Bovine Meat and Milk Factor-like Sequences Are Frequently Detected in Renal Cell Carcinoma Tissues.

Previous studies have indicated a potential role of diet in the pathogenesis of renal cell carcinoma (RCC). Recently, circular bovine meat and milk factor (BMMF) DNAs have been identified in peritumoral tissues of human colon and breast cancers. Here, we investigated the prevalence of the DNA of these novel human pathogenic infectious agents in RCC and adjacent peritumoral renal tissues. DNA was extracted from formalin-fixed and paraffin-embedded (FFPE) RCC and peritumoral kidney tissues, including a test (n = 11) and a validation (n = 152) collection. BMMF1 and BMMF2 consensus primers were designed to screen for the presence of BMMF1- and BMMF2-like DNA. In addition, BMMF-specific PCR was performed on selected cases to test for the presence of additional regions of BMMF1 and BMMF2 genomes. A reference collection of hepatocellular carcinomas (HCCs; n = 60) and adjacent peritumoral liver tissues (n = 50) was also included. Our results demonstrated that BMMF1 and BMMF2 DNAs are frequently found in human RCC tissues and are particularly more prevalent in peritumoral kidney tissues. Of note, BMMF1 and BMMF2 genotype heterogeneity was higher in peritumoral kidney tissues compared to RCC tissues. This is the first study to directly test human FFPE tissues for BMMF1- and BMMF2-like DNA using consensus PCR and demonstrate BMMF DNA in neoplastic and peritumoral kidney tissues. The findings are in line with the recently proposed indirect etiopathogenetic role of BMMFs in, e.g., colorectal carcinogenesis. Follow-up studies are needed to explore the potential role of BMMFs in the etiopathogenesis of RCC.

Abdominal inflammatory myofibroblastic tumour: Clinicopathological and molecular analysis of 20 cases, highlighting potential therapeutic targets.

AIMS: Inflammatory myofibroblastic tumour (IMT) is a rare mesenchymal neoplasm of intermediate malignant potential, occurring at any age and at multiple sites. Epithelioid inflammatory myofibroblastic sarcoma (EIMS) is an aggressive subtype of IMT, typically involving the abdomen. Most IMTs harbour kinase gene fusions, especially involving ALK and ROS1, but 20-30% of IMTs show no detectable translocations. The aim of this study is to further delineate clinicopathological and molecular characteristics of abdominal IMT and discover potential new therapeutic targets. METHODS AND RESULTS: In 20 IMTs, including four EIMS, RNA fusion analysis was performed, followed by multiplex DNA analysis if no ALK or ROS1 fusion was detected. Fourteen IMTs (70.0%) had an ALK translocation and the fusion partner was identified in 11, including a RRBP1::ALK fusion, not previously described in classical (non-EIMS) IMT. RANBP2::ALK fusion was demonstrated in all EIMS. One IMT had a ROS1 fusion. In all ALK/ROS1 translocation-negative IMTs mutations or fusions - as yet unreported in primary IMT - were found in genes related to the receptor tyrosine kinase (RTK)/PI3K/AKT pathway. Three of four patients with EIMS died of disease [mean survival 8 months (4-15 months)], whereas only one of 14 classical IMT patients succumbed to disease [mean follow-up time 52 months (2-204 months); P < 0.01]. CONCLUSION: This study shows the wide clinical spectrum of abdominal IMTs and affirms the poor prognosis of EIMS, raising discussion about its status as IMT subtype. Furthermore, the newly detected alterations of the RTK/PI3K/AKT pathway expand the molecular landscape of IMTs and provide potential therapeutic targets.

An international multi-institutional validation study of the algorithm for prostate cancer detection and Gleason grading.

Pathologic examination of prostate biopsies is time consuming due to the large number of slides per case. In this retrospective study, we validate a deep learning-based classifier for prostate cancer (PCA) detection and Gleason grading (AI tool) in biopsy samples. Five external cohorts of patients with multifocal prostate biopsy were analyzed from high-volume pathology institutes. A total of 5922 H&E sections representing 7473 biopsy cores from 423 patient cases (digitized using three scanners) were assessed concerning tumor detection. Two tumor-bearing datasets (core n = 227 and 159) were graded by an international group of pathologists including expert urologic pathologists (n = 11) to validate the Gleason grading classifier. The sensitivity, specificity, and NPV for the detection of tumor-bearing biopsies was in a range of 0.971-1.000, 0.875-0.976, and 0.988-1.000, respectively, across the different test cohorts. In several biopsy slides tumor tissue was correctly detected by the AI tool that was initially missed by pathologists. Most false positive misclassifications represented lesions suspicious for carcinoma or cancer mimickers. The quadratically weighted kappa levels for Gleason grading agreement for single pathologists was 0.62-0.80 (0.77 for AI tool) and 0.64-0.76 (0.72 for AI tool) for the two grading datasets, respectively. In cases where consensus for grading was reached among pathologists, kappa levels for AI tool were 0.903 and 0.855. The PCA detection classifier showed high accuracy for PCA detection in biopsy cases during external validation, independent of the institute and scanner used. High levels of agreement for Gleason grading were indistinguishable between experienced genitourinary pathologists and the AI tool.

