The Concept of Stroma AReactive Invasion Front Areas (SARIFA) as a new prognostic biomarker for lipid-driven cancers holds true in pancreatic ductal adenocarcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a 'difficult-to-treat' entity. To forecast its prognosis, we introduced a new biomarker, SARIFA (stroma areactive invasion front areas), which are areas at the tumour invasion front lacking desmoplastic stroma reaction upon malignant invasion in the surrounding tissue, leading to direct contact between tumour cells and adipocytes. SARIFA showed its significance in gastric and colorectal carcinoma, revealing lipid metabolism alternations that promote tumour progression. METHODS: We reviewed the SARIFA status of 166 PDAC cases on all available H&E-stained tumour slides from archival Whipple-resection specimens. SARIFA positivity was defined as SARIFA detection in at least 66% of the available slides. To investigate alterations in tumour metabolism and microenvironment, we performed immunohistochemical staining for FABP4, CD36 and CD68. To verify and quantify a supposed delipidation of adipocytes, adipose tissue was digitally morphometrised. RESULTS: In total, 53 cases (32%) were classified as SARIFA positive and 113 (68%) as SARIFA negative. Patients with SARIFA-positive PDAC showed a significantly worse overall survival compared with SARIFA-negative cases (median overall survival: 11.0 months vs. 22.0 months, HR: 1.570 (1.082-2.278), 95% CI, p = 0.018), which was independent from other prognostic markers (p = 0.014). At the invasion front of SARIFA-positive PDAC, we observed significantly higher expression of FABP4 (p < 0.0001) and higher concentrations of CD68+ macrophages (p = 0.031) related to a higher risk of tumour progression. CD36 staining showed no significant expression differences. The adipocyte areas at the invasion front were significantly smaller, with mean values of 4021 ± 1058 µm2 and 1812 ± 1008 µm2 for the SARIFA-negative and -positive cases, respectively (p < 0.001). CONCLUSIONS: SARIFA is a promising prognostic biomarker for PDAC. Its assessment is characterised by simplicity and low effort. The mechanisms behind SARIFA suggest a tumour-promoting increased lipid metabolism and altered immune background, both showing new therapeutic avenues.

SARIFA as a new histopathological biomarker is associated with adverse clinicopathological characteristics, tumor-promoting fatty-acid metabolism, and might predict a metastatic pattern in pT3a prostate cancer.

BACKGROUND: Recently, we introduced Stroma-AReactive-Invasion-Front-Areas (SARIFA) as a novel hematoxylin-eosin (H&E)-based histopathologic prognostic biomarker for various gastrointestinal cancers, closely related to lipid metabolism. To date, no studies on SARIFA, which is defined as direct tumor-adipocyte-interaction, beyond the alimentary tract exist. Hence, the objective of our current investigation was to study the significance of SARIFA in pT3a prostate cancer (PCa) and explore its association with lipid metabolism in PCa as lipid metabolism plays a key role in PCa development and progression. METHODS: To this end, we evaluated SARIFA-status in 301 radical prostatectomy specimens and examined the relationship between SARIFA-status, clinicopathological characteristics, overall survival, and immunohistochemical expression of FABP4 and CD36 (proteins closely involved in fatty-acid metabolism). Additionally, we investigated the correlation between SARIFA and biochemical recurrence-free survival (BRFS) and PSMA-positive recurrences in PET/CT imaging in a patient subgroup. Moreover, a quantitative SARIFA cut-off was established to further understand the underlying tumor biology. RESULTS: SARIFA positivity occurred in 59.1% (n = 178) of pT3a PCas. Our analysis demonstrated that SARIFA positivity is strongly associated with established high-risk features, such as R1 status, extraprostatic extension, and higher initial PSA values. Additionally, we observed an upregulation of immunohistochemical CD36 expression specifically at SARIFAs (p = 0.00014). Kaplan-Meier analyses revealed a trend toward poorer outcomes, particularly in terms of BRFS (p = 0.1). More extensive tumor-adipocyte interaction, assessed as quantity-dependent SARIFA-status on H&E slides, is also significantly associated with high-risk features, such as lymph node metastasis, and seems to be associated with worse survival outcomes (p = 0.16). Moreover, SARIFA positivity appeared to be linked to more distant lymph node and bone metastasis, although statistical significance was slightly not achieved (both p > 0.05). CONCLUSIONS: This is the first study to introduce SARIFA as easy-and-fast-to-assess H&E-based biomarker in locally advanced PCa. SARIFA as the histopathologic correlate of a distinct tumor biology, closely related to lipid metabolism, could pave the way to a more detailed patient stratification and to the development of novel drugs targeting lipid metabolism in pT3a PCa. On the basis of this biomarker discovery study, further research efforts on the prognostic and predictive role of SARIFA in PCa can be designed.

