BACKGROUND:Cancer immune evasion is critical in non-small cell lung cancer (NSCLC) and has been targeted by immunotherapy. High soluble (s)PD-L1 is associated with reduced survival and treatment failure in advanced stages. Here we evaluated the effects of sPD-L1 on T cells, relapse free survival, and overall survival in early stage NSCLC. METHODS:In vitro T cell stimulation was performed in the presence of sPD-L1 to evaluate its immunomodulatory activity. Data from The Cancer Genome Atlas (TCGA) were investigated for PD-L1 splice variants and enzymes involved in proteolytic cleavage (i.e. ADAM10). Plasma from 74 NSCLC (stage IA-IIIB), as well as an additional 73 (control cohort) patients was collected prior to curative surgery. Thereafter sPD-L1 levels from an immunosorbent assay were correlated with patient outcome. RESULTS:In vitro sPD-L1 inhibited IFN-γ production and proliferation of T cells and induced a terminal effector CD4 T cell subtype expressing CD27. Data from the TCGA demonstrated that elevated mRNA levels of ADAM10 is a negative predictor of outcome in NSCLC patients. To investigate the clinical relevance of these in vitro and TCGA findings, we quantified sPD-L1 in the plasma of early-stage NSCLC patients. In the first cohort we found significantly higher sPD-L1 levels in relapsing NSCLC patients, with a multivariate analysis revealing high sPD-L1 (>1000 pg/mL) as an independent predictor of survival. However, these findings could not be validated in two independent control cohorts. DISCUSSION:Although in vitro and TCGA data support the suppressive effect of sPD-L1 we were unable to translate this in our clinical setting. These results may be due to the small patient number and their heterogeneity as well as the lack of a standardized sPD-L1 ELISA. Our inconclusive results regarding the value of sPD-L1 in early stage NSCLC warrant assay validation and further investigation in larger (neo-)adjuvant trials.

Cystic echinococcosis (CE) is a global health concern caused by cestodes, posing diagnostic challenges due to nonspecific symptoms and inconclusive radiographic results. Diagnosis relies on histopathological evaluation of affected tissue, demanding comprehensive tools. In this retrospective case study, Fourier transform infrared microscopy was explored for detecting and identifying CE through biochemical changes in human tissue sections. Tissue samples from 11 confirmed CE patients were analyzed. Archived FFPE blocks were cut and stained, and then CE-positive unstained sections were examined using Fourier transform infrared microscopy post-deparaffinization. Results revealed the method's ability to distinguish echinococcus elements from human tissue, irrespective of organ type. This research showcases the potential of mid-infrared microscopy as a valuable diagnostic tool for CE, offering promise in enhancing diagnostic precision in the face of the disease's complexities.

Introduction: We analyzed the expression of PD-L1 in human lymphomas using hyperspectral imaging (HSI) compared to visual assessment (VA) and conventional digital image analysis (DIA) to strengthen further the value of HSI as a tool for the evaluation of brightfield-based immunohistochemistry (IHC). In addition, fluorescent multiplex immunohistochemistry (mIHC) was used as a second detection method to analyze the impact of a different detection method. Material and methods: 18 cases (6 follicular lymphomas and 12 diffuse large B-cell lymphomas) were stained for PD-L1 by IHC and for PD-L1, CD3, and CD8 by fluorescent mIHC. The percentage of positively stained cells was evaluated with VA, HSI, and DIA for IHC and VA and DIA for mIHC. Results were compared between the different methods of detection and analysis. Results: An overall high concordance was found between VA, HSI, and DIA in IHC (Cohens Kappa = 0.810(VA)(/)(HSI), 0.710 (VA/DIA), and 0.516 (HSI)(/)(DIA)) and for VA(mIHC) versus DIA(mIHC) (Cohens Kappa = 0.894). Comparing IHC and mIHC general agreement differed depending on the methods compared but reached at most a moderate agreement (Cohens Kappa between 0.250 and 0.483). This is reflected by the significantly higher percentage of PD-L1+ cells found with mIHC (p(Friedman) = 0.014). Conclusion: Our study shows a good concordance for the different analysis methods. Compared to VA and DIA, HSI proved to be a reliable tool for assessing IHC. Understanding the regulation of PD-L1 expression will further enlighten the role of PD-L1 as a biomarker. Therefore it is necessary to develop an instrument, such as HSI, which can offer a reliable and objective evaluation of PD-L1 expression.

