treatments are used in conjunction with PD-L1 diagnostic immunohistochemistry assays. We developed a computer-aided automated image analysis with customized PD-L1 scoring algorithm that was evaluated via correlation with manual pathologist scores and used to determine comparability across PD-L1 immunohistochemistry assays. The image analysis scoring algorithm was developed to quantify the percentage of PD-L1 positive tumor cells on scans of whole-slide images of archival tumor samples from commercially available non-small cell lung cancer cases, stained with four immunohistochemistry PD-L1 assays (Ventana SP263 and SP142 and Dako 22C3 and 28-8). The scans were co-registered and tumor and exclusion annotations aligned to ensure that analysis of each case was restricted to comparable tissue areas. Reference pathologist scores were available from previous studies. F1, a statistical measure of precision and recall, and overall percentage agreement scores were used to assess concordance between pathologist and image analysis scores and between immunohistochemistry assays. In total, 471 PD-L1-evalulable samples were amenable to image analysis scoring. Image analysis and pathologist scores were highly concordant, with F1 scores ranging from 0.8 to 0.9 across varying matched PD-L1 cutoffs. Based on F1 and overall percentage agreement scores (both manual and image analysis scoring), the Ventana SP263 and Dako 28-8 and 22C3 assays were concordant across a broad range of cutoffs; however, the Ventana SP142 assay showed very different characteristics. In summary, a novel automated image analysis scoring algorithm was developed that was highly correlated with pathologist scores. The algorithm permitted quantitative comparison of existing PD-L1 diagnostic assays, confirming previous findings that indicate a high concordance between the Ventana SP263 and Dako 22C3 and 28-8 PD-L1 immunohistochemistry assays.
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http://dx.doi.org/10.1038/s41379-019-0349-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7051919PMC
March 2020

Automated image analysis of NSCLC biopsies to predict response to anti-PD-L1 therapy.

J Immunother Cancer 2019 05 6;7(1):121. Epub 2019 May 6.

AstraZeneca, Gaithersburg, MD, 20878, USA.

Background: Immune checkpoint therapies (ICTs) targeting the programmed cell death-1 (PD1)/programmed cell death ligand-1 (PD-L1) pathway have improved outcomes for patients with non-small cell lung cancer (NSCLC), particularly those with high PD-L1 expression. However, the predictive value of manual PD-L1 scoring is imperfect and alternative measures are needed. We report an automated image analysis solution to determine the predictive and prognostic values of the product of PD-L1+ cell and CD8+ tumor infiltrating lymphocyte (TIL) densities (CD8xPD-L1 signature) in baseline tumor biopsies.

Methods: Archival or fresh tumor biopsies were analyzed for PD-L1 and CD8 expression by immunohistochemistry. Samples were collected from 163 patients in Study 1108/NCT01693562, a Phase 1/2 trial to evaluate durvalumab across multiple tumor types, including NSCLC, and a separate cohort of 199 non-ICT- patients. Digital images were automatically scored for PD-L1+ and CD8+ cell densities using customized algorithms applied with Developer XD™ 2.7 software.

Results: For patients who received durvalumab, median overall survival (OS) was 21.0 months for CD8xPD-L1 signature-positive patients and 7.8 months for signature-negative patients (p = 0.00002). The CD8xPD-L1 signature provided greater stratification of OS than high densities of CD8+ cells, high densities of PD-L1+ cells, or manually assessed tumor cell PD-L1 expression ≥25%. The CD8xPD-L1 signature did not stratify OS in non-ICT patients, although a high density of CD8+ cells was associated with higher median OS (high: 67 months; low: 39.5 months, p = 0.0009) in this group.

Conclusions: An automated CD8xPD-L1 signature may help to identify NSCLC patients with improved response to durvalumab therapy. Our data also support the prognostic value of CD8+ TILS in NSCLC patients who do not receive ICT.

Trial Registration: ClinicalTrials.gov identifier: NCT01693562 . Study code: CD-ON-MEDI4736-1108. Interventional study (ongoing but not currently recruiting). Actual study start date: August 29, 2012. Primary completion date: June 23, 2017 (final data collection date for primary outcome measure).
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http://dx.doi.org/10.1186/s40425-019-0589-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6501300PMC
May 2019

Measuring multiple parameters of CD8+ tumor-infiltrating lymphocytes in human cancers by image analysis.

J Immunother Cancer 2018 03 6;6(1):20. Epub 2018 Mar 6.

Professional Services, Definiens AG, Bernhard-Wicki-Strasse 5, 80636, Munich, Germany.

Background: Immuno-oncology and cancer immunotherapies are areas of intense research. The numbers and locations of CD8+ tumor-infiltrating lymphocytes (TILs) are important measures of the immune response to cancer with prognostic, pharmacodynamic, and predictive potential. We describe the development, validation, and application of advanced image analysis methods to characterize multiple immunohistochemistry-derived CD8 parameters in clinical and nonclinical tumor tissues.