Alterations in Peripheral Lymphocyte Subsets under Immunochemotherapy in Stage IV SCLC Patients: Th17 Cells as Potential Early Predictive Biomarker for Response.

UICC stage IV small-cell lung cancer (SCLC) is a highly aggressive malignancy without curative treatment options. Several randomized trials have demonstrated improved survival rates through the addition of checkpoint inhibitors to first-line platin-based chemotherapy. Consequently, a combination of chemo- and immunotherapy has become standard palliative treatment. However, no reliable predictive biomarkers for treatment response exist. Neither PD-L1 expression nor tumor mutational burden have proven to be effective predictive biomarkers. In this study, we compared the cellular immune statuses of SCLC patients to a healthy control cohort and investigated changes in peripheral blood B, T, and NK lymphocytes, as well as several of their respective subsets, during treatment with immunochemotherapy (ICT) using flow cytometry. Our findings revealed a significant decrease in B cells, while T cells showed a trend to increase throughout ICT. Notably, high levels of exhausted CD4+ and CD8+ cells, alongside NK subsets, increased significantly during treatment. Furthermore, we correlated decreases/increases in subsets after two cycles of ICT with survival. Specifically, a decrease in Th17 cells indicated a better overall survival. Based on these findings, we suggest conducting further investigation into Th17 cells as a potential early predictive biomarkers for response in patients receiving palliative ICT for stage IV SCLC.

Alterations of circulating lymphocyte subsets in patients with colorectal carcinoma.

INTRODUCTION: Cellular immune response to cancer is known to be of great importance for tumor control. Moreover, solid tumors influence circulating lymphocytes, which has been shown for several types of cancer. In our prospective study we elucidate changes in lymphocyte subsets in patients with colorectal carcinoma compared to healthy volunteers. METHODS: Flow cytometry was performed at diagnosis of colon carcinoma to analyze B cells, T cells and NK cells including various subtypes of each group. Univariate and multivariate analyses including age, gender, tumor stage, sidedness and microsatellite instability status (MSI) were performed. RESULTS: Forty-seven patients and 50 healthy volunteers were included. Median age was 65 years in patients and 43 years in the control group. Univariate analysis revealed lower total lymphocyte counts, lower CD4 + cells, CD8 + cells, B cells and NKs including various of their subsets in patients. In multivariate analysis patients had inferior values of B cells, CD4 + cells and NK cells and various subsets, regardless of age and gender. Naïve, central memory and HLADR + CD8 + cells showed an increase in patients whereas all other altered subsets declined. MSI status had no influence on circulating lymphocytes except for higher effector memory CD8 + cells in MSI-high patients. Localization in the left hemicolon led to higher values of total cytotoxic T cells and various T cell subsets. CONCLUSION: We found significant changes in circulating lymphocyte subsets in colon carcinoma patients, independent of physiological alterations due to gender or age. For some lymphocyte subsets significant differences according to tumor localization or MSI-status could be seen.

Chemotherapy markedly reduces B cells but not T cells and NK cells in patients with cancer.