The present study presents an alternative analytical workflow that combines mid-infrared (MIR) microscopic imaging and deep learning to diagnose human lymphoma and differentiate between small and large cell lymphoma. We could show that using a deep learning approach to analyze MIR hyperspectral data obtained from benign and malignant lymph node pathology results in high accuracy for correct classification, learning the distinct region of 3900 cm-1 to 850 cm-1. The accuracy is above 95% for every pair of malignant lymphoid tissue and still above 90% for the distinction between benign and malignant lymphoid tissue for binary classification. These results demonstrate that a preliminary diagnosis and subtyping of human lymphoma could be streamlined by applying a deep learning approach to analyze MIR spectroscopic data.

IntroductionTo implement Hyperspectral Imaging (HSI) as a tool for quantifying inflammatory cells in tissue specimens by the example of myocarditis in a collective of forensic patients.Material and methods44 consecutive patients with suspected myocardial inflammation at autopsy, diagnosed between 2013 and 2018 at the Institute of ForensicMedicine, Medical University of Innsbruck, were selected for this study. Using the IMEC SNAPSCAN camera, visible and near infrared hyperspectral images were collected from slides stained with CD3 and CD45 to assess quantity and spatial distribution of positive cells. Results were compared with visual assessment (VA) and conventional digital image analysis (DIA).ResultsFinally, specimens of 40 patients were evaluated, of whom 36 patients (90%) suffered from myocarditis, two patients (5%) had suspected healing/healed myocarditis, and two did no have myocarditis (5%). The amount of CD3 and CD45 positive cells did not differ significantly between VA, HSI, and DIA (pVA/HSI/DIA = 0.46 for CD3 and 0.81 for CD45). Coheńs Kappa showed a very high correlation between VA versus HSI, VA versus DIA, and HSI versus DIA for CD3 (Coheńs Kappa = 0.91, 1.00, and 0.91, respectively). For CD45 an almost as high correlation was seen for VA versus HSI and HSI versus DIA (Coheńs Kappa = 0.75 and 0.70) and VA versus DIA (Coheńs Kappa = 0.89).ConclusionHSI is a reliable and objective method to count inflammatory cells in tissue slides of suspected myocarditis. Implementation of HSI in digital pathology might further expand the possibility of a sophisticated method.

Information, archives, and intelligent artificial systems are part of everyday life in modern medicine. They already support medical staff by mapping their workflows with shared availability of cases’ referral information, as needed for example, by the pathologist, and this support will be increased in the future even more. In radiology, established standards define information models, data transmission mechanisms, and workflows. Other disciplines, such as pathology, cardiology, and radiation therapy, now define further demands in addition to these established standards. Pathology may have the highest technical demands on the systems, with very complex workflows, and the digitization of slides generating enormous amounts of data up to Gigabytes per biopsy. This requires enormous amounts of data to be generated per biopsy, up to the gigabyte range. Digital pathology allows a change from classical histopathological diagnosis with microscopes and glass slides to virtual microscopy on the computer, with multiple tools using artificial intelligence and machine learning to support pathologists in their future work.