Methods: Commercial resection tumors from nine cancer types, and paired screening/on-drug biopsies of non-small-cell lung carcinoma (NSCLC) patients enrolled in a phase 1/2 clinical trial investigating the PD-L1 antibody therapy durvalumab (NCT01693562), were immunostained for CD8. Additional NCT01693562 samples were immunostained with a CD8/PD-L1 dual immunohistochemistry assay. Whole-slide scanning was performed, tumor regions were annotated by a pathologist, and images were analyzed with customized algorithms using Definiens Developer XD software. Validation of image analysis data used cell-by-cell comparison to pathologist scoring across a range of CD8+ TIL densities of all nine cancers, relying primarily on 95% confidence in having at least moderate agreement regarding Lin concordance correlation coefficient (CCC = 0.88-0.99, CCC_lower = 0.65-0.96).

Results: We found substantial variability in CD8+ TILs between individual patients and across the nine types of human cancer. Diffuse large B-cell lymphoma had several-fold more CD8+ TILs than some other cancers. TIL densities were significantly higher in the invasive margin versus tumor center for carcinomas of head and neck, kidney and pancreas, and NSCLC; the reverse was true only for prostate cancer. In paired patient biopsies, there were significantly increased CD8+ TILs 6 weeks after onset of durvalumab therapy (mean of 365 cells/mm over baseline; P = 0.009), consistent with immune activation. Image analysis accurately enumerated CD8+ TILs in PD-L1+ regions of lung tumors using the dual assay and also measured elongate CD8+ lymphocytes which constituted a fraction of overall TILs.

Conclusions: Validated image analysis accurately enumerates CD8+ TILs, permitting comparisons of CD8 parameters among tumor regions, individual patients, and cancer types. It also enables the more complex digital solutions needed to better understand cancer immunity, like analysis of multiplex immunohistochemistry and spatial evaluation of the various components comprising the tumor microenvironment.

Trial Registration: ClinicalTrials.gov identifier: NCT01693562 . Study code: CD-ON-MEDI4736-1108. Interventional study (ongoing but not currently recruiting). Actual study start date: August 29, 2012. Primary completion date: June 23, 2017 (final data collection date for primary outcome measure).
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http://dx.doi.org/10.1186/s40425-018-0326-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5839005PMC
March 2018

Kindlin-2 cooperates with talin to activate integrins and induces cell spreading by directly binding paxillin.

Elife 2016 Jan 27;5:e10130. Epub 2016 Jan 27.

Department of Molecular Medicine, Max Planck Institute of Biochemistry, Martinsried, Germany.

Integrins require an activation step prior to ligand binding and signaling. How talin and kindlin contribute to these events in non-hematopoietic cells is poorly understood. Here we report that fibroblasts lacking either talin or kindlin failed to activate β1 integrins, adhere to fibronectin (FN) or maintain their integrins in a high affinity conformation induced by Mn(2+). Despite compromised integrin activation and adhesion, Mn(2+) enabled talin- but not kindlin-deficient cells to initiate spreading on FN. This isotropic spreading was induced by the ability of kindlin to directly bind paxillin, which in turn bound focal adhesion kinase (FAK) resulting in FAK activation and the formation of lamellipodia. Our findings show that talin and kindlin cooperatively activate integrins leading to FN binding and adhesion, and that kindlin subsequently assembles an essential signaling node at newly formed adhesion sites in a talin-independent manner.
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http://dx.doi.org/10.7554/eLife.10130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4749545PMC
January 2016

Kindlin-1 controls Wnt and TGF-β availability to regulate cutaneous stem cell proliferation.

Nat Med 2014 Apr 30;20(4):350-9. Epub 2014 Mar 30.

Department of Molecular Medicine, Max Planck Institute of Biochemistry, Martinsried, Germany.

Kindlin-1 is an integrin tail binding protein that controls integrin activation. Mutations in the FERMT-1 gene, which encodes for Kindlin-1, lead to Kindler syndrome in man, which is characterized by skin blistering, premature skin aging and skin cancer of unknown etiology. Here we show that loss of Kindlin-1 in mouse keratinocytes recapitulates Kindler syndrome and also produces enlarged and hyperactive stem cell compartments, which lead to hyperthickened epidermis, ectopic hair follicle development and increased skin tumor susceptibility. Mechanistically, Kindlin-1 controls keratinocyte adhesion through β1-class integrins and proliferation and differentiation of cutaneous epithelial stem cells by promoting α(v)β(6) integrin-mediated transforming growth factor-β (TGF-β) activation and inhibiting Wnt-β-catenin signaling through integrin-independent regulation of Wnt ligand expression. Our findings assign Kindlin-1 the previously unknown and essential task of controlling cutaneous epithelial stem cell homeostasis by balancing TGF-β-mediated growth-inhibitory signals and Wnt-β-catenin-mediated growth-promoting signals.
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http://dx.doi.org/10.1038/nm.3490DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3982140PMC
April 2014

Integrin-linked kinase at a glance.