Chemotherapy is still the backbone of systemic treatment in the majority of cancers. However, immunotherapies, especially those based on checkpoint inhibition, are additional therapy options for many. For this, functional T cells are a mandatory requirement. The aim of this prospective study was to investigate the influence of chemotherapy on the cellular immune status of individual patients. Peripheral blood samples of 26 patients with solid malignancies undergoing chemotherapy were analyzed for lymphocyte populations and their subsets in a longitudinal approach. Chemotherapy decreased total B lymphocyte counts [median value (25-75 percentile): before chemotherapy 76/µl (39-160) vs. after chemotherapy 49/µl (24-106); p = 0.001]. Among B cells, specific subsets decreased particularly [naïve B cells (49/µl (21-111) vs. 25/µl (13-56); p = 0.001], memory B cells [3/µl (2-8) vs. 2/µl (1-4); p = 0.001], and class-switched B cells [11/µl (6-20) vs. 6/µl (3-12); p = 0.011]. In contrast, chemotherapy had no influence on the total numbers of CD4 + and CD8 + T lymphocytes or on their subsets (T helper cells 1, 2, and 17 as well as cytotoxic T cells in early, intermediate, late, terminal effector and exhausted status as well as both T-cell types with naïve, center memory, effector memory, activated, or regulatory phenotype). Furthermore, the count of natural killer (NK) lymphocytes showed no significant change before and after chemotherapy. In summary, this study shows a decrease of B lymphocytes during systemic chemotherapy, but no relevant effect on T lymphocytes, NK lymphocytes and their subsets. This could support the idea of an effective additive T-cell-dependent immunotherapy to chemotherapy.

All-Body-Cavity (ABC)-scopy-An approach for a feasible method of minimally invasive autopsy to allow for postmortem tissue sampling in cases where a conventional autopsy is denied.

OBJECTIVES: The decreasing autopsy numbers in many western countries have been partially attributed to the invasiveness of the autopsy, which causes relatives to decline postmortem examination. This issue has been addressed by developing methods of minimally or non-invasive autopsy, which could be shown to increase acceptance for autopsies. The aim of this study is to compare the All-Body-Cavity-scopy (ABC-scopy) to conventional autopsies for diagnostic accuracy. METHODS: The ABC-scopy is an endoscopic approach for minimally invasive autopsy involving laparoscopic and thoracoscopic evaluation of the accessible organs, followed by excision biopsies of relevant organs and conspicuous findings. The method was performed in 10 cases on deceased patients scheduled for autopsy, each followed by a conventional autopsy. RESULTS: The results gathered from ABC-scopy through observation and histopathological evaluation provided an acceptable diagnostic accuracy in 9 out of 10 autopsies when compared to those of the conventional autopsy for diagnostic findings. CONCLUSIONS: The ABC-scopy is a feasible approach for minimally invasive autopsy that provides acceptable diagnostic value. Despite its minimally invasive nature, the procedure enables representative histology through providing large size excision biopsies from intraabdominal and thoracic organs, which is especially useful for examining disseminated diseases such as metastasized tumors.

Alterations in Natural Killer Cells in Colorectal Cancer Patients with Stroma AReactive Invasion Front Areas (SARIFA).

BACKGROUND: Recently, our group introduced Stroma AReactive Invasion Front Areas (SARIFA) as an independent prognostic predictor for a poorer outcome in colon cancer patients, which is probably based on immunologic alterations combined with a direct tumor-adipocyte interaction: the two together reflecting a distinct tumor biology. Considering it is already known that peripheral immune cells are altered in colorectal cancer (CRC) patients, this study aims to investigate the changes in lymphocyte subsets in SARIFA-positive cases and correlate these changes with the local immune response. METHODS: Flow cytometry was performed to analyze B, T, and natural killer (NK) cells in the peripheral blood (PB) of 45 CRC patients. Consecutively, lymphocytes in PB, tumor-infiltrating lymphocytes (TILs), and CD56+ and CD57+ lymphocytes at the invasion front and the tumor center were compared between patients with SARIFA-positive and SARIFA-negative CRCs. RESULTS: Whereas no differences could be observed regarding most PB lymphocyte populations as well as TILs, NK cells were dramatically reduced in the PB of SARIFA-positive cases. Moreover, CD56 and CD57 immunohistochemistry suggested SARIFA-status-dependent changes regarding NK cells and NK-like lymphocytes in the tumor microenvironment. CONCLUSION: This study proves that our newly introduced biomarker, SARIFA, comes along with distinct immunologic alterations, especially regarding NK cells.

