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CD8+ cytotoxic T-lymphocytes are essential components of the anti-tumor immunity. To better understand the expansion of CD8+ T-cells we used multiplex fluorescence immunohistochemistry to study Ki67+CD8+ cells in normal lymphoid tissues, selected inflammatory diseases and cancers in 41 large sections/ microenvironment tissue microarrays (TMAs) as well as 765 samples in a conventional TMA format. The evaluation of more than 20 different compartments of normal lymphoid tissues revealed that the percentage of proliferating (ki67+) CD8+ cells did commonly not exceed 3%. In inflammations, the percentage of Ki67+CD8+ cells was more variable and higher compared to normal tissues. In cancers, the percentage of Ki67+CD8+ cells was higher in the tumor center than at the invasive margin. In the tumor center of 765 colorectal cancers, the density of Ki67+CD8+ cells and the percentage of proliferating CD8+ cytotoxic T-cells was significantly associated with microsatellite instability (p

Secreted frizzled-related protein 4 (SFRP4) controls WNT signaling and is thought to play a role for tumor aggressiveness. Here, we analyzed a tissue microarray containing 11,152 prostate cancers with pathological, clinical and molecular data by immunohistochemistry. SFRP4 expression was higher in cancer than in non-neoplastic acinar cells. SFRP4 staining was seen in 64.9% of tumors and classified as weak in 33.2%, moderate in 23.9% and strong in 7.8% of cancers. SFRP4 overexpression was linked to advanced tumor stage, high classical/quantitative Gleason grade (p

BackgroundMalnutrition caused by protein and vitamin deficiencies is a significant negative prognostic factor in surgical wound healing disorders and infections. Particularly in elective surgery, preoperative compensation of deficiencies is advisable to avoid negative postoperative consequences. This study examined the nutritional and protein balance of patients with periprosthetic hip and knee joint infections.Material and methodsPatients with periprosthetic hip or knee joint infections constituted the study group (SG). Control group I (CG I) included patients with primary implants and CG II included patients who required revision surgery because of aseptic loosening. Relevant nutritional and protein parameters were determined via analysis of peripheral venous blood samples. In addition, a questionnaire was used to evaluate the nutritional and eating patterns of all patients. The nutritional risk screening (NRS) 2002 score and body mass index (BMI) were also calculated for all participants.ResultsDifferences were found in the albumin level (SG: 36.23 ± 7.34, CG I: 44.37 ± 3.32,p p p = 0.004, CG II: 71.22 ± 5.21,p = 0.004). The number of patients with lowered albumin levels (SG 19/61, CG I 1/78, CG II 2/55) and total protein in serum (SG: 12/61, CG I 5/78, CG II 2/55) also showed considerable variation. The number of patients with a NRS 2002 score ≥3 differed significantly between SG and both CGs (SG: 5/61, CG I 1/78, CG II 0/55); however, these differences could not be confirmed using BMI.ConclusionAs expected, lowered albumin and total protein levels were observed in PJI due to the acute phase reaction. The NRS can be performed to exclude nutritional deficiency, which cannot be excluded based on BMI. In cases of periprosthetic joint infection it is reasonable to compensate the nutritional deficiency with dietary supplements.

Purpose Immune checkpoint inhibitors have recently been approved by the US FDA as first and/or second line therapy in a subset of cancer types. Recent evidence suggests that the quantity of tumor infiltrating lymphocytes (TILs) influences the likelihood of response to immune checkpoint inhibitors. Here, we set out to assess the density of CD8+ lymphocytes in a wide range of different cancer types and subtypes. Methods The density of CD8+ lymphocytes was compared across different cancer types using tissue microarrays (TMAs) composed of up to 50 tumor samples each from 84 different cancer types and subtypes. In total 2652 cancers and 608 normal tissues were successfully analyzed by CD8 immunohistochemistry followed by automated image analysis of digitized slides. Results We found that the median CD8+ lymphocyte counts ranged from 6 cells/mm2 in pleomorphic adenoma up to 1573 cells/mm2 in Hodgkin’s lymphoma. The CD8 counts were generally lower in normal tissues compared to cancer tissues. Blood vessels of the spleen were the only non-lymphatic tissue staining positive for CD8. Tumor types approved for checkpoint inhibitor therapy, including malignant melanoma (81), muscle invasive urothelial carcinoma (119), small cell lung cancer (120), clear cell renal cell cancer (153), squamous cell carcinoma (189) and adenocarcinoma of the lung (328) as well as Hodgkin’s lymphoma (1573) were all ranking among the upper half of our list. Comparably high CD8 densities (median cells/mm2) were also found in several rare and aggressive cancer types including Merkel cell carcinoma (70), angiosarcoma (95), anaplastic thyroid cancer (156) and embryonal carcinoma of the testis (186). In 73 of the 84 analyzed cancer types, the highly variable CD8 counts occasionally exceeded the average CD8 count of tumors for which checkpoint inhibitors have been approved. Conclusion These data support the concept that among most tumor types at least some individual cancers may benefit from treatment with immune checkpoint inhibitors.