J Cell Sci 2012 Apr;125(Pt 8):1839-43

Max Planck Institute of Biochemistry, Department of Molecular Medicine, Martinsried, Germany.

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http://dx.doi.org/10.1242/jcs.093864DOI Listing
April 2012

Sorting nexin 17 prevents lysosomal degradation of β1 integrins by binding to the β1-integrin tail.

Nat Cell Biol 2012 May 6;14(6):584-92. Epub 2012 May 6.

Department of Molecular Medicine, Max Planck Institute for Biochemistry, 82152 Martinsried, Germany.

Integrin functions are controlled by regulating their affinity for ligand, and by the efficient recycling of intact integrins through endosomes. Here we demonstrate that the Kindlin-binding site in the β1-integrin cytoplasmic domain serves as a molecular switch enabling the sequential binding of two FERM-domain-containing proteins in different cellular compartments. When β1 integrins are at the plasma membrane, Kindlins control ligand-binding affinity. However, when they are internalized, Kindlins dissociate from integrins and sorting nexin 17 (SNX17) is recruited to free β1-integrin tails in early endosomes to prevent β1-integrin degradation, leading to their recycling back to the cell surface. Our results identify SNX17 as a β1-integrin-tail-binding protein that interacts with the free Kindlin-binding site in endosomes to stabilize β1 integrins, resulting in their recycling to the cell surface where they can be reused.
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http://dx.doi.org/10.1038/ncb2501DOI Listing
May 2012

Adenovirus-based virotherapy enabled by cellular YB-1 expression in vitro and in vivo.

Cancer Gene Ther 2009 Oct 10;16(10):753-63. Epub 2009 Apr 10.

Institute of Experimental Oncology and Therapeutics, Klinikum Rechts der Isar, Technische Universitaet Muenchen, Muenchen 81675, Germany.

We have earlier described the oncolytic adenovirus vector dl520 that was rendered cancer-specific by deletion of the transactivation domain CR3 of the adenoviral E1A13S protein; this deletion causes antitumor activity in drug-resistant cells displaying nuclear YB-1 expression. We hypothesized that the anticancer activity of dl520 could be further improved by introducing the RGD motif in the fiber knob and by deletion of the adenoviral E1B19K protein (Ad-Delo3-RGD). In this study, the in vitro and in vivo antitumor activity of Ad-Delo3-RGD was investigated focussing on two pancreatic cancer cell lines MiaPaCa-2 and BxPC3 alone and in combination with cytotoxic drugs. Furthermore, luciferin-based bioluminescence imaging was established to study the therapeutic response in vivo. In addition, to confirm the specificity of Ad-Delo3-RGD for YB-1 a tetracycline-inducible anti-YB-1 shRNA-expressing cell variant EPG85-257RDB/tetR/YB-1 was used. This TetON regulatable expression system allows us to measure adenoviral replication by real-time PCR in the absence of YB-1 expression. The results confirmed the YB-1 dependency of Ad-Delo3-RGD and showed that Ad-Delo3-RGD has potent activity against human pancreatic cancer cells in vitro and in vivo, which was augmented by the addition of paclitaxel. However, although high replication capacity was measured in vitro and in vivo, complete tumor regression was not achieved, indicating the need for further improvements to treat pancreatic cancer effectively.
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http://dx.doi.org/10.1038/cgt.2009.20DOI Listing
October 2009

Loss of Kindlin-1 causes skin atrophy and lethal neonatal intestinal epithelial dysfunction.

PLoS Genet 2008 Dec 5;4(12):e1000289. Epub 2008 Dec 5.

Department of Molecular Medicine, Max-Planck Institute of Biochemistry, Martinsried, Germany.

Kindler Syndrome (KS), characterized by transient skin blistering followed by abnormal pigmentation, skin atrophy, and skin cancer, is caused by mutations in the FERMT1 gene. Although a few KS patients have been reported to also develop ulcerative colitis (UC), a causal link to the FERMT1 gene mutation is unknown. The FERMT1 gene product belongs to a family of focal adhesion proteins (Kindlin-1, -2, -3) that bind several beta integrin cytoplasmic domains. Here, we show that deleting Kindlin-1 in mice gives rise to skin atrophy and an intestinal epithelial dysfunction with similarities to human UC. This intestinal dysfunction results in perinatal lethality and is triggered by defective intestinal epithelial cell integrin activation, leading to detachment of this barrier followed by a destructive inflammatory response.
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http://dx.doi.org/10.1371/journal.pgen.1000289DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2585060PMC
December 2008