Fatal cases after Omicron BA.1 and BA.2 infection: Results of an autopsy study.

OBJECTIVES: Omicron lineages BA.1/2 are considered to cause mild clinical courses. Nevertheless, fatal cases after those infections are recognized but little is known about risk factors. METHODS: A total of 23 full and three partial autopsies in deceased with known Omicron BA.1/2 infections have been consecutively performed. The investigations included histology, blood analyses, and molecular virus detection. RESULTS: COVID-19-associated diffuse alveolar damage was found in only eight cases (31%). This rate is significantly lower compared with previous studies, including non-Omicron variants, where rates between 69% and 92% were observed. Neither vaccination nor known risk factors were significantly associated with a direct cause of death by COVID-19. Only those patients who were admitted to the clinic because of COVID-19 but not for other reasons had a significant association with a direct COVID-19 -caused death (P >0.001). CONCLUSION: Diffuse alveolar damage still occurred in the Omicron BA.1/BA.2 era but at a considerably lower frequency than seen with previous variants of concern. None of the known risk factors discriminated the cases with COVID-19-caused death from those that died because of a different disease. Therefore, the host's genomics might play a key role in this regard. Further studies should elucidate the existence of such a genomic risk factor.

Circulating Lymphocytes Reflect the Local Immune Response in Patients with Colorectal Carcinoma.

Tumor-infiltrating lymphocytes (TILs) correlate with the number and size of the surrounding lymph nodes in patients with colorectal carcinoma (CRC) and reflect the quality of the antitumor immune response. In this prospective study, we analyzed whether this response correlated with the circulating lymphocytes in peripheral blood (PB). In 47 patients with newly diagnosed CRC, flow cytometry was performed to analyze the B cells, T cells, NK cells, and a variety of their subsets in PB. The results were correlated with TILs in the resected tumor and with the number and size of the surrounding lymph nodes in nodal negative (N- patients (LN5: number of lymph nodes measuring ≥5 mm) and the metastasis-to-lymph node size ratio (MSR) in nodal positive patients (N+). Differences between the number of TILs could be seen between N+ and N- patients, dependent on the LN5 and MSR categories, with higher values in N- cases and in patients with a higher LN5 category or a lower MSR. Additionally, higher values of various circulating lymphocyte subgroups were observed in these patients. For the total PB lymphocytes, CD8 cells, and some of their subgroups, a positive correlation with the TILs was found. This study shows that circulating lymphocytes-in particular, cytotoxic T cells-correlate with the local antitumor immune response displayed by TILs and lymph node activation. Our findings indicate that local and generalized antitumor immune responses are concordant with their different components.

Concurrent loss of MLH1, PMS2 and MSH6 immunoexpression in digestive system cancers indicating a widespread dysregulation in DNA repair processes.