Developmental and epileptic encephalopathies are devastating disorders characterized by epilepsy, intellectual disability, and other neuropsychiatric symptoms, for which available treatments are largely ineffective. Following a precision medicine approach, we show for KCNA2-encephalopathy that the K+ channel blocker 4-aminopyridine can antagonize gain-of-function defects caused by variants in the KV1.2 subunit in vitro, by reducing current amplitudes and negative shifts of steady-state activation and increasing the firing rate of transfected neurons. In n-of-1 trials carried out in nine different centers, 9 of 11 patients carrying such variants benefitted from treatment with 4-aminopyridine. All six patients experiencing daily absence, myoclonic, or atonic seizures became seizure-free (except some remaining provoked seizures). Two of six patients experiencing generalized tonic-clonic seizures showed marked improvement, three showed no effect, and one worsening. Nine patients showed improved gait, ataxia, alertness, cognition, or speech. 4-Aminopyridine was well tolerated up to 2.6 mg/kg per day. We suggest 4-aminopyridine as a promising tailored treatment in KCNA2-(gain-of-function)–encephalopathy and provide an online tool assisting physicians to select patients with gain-of-function mutations suited to this treatment.

Background Presence of small (tertiary) Gleason 5 pattern is linked to a higher risk of biochemical recurrence in prostate cancer. It is unclear, however, how to integrate small Gleason 5 elements into clinically relevant Gleason grade groups. Objective To analyze the prognostic impact of Gleason 5 patterns in prostate cancer and to develop a method for integrating tertiary Gleason 5 patterns into a quantitative Gleason grading system. Design, setting, and participants Prostatectomy specimens from 13 261 consecutive patients and of 3295 matched preoperative biopsies were available. Percentages of Gleason 3, 4, and 5 had been recorded for each cancer. Outcome measurements and statistical analysis: Results and limitations Our data demonstrate that minimal Gleason 5 areas have strong prognostic impact in Gleason 7 carcinomas, while further expansion of the Gleason 5 pattern population has less impact. We thus defined an integrated quantitative Gleason score (IQ-Gleason) by adding a lump score of 10 to the percentage of unfavorable Gleason pattern (Gleason 4/5) if any Gleason 5 was present and by adding another 7.5 points in case of a Gleason 5 fraction >20%. There was a continuous increase of the risk of prostate-specific antigen recurrence with increasing IQ-Gleason. This was also true for subgroups with identical Cancer of the Prostate Risk Assessment Postsurgical scores ( p p Conclusions The IQ-Gleason represents a simple and efficient approach for combining both quantitative Gleason grading and tertiary Gleason grades in one highly prognostic numerical variable. Patient summary Prostatectomy specimens (13 261) were analyzed to estimate the relevance of small Gleason 5 elements in prostate cancers. Even the smallest Gleason 5 areas markedly increased the risk of prostate-specific antigen recurrence after surgery. Larger fractions of Gleason 5 patterns had less further impact on prognosis. Based on this, a numerical Gleason score (integrated quantitative Gleason score) was defined by the percentages of Gleason 4 and 5 patterns, enabling a refined estimate of patient prognosis.

Background: Remodelling and spacing factor 1 (RSF1) is involved in the regulation of chromatin remodelling and represents a potential therapeutic target. High RSF1 expression has been linked to adverse tumour features in many cancer types, but its role in prostate cancer is uncertain. Methods: In this study, RSF1 expression was analysed by immunohistochemistry on a tissue microarray with 17,747 prostate cancers. Results: Nuclear RSF1 staining of 16,456 interpetable cancers was considered strong, moderate, weak and negative in 25.2%, 48.7%, 5.3% and 20.8% of cancers respectively. Positive RSF1 expression was associated with advanced tumour stage, high Gleason grade, lymph node metastasis (p p p p Conclusion: In summary, the results of our study identify RSF1 as an independent prognostic marker in prostate cancer with a particularly strong role in ERG negative cases.