ZusammenfassungDie Möglichkeit einer Tumorerkrankung auf Basis eines familiären Tumorprädispositionssyndroms muss bei jeder Krebsdiagnose in Betracht gezogen werden. Die Erfassung erkrankter „Index“-PatientInnen ist entscheidend für die Ermittlung des Risikos für Neu- oder Wiedererkrankungen bei den Betroffenen wie auch für das Auftreten von Tumoren bei bisher gesunden Verwandten. Die Erfassung von PatientInnen mit familiärer Tumorprädisposition erlaubt es, Betroffene in Vorsorgeprogramme zur Senkung von Morbidität und Letalität aufzunehmen. Für das erbliche Brust- und Eierstockkrebssyndrom besteht in Österreich ein breites Bewusstsein. Dadurch wird eine zufriedenstellende Erfassung der PatientInnen erreicht. Das ist für das Lynch-Syndrom, welches bei 2–3 % aller Kolorektal- und Endometriumkarzinome vorliegt, leider nicht der Fall. Um die Identifizierung von Lynch-Syndrom-PatientInnen zu verbessern, empfiehlt die Österreichische Arbeitsgemeinschaft Pathologie-Humangenetik (die Österreichische Arbeitsgemeinschaft Pathologie-Humangenetik setzt sich aus jeweils fünf Delegierten der Österreichischen Gesellschaft für Klinische Pathologie und Molekularpathologie und der Österreichischen Gesellschaft für Humangenetik zusammen) in diesem Konsensus-Statement eine diagnostische Strategie, die möglichst alle Lynch-Syndrom-assoziierten Kolorektal- und Endometriumkarzinome im Rahmen der pathologischen Tumorbeurteilung erfasst. Durch eine darauf basierende systematische Zuweisung von PatientInnen mit Verdacht auf Lynch-Syndrom an ein Zentrum für medizinische Genetik zur genetischen Beratung und weiterführenden genetischen Diagnostik wird sichergestellt, dass auch Familienangehörige mit Lynch-Syndrom erfasst werden.

A familial tumor predisposition syndrome must be considered a possibility in every cancer diagnosis. The detection of affected "index" patients is crucial for determining the risk of new or recurrent disease in the affected individuals as well as for the occurrence of tumors in previously healthy relatives. The registration of patients with familial tumor predisposition makes it possible to include them in screening programs to reduce morbidity and mortality. In Austria, there is already broad public awareness of hereditary breast and ovarian cancer syndromes. Thus, satisfactory detection of patients is achieved. Unfortunately, this is not the case for Lynch syndrome, which is present in 2-3% of all colorectal and endometrial cancers. To improve the identification of patients with Lynch syndrome, the Austrian Working Group Pathology-Human Genetics (the Austrian Working Group Pathology-Human Genetics comprises five delegates each from the Austrian Society of Clinical Pathology and Molecular Pathology and the Austrian Society of Human Genetics) recommends in this consensus statement a diagnostic strategy that covers as far as possible all colorectal and endometrial cancers associated with Lynch syndrome as part of the pathological tumor assessment. Systematically referring patients with suspected Lynch syndrome to medical genetics centers for genetic counselling and further genetic diagnostic workup ensures that family members with Lynch syndrome are also identified.

Our research group has recently shown that Borrelia burgdorferi, the Lyme disease bacterium, is capable of forming biofilms in Borrelia-infected human skin lesions called Borrelia lymphocytoma (BL). Biofilm structures often contain multiple organisms in a symbiotic relationship, with the goal of providing shelter from environmental stressors such as antimicrobial agents. Because multiple co-infections are common in Lyme disease, the main questions of this study were whether BL tissues contained other pathogenic species and/or whether there is any co-existence with Borrelia biofilms. Recent reports suggested Chlamydia-like organisms in ticks and Borrelia-infected human skin tissues; therefore, Chlamydia-specific polymerase chain reaction (PCR) analyses were performed in Borrelia-positive BL tissues. Analyses of the sequence of the positive PCR bands revealed that Chlamydia spp. DNAs are indeed present in these tissues, and their sequences have the best identity match to Chlamydophila pneumoniae and Chlamydia trachomatis. Fluorescent immunohistochemical and in situ hybridization methods demonstrated the presence of Chlamydia antigen and DNA in 84% of Borrelia biofilms. Confocal microscopy revealed that Chlamydia locates in the center of Borrelia biofilms, and together, they form a well-organized mixed pathogenic structure. In summary, our study is the first to show Borrelia-Chlamydia mixed biofilms in infected human skin tissues, which raises the questions of whether these human pathogens have developed a symbiotic relationship for their mutual survival.