Immunohistochemical analysis of mismatch repair (MMR) protein expression is widely used to identify tumors with a deficient MMR (dMMR). MMR proteins (MLH1/PMS2 and MSH2/MSH6) work as functional heterodimers, which usually leads to the loss of expression in only one functional MMR heterodimer. Recently, there have been studies showing the simultaneous loss of immunoexpression in proteins of both heterodimers. Yet, this phenomenon has been rarely investigated. In this study, we retrospectively considered cases of different digestive system cancers (gastric cancer, ampullary cancer, small bowel cancer, colorectal cancer), which were immunohistochemically tested for dMMR within a 4-year period at our university hospital (n=352). Of the 103 cases showing dMMR, 5 cases (1.4% of all, 5.1% of dMMR cases) showed a concurrent loss of MLH1, PMS2 and MSH6 immunoexpression, whereas in the other 98 dMMR cases only one MMR heterodimer was affected. MLH1-/PMS2-/MSH6- cancer cases almost arose throughout the entire digestive tract: from the gastric antrum to the left colic flexur. To provide a comprehensive molecular characterization of this MLH1-/PMS2-/MSH6- immunophenotype, tumors were analyzed for microsatellite instability, MLH1 promotor hypermethylation and BRAF exon 15 status. Furthermore, we performed next-generation sequencing focusing on genes related to DNA repair. Here, we could detect pathogenic germline variants as well as multiple sporadic mutations in different genes involved in MMR and homologous recombination repair (HRR) respectively. The affected MMR/HRR-related genes were: ATM, BARD1, BRCA1, CDK12, CHEK1, CHEK2, FANCA, MLH1, MSH6, PALB2, TP53. Considering the biologic function of HRR/MMR proteins as potential drug targets and the low frequency of most of these mutations in digestive system cancers in general, their common occurrence in our MLH1-/PMS2-/MSH6- cases seems to be even more noteworthy, highlighting the need for recognition, awareness and further investigation of this unusual IHC staining pattern.

Can low autopsy rates be increased? Yes, we can! Should postmortem examinations in oncology be performed? Yes, we should! A postmortem analysis of oncological cases.

Ever declining autopsy rates have been a concern of pathologists as well as clinicians for decades. Notably, in the field of oncology, data on autopsies and discrepancies between clinical and autoptic diagnoses are particularly scarce. In this retrospective study, we show the effect of a simple catalog of measures consisting of a different approach to obtain consent for autopsy, structured conferencing, and systematic teaching of residents, as well as a close collaboration between clinicians and pathologists on the numbers of autopsies, especially of oncological patients. Additionally, postmortem examination protocols from the years 2015 until 2019 were analyzed, regarding rates of discrepancies between clinical and autoptic causes of death in this category of patients. Autopsy numbers could be significantly increased from a minimum in 2014 (60 autopsies) to a maximum in 2018 (142 autopsies) (p < 0.0001). In the 67 autopsies of oncological cases, a high rate of 51% of major discrepancy between clinical and autoptic causes of death could be detected. In contrast to the general reported decline of autopsy rates, we present rising autopsy numbers over the past 5 years with an increasing number of oncological cases who underwent a postmortem examination. The high percentage of major discrepancies between clinical and autopsy diagnosis is in contrast to an expected decrease of major discrepancies in times of precise diagnostic methods and underlines the importance of autopsies to ensure high quality in diagnostics and therapy not only in the field of oncology.

Allogeneic stem cell transplantation for AML patients with RUNX1 mutation in first complete remission: a study on behalf of the acute leukemia working party of the EBMT.

Acute myeloid leukemia with runt-related transcription factor 1 gene mutation (RUNX1+ AML) is associated with inferior response rates and outcome after conventional chemotherapy. We performed a retrospective, registry-based analysis to elucidate the prognostic value of RUNX1 mutation after allogeneic stem cell transplantation (alloSCT). All consecutive adults undergoing alloSCT for AML in first complete remission (CR1) between 2013 and 2019 with complete information on conventional cytogenetics and RUNX1 mutational status were included. Endpoints of interest were cumulative relapse incidence, non-relapse mortality, overall and leukemia-free survival (OS/LFS), and GvHD-free/relapse-free survival. A total of 674 patients (183 RUNX1+, 491 RUNX1-) were identified, with >85% presenting as de novo AML. Median follow-up was 16.4 (RUNX1+) and 21.9 (RUNX1-) months. Survival rates showed no difference between RUNX1+ and RUNX1- patients either in univariate or multivariate analysis (2-year OS: 67.7 vs. 66.1%, p = 0.7; 2-year LFS: 61.1 vs. 60.8%, p = 0.62). Multivariate analysis identified age, donor type and poor cytogenetics as risk factors for inferior outcome. Among patients with RUNX+ AML, older age, reduced intensity conditioning and minimal residual disease at alloSCT predicted inferior outcome. Our data provide evidence that the negative influence of RUNX1 mutations in patients with AML can be overcome by transplantation in CR1.