Giant mitochondria in human liver disease.

This thematic review aims to provide an overview of the current state of knowledge about the occurrence of giant mitochondria or megamitochondria in liver parenchymal cells. Their presence and accumulation are considered to be a major pathological hallmark of the health and fate of liver parenchymal cells that leads to overall tissue deterioration and eventually results in organ failure. The first description on giant mitochondria dates back to the 1960s, coinciding with the availability of the first generation of electron microscopes in clinical diagnostic laboratories. Detailed accounts on their ultrastructure have mostly been described in patients suffering from alcoholic liver disease, chronic hepatitis, hepatocellular carcinoma and non-alcoholic fatty liver disease. Interestingly, from this extensive literature survey, it became apparent that giant mitochondria or megamitochondria present themselves with or without highly organised crystal-like intramitochondrial inclusions. The origin, formation and potential role of giant mitochondria remain to-date largely unanswered. Likewise, the biochemical composition of the well-organised crystal-like inclusions and their possible impact on mitochondrial function is unclear. Herein, concepts about the possible mechanism of their formation and three-dimensional architecture will be approached. We will furthermore discuss their importance in diagnostics, including future research outlooks and potential therapeutic interventions to cure liver disease where giant mitochondria are implemented.

Scoring Systems for Immunohistochemistry in Urothelial Carcinoma.

Immunohistochemistry is widely used in diagnostic and scientific analysis of urothelial carcinoma. Objective interpretation of staining results is mandatory for accuracy and comparability in diagnostic and therapeutic patient care as well as research.Herein we summarize and explain standardized microscopic evaluation and scoring approaches for immunohistochemical stainings. We focus on commonly used and generally feasible approaches for different cellular compartments and comment on their utility in diagnostics and research practice.

Relationship between the Warburg effect in tumour cells and the tumour microenvironment in colorectal cancer patients: Results from a large multicentre study.

Colorectal cancer (CRC) remains one of the most prevalent and deadly cancers worldwide. The tumour-node-metastasis stage (TNM) is currently the most clinically important tool to predict prognosis for CRC patients. However, patients with the same TNM stage can have different prognoses. The metabolic status of tumour cells (Warburg-subtype) has been proposed as potential prognostic factor in CRC. However, potential biological mechanisms underlying the relationship between Warburg-subtype and prognosis have not been investigated in detail. One potential mechanism could be that the metabolic status of tumour cells affects the tumour microenvironment (TME). Our objective was to investigate the relationship between Warburg-subtypes and the TME. Haematoxylin/Eosin stained tumour tissue microarray cores from 2171 CRC patients from the Netherlands Cohort Study were semi quantitatively assessed for tumour infiltrating lymphocytes (TILs) and relative tumour stroma content. 5745 cores were assessed by putting each core in one of four categories for both TILs and stroma. The relationship between Warburg-subtype, TILs, and tumour stroma content was investigated. The frequency of CRC in the different TIL categories was (n, %): very low (2538, 44.2), low (2463, 42.9), high (722, 12.6), and very high (22, 0.4). The frequency of CRC in the different tumour stroma content categories was: ≤ 25% (2755, 47.9), > 25% ≤ 50% (1553, 27) > 50% ≤ 75% (905, 15.8), and > 75% (532, 9.3). There was neither an association between Warburg-subtype and tumour stroma content (p = 0.229) nor between Warburg-subtype and TILs (p = 0.429). This is the first study to investigate the relationship between Warburg-subtypes and the TME in a large population-based series of CRC patients. Our data suggest that the prognostic value of Warburg-subtypes cannot be directly attributed to differences in TILs or tumour stroma content. Our results require confirmation in an independent series.

Histologic re­evaluation of a population­based series of renal cell carcinomas from The Netherlands Cohort Study according to the 2022 ISUP/WHO classification.

The aim of the present study was to re-evaluate 457 renal cell carcinoma (RCC) cases from the Netherlands Cohort Study on Diet and Cancer (NLCS), a large population-based cohort, according to the new 2022 ISUP, Genitourinary Pathology Society and World Health Organisation (WHO) classifications to assess whether newly recognized subtypes of RCC could be found among these cases. These cases were initially evaluated according to the 2004 WHO classification, the Fuhrman grading system and the 3rd version of the Tumor-Node-Metastasis