Rational: Within the evolving area of immune checkpoint inhibitory therapies predictive biomarkers are still rare and intricate in clinical routine, even though they are highly warranted to improve patient outcome and cost effectiveness.

New developments in Mid-infrared microscopic imaging instrumentation and data analysis have turned this method into a conventional technique. This imaging method offers a global analysis of samples, with a resolution close to the cellular level enabling the acquisition of local molecular expression profiles. It is possible to get chemo-morphological information about the tissue status, which represents an essential benefit for future analytical interpretation of pathological changes of tissue. In this review, we give an overview of Mid-infrared microscopic imaging and its applications in clinical research.

### A1 Intratumoral immunotherapy. B16-F10 murine melanoma model #### J. Ženka1, V. Caisova1, O. Uher1, P. Nedbalova1, K. Kvardova1, K. Masakova1, G. Krejcova1, L. Paďoukova1, I. Jochmanova2, K. I. Wolf3, J. Chmelař1, J. Kopecký1 ##### 1Department of Medical Biology, Faculty of

Belatacept (cytotoxic T-lymphocyte-associated protein 4 Ig) is an emerging treatment in kidney transplantation. Lack of nephrotoxicity and possibly an inhibitory effect on the development of donor-specific antibodies (DSAs) make it an interesting agent in hand transplantation. To reduce calcineurin inhibitor immunosuppression and preserve kidney function, we have added belatacept to the therapeutic regimen of 4 hand-transplanted patients at month 4 and at 6, 9, and 13 years after hand-forearm transplantation. Patients received 5 mg/kg belatacept every 2 weeks, and the dosing interval was extended to 4 weeks after 5 applications. Belatacept was initially well tolerated in all cases. Two patients were weaned to a low-dose tacrolimus monotherapy together with monthly belatacept applications. One patient is taking belatacept with lowered tacrolimus and sirolimus trough levels. A fourth patient had significant levels of DSAs at time of conversion and progressed to a severe necrotizing rejection early despite an unaltered baseline immunosuppression. Finger skin necrosis and histologic signs of severe chronic allograft vasculopathy eventually led to amputation of the graft. Implementation of belatacept can be beneficial in hand transplantation. However, our findings indicated both potential and caution and reflection of the immunologic state at the time of conversion.

Wir berichten über eine 21-jährige Patientin mit seit 3 Jahren bestehenden Krusten und Erosionen an der Kopfhaut, die nach Therapiebeginn mit Adalimumab wegen Morbus Crohn auftraten. Mittels klinikopathologischer Korrelation wurde die Diagnose Pityriasis amiantacea bei Folliculitis decalvans gestellt. Topische und antibiotische systemische Therapie zeigten ein rasches Ansprechen. Das Auftreten von Pityriasis amiantacea und Folliculitis decalvans bei Anti-TNF (Tumor-Nekrose-Faktor)-α-Therapie ist bemerkenswert und unterstreicht die ambivalente Rolle von TNF-α bei Erkrankungen mit immunologischen Fehlfunktionen und Infektionen.

MIR microscopic imaging with subsequent PCA clustering is a very promising tool for the detection and identification of mucormycetes in pathology routine laboratories.

### A1 Intratumoral immunotherapy. B16-F10 murine melanoma model #### J. Ženka1, V. Caisová1, O. Uher1, P. Nedbalová1, K. Kvardová1, K. Masáková1, G. Krejčová1, L. Paďouková1, I. Jochmanová2, K. I. Wolf3, J. Chmelař1, J. Kopecký1 ##### 1Department of Medical Biology, Faculty of