Frequency, localization, and types of gastrointestinal stromal tumor-associated neoplasia.

BACKGROUND: In recent years, increasing evidence of second neoplasms associated with gastrointestinal stromal tumors (GIST) has been found. Numerous case reports, mostly retrospective studies and a few reviews, have been published. To our knowledge, however, no systematic review or meta-analysis of the existing data has been performed so far. AIM: To prepare a compilation, as complete as possible, of all reported second tumor entities that have been described in association with GIST and to systematically analyze the published studies with regard to frequency, localization, and types of GIST-associated neoplasms. METHODS: The MEDLINE and EBSCO databases were searched for a combination of the keywords GIST/secondary, synchronous, coincident/tumor, neoplasm, and relevant publications were selected by two independent authors. RESULTS: Initially, 3042 publications were found. After deletion of duplicates, 1631 remained, and 130 papers were selected; 22 of these were original studies with a minimum of 20 patients, and 108 were case reports. In the 22 selected studies, comprising a total number of 12050 patients, an overall rate of GIST-associated neoplasias of 20% could be calculated. Most second neoplasias were found in the gastrointestinal tract (32%) and in the male and female urogenital tract (30%). The specific risk scores of GISTs associated with other tumors were significantly lower than those without associated neoplasias. CONCLUSION: In this first systematic review, we could confirm previously reported findings of a more than coincidental association between GIST and other neoplasias. The question whether there is an underlying causal association will need further investigation. Our data suggest that even GIST with a very low risk of disease progression should prompt screening for second neoplasia and subsequent frequent controls or extended staging.

Human bone marrow contains high levels of extracellular vesicles with a tissue-specific subtype distribution.

INTRODUCTION: Extracellular vesicles (EV) are shed from a broad variety of cells and play an important role in activation of coagulation, cell to cell interaction and transport of membrane components. They are usually measured as circulating EV in peripheral blood (PB) and other body fluids. However, little is known about the distribution, presence and impact of EV and their subpopulations in bone marrow (BM). In our study, we focused on the analysis of different EV subtypes in human BM as compared to EV subsets in PB. METHODS: EV in BM and PB from 12 healthy stem cell donors were measured by flow-cytometry using Annexin V and cell-specific antibodies for hematopoietic stem cells, leucocytes, platelets, red blood cells, and endothelial cells. Additionally, concentrations of tissue factor-bearing EV were evaluated. RESULTS: High numbers of total EV were present in BM (median value [25-75 percentile]: 14.8 x10(9)/l [8.5-19.3]). Non-significantly lower numbers of total EV were measured in PB (9.2 x10(9)/l [3.8-14.5]). However, distribuation of EV subtypes showed substantial differences between BM and PB: In PB, distribution of EV fractions was similar as previously described. Most EV originated from platelets (93.9%), and only few EV were derived from leucocytes (4.5%), erythrocytes (1.8%), endothelial cells (1.0%), and hematopoietic stem cells (0.7%). In contrast, major fractions of BM-EV were derived from red blood cells or erythropoietic cells (43.2%), followed by megacaryocytes / platelets (27.6%), and by leucocytes as well as their progenitor cells (25,7%); only low EV proportions originated from endothelial cells and hematopoietic stem cells (2.0% and 1.5%, respectively). Similar fractions of tissue factor-bearing EV were found in BM and PB (1.3% and 0.9%). CONCULSION: Taken together, we describe EV numbers and their subtype distribution in the BM compartment for the first time. The tissue specific EV distribution reflects BM cell composition and favours the idea of a BM-PB barrier existing not only for cells, but also for EV.