Checkpoint kinase 2 (CHK2) is a serine-threonine kinase with a role in DNA repair, cell cycle arrest or apoptosis in response to DNA damage. Both reduced and increased CHK2 expression has been described in different tumour types with impact on patient prognosis. To evaluate prevalence and significance of altered CHK2 expression in prostate cancer, a tissue microarray containing 17,747 tumours was analysed by immunohistochemistry. Nuclear CHK2 immunostaining was absent or weak in benign prostate epithelium but often more prominent in cancers. CHK2 immunostaining was considered weak in 38.8%, moderate in 33.6% and strong in 11.2% of prostate cancers. High CHK2 expression was strongly associated with TMPRSS2:ERG fusions (p0.0001). Subgroup analysis of ERG positive and negative cancers revealed that high CHK2 staining was significantly linked to advanced tumour stage, high Gleason score, positive nodal status, positive surgical margin, high preoperative PSA (p0.0001 each) and early prostate-specific antigen (PSA) recurrence (p=0.0001) in the subset of ERG negative cancers, while most of these associations were absent in ERG positive cancers. In ERG negative cancers, high CHK2 expression was an independent predictor of patient prognosis, even if parameters were included that were only available postoperatively. High CHK2 expression was also linked to presence of chromosomal deletions, high level of androgen receptor expression, positive p53 immunostaining, and high Ki-67 labelling index. These provide further in vivo evidence for previously described functional interactions. In summary, high CHK2 expression is linked to adverse tumour features and independently predicts early biochemical recurrence in ERG negative prostate cancer. CHK2 measurement, either alone or in combination, might be of clinical utility in this prostate cancer subgroup.

SAM pointed domain-containing Ets transcription factor (SPDEF), a member of the ETS transcription factor family, has been associated with prostate cancer development; however, its role in tumour development and progression is controversial. In the present study, SPDEF expression was analysed on a tissue microarray with >12,000 prostate cancer samples. SPDEF expression levels were higher in most prostate cancer samples than in normal prostate epithelium, suggesting SPDEF was upregulated in cancer. Nuclear SPDEF expression was identified in 80% of prostate cancer samples, and considered weak in 26.4%, moderate in 40.1% and strong in 13.5% of cases. SPDEF positivity was significantly associated with tumour stage, Gleason grade, lymph node metastasis and PSA recurrence (all P

Background Gleason grading is the strongest prognostic parameter in prostate cancer. Gleason grading is categorized as Gleason ≤6, 3+4, 4+3, 8, and 9–10, but there is variability within these subgroups. For example, Gleason 4 components may range from 5–45% in a Gleason 3+4=7 cancer. Objective To assess the clinical relevance of the fractions of Gleason patterns. Design, setting, and participants Prostatectomy specimens from 12823 consecutive patients and of 2971 matched preoperative biopsies for which clinical data with an annual follow-up between 2005 and 2014 were available from the Martini-Klinik database. Outcome measurements and statistical analysis To evaluate the utility of quantitative grading, the fraction of Gleason 3, 4, and 5 patterns seen in biopsies and prostatectomies were recorded. Gleason grade fractions were compared with prostatectomy findings and prostate-specific antigen recurrence. Results and limitations Our data suggest a striking utility of quantitative Gleason grading. In prostatectomy specimens, there was a continuous increase of the risk of prostate-specific antigen recurrence with increasing percentage of Gleason 4 fractions with remarkably small differences in outcome at clinically important thresholds (0% vs 5%; 40% vs 60% Gleason 4), distinguishing traditionally established prognostic groups. Also, in biopsies, the quantitative Gleason scoring identified various intermediate risk groups with respect to Gleason findings in corresponding prostatectomies. Quantitative grading may also reduce the clinical impact of interobserver variability because borderline findings such as tumors with 5%, 40%, or 60% Gleason 4 fractions and very small Gleason 5 fractions (with pivotal impact on the Gleason score) are disclaimed. Conclusions Quantitative Gleason pattern data should routinely be provided in addition to Gleason score categories, both in biopsies and in prostatectomy specimens. Patient summary Gleason score is the most important prognostic parameter in prostate cancer, but prone to interobserver variation. The results of our study show that morphological aspects that define the Gleason grade in prostate cancer represent a continuum. Quantitation of Gleason patterns provides clinically relevant information beyond the traditional Gleason grading categories ≤3+3, 3+4, 4+3, 8, 9–10. Quantitative Gleason scoring can help to minimize variations between different pathologists and substantially aid in optimized therapy decision-making.

Small-cell cancer of the urinary bladder is a rare but highly aggressive disease. It is currently unclear whether immune checkpoint therapies that have been approved for urothelial carcinomas will also be efficient in small-cell carcinomas. In this study, we analyzed potential predictors of response including PD-L1 expression and the quantity and location of tumor-infiltrating lymphocytes (TILs) in 12 small-cell and 69 “classical” urothelial cancers by immunohistochemistry. The analysis revealed that small-cell carcinomas were characterized by the virtual absence of PD-L1 expression and an “immune-excluded” phenotype with only a few TILs in the center of the tumor (CT). In small-cell carcinomas, the average immune cell density in the CT (CD3: 159±206, CD8: 87±169 cells/mm2) was more than 3 times lower than that in the urothelial carcinomas (CD3: 625±800, p<0.001; CD8: 362±626 cells/mm2, p=0.004) while there was no significant difference in the immune cell density at the invasive margin (IM) (small-cell carcinomas CD3: 899±733, CD8: 404±433 cells/mm2; urothelial carcinomas CD3: 1167±1206, p=0.31; CD8: 582±864 cells/mm2, p=0.27). Positive PD-L1 staining was found in 39% of urothelial cancers, but in only 8% of small-cell bladder cancer cases (p=0.04). Concordant with these data, a sharp decrease of PD-L1 positivity from >80% to 0% positive cells and of TILS in the CT from 466-1063 CD3-positive cells/mm2 to 50-109 CD3-positive cells/mm2 was observed in two cancers with clear-cut progression from “classical” urothelial to small-cell carcinoma. In conclusion, these data demonstrate that small-cell bladder cancer commonly exhibits an immune-excluded phenotype.

Evidence suggests that the quantity of cytotoxic lymphocytes influences the likelihood for a successful application of immune checkpoint inhibitors. To compare the density of CD8+lymphocytes across various different tumor types, a tissue microarray (TMA) composed of up to 50 tumor samples each from 85 different cancer types and subtypes was analyzed. A total of 2652 cancers and 608 normal tissues were successfully analyzed by CD8 immunohistochemistry followed by automated image analysis of digitized slides. The median number of CD8+lymphocytes ranged from 6 cells/mm2in pleomorphic adenoma up to 1573 cells/mm2in Hodgkin’s lymphoma. CD8 counts were generally lower in normal tissues. Blood vessels of the spleen was the only non-lymphatic tissue staining for CD8.In solid tumors, highest CD8 densities (cells/mm2) were found in seminoma (median: 424), Warthin’s tumor (median: 425), squamous cell cervical cancer (median 468), medullary breast cancer (median: 657) and thymoma (median: 889).Tumor types approved for therapy with checkpoint inhibitors such malignant melanoma (median: 81), muscle invasive urothelial carcinomas (median: 119), small cell lung cancer (median: 120), clear cell kidney cancer (median: 153), squamous cell cancer (median: 189) and adenocarcinoma of the lung (median: 328) as well as Hodgkin’s lymphoma (median:1573) were all ranking among the upper half of our list. Comparably high CD8 densities (cells/mm2) were also found for several rare and aggressive cancer types including Merkel cell carcinoma (median: 70), angiosarcoma (median: 95), anaplastic thyroid cancer (median: 156), anal carcinoma (median: 104), squamous cell carcinoma of the vagina (median: 128) and embryonal carcinoma of the testis (median: 186). The CD8 cell count was highly variable within tumor types. In 73 of 84 analyzed cancer types, the CD8 count at least occasionally exceeded the average CD8 count of tumors for which checkpoint inhibitors have been approved.These data support the concept, that in most tumor types at least some individual cancers may benefit from treatment with immune checkpoint inhibitors. Citation Format: Niclas C. Blessin, Florian Lutz, Patrick Spriesterbach, Wenchao Li, Tim Mandelkow, Vera Nickelsen, Ronald Simon, Claudia Hube-Magg, Florian Viehweger, Maximillian Lennartz, Christoph Fraune, Kristine Fischer, Katharina Möller, Stefan Steurer, Jacob R. Izbicki, Guido Sauter, Sarah Minner, Frank Jacobsen, Andreas M. Luebke, Franziska Büscheck, Doris Höflmayer, Waldemar Wilczak, Eike Burandt, Andrea Hinsch. Distribution of CD8+cytotoxic lymphocytes in human neoplasms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4567.

Since several decades, prostate specific antigen (PSA) is regarded as a specific marker for healthy and neoplastic prostate tissues, and PSA level changes in blood and tissue are discussed as prognostic markers of prostate cancer. Using a custom-made mouse monoclonal anti-PSA antibody, we studied expression of PSA in a tissue microarray (TMAs) comprising 17,456 prostate cancers with long-term follow-up data. PSA staining was typically present in the cytoplasm and pronounced at the apical cell pole. PSA expression was often reduced (15%) or even completely lost (1%). Reduction of PSA expression was significantly linked to adverse features of prostate cancer, including advanced stage, high classical and quantitative Gleason grade, presence of lymph node metastasis, a positive surgical margin, increased cell proliferation, and reduced time to recurrence (p Citation Format: Ronald Simon, Martina Kluth, Kristine Fischer, Sarah Bonk, Claudia Hube-Magg, Doris Höflmayer, Maximilian Lennartz, Florian Lutz, Guido Sauter, Franziska Büscheck, Ria Uhlig, David Dum, Andreas M. Luebke, Corinna Wittmer, Frank Jacobsen, Eike Burandt, Stefan Steurer, Waldemar Wilczak, Sarah Minner. Prostate specific antigen (PSA) as a diagnostic and prognostic marker of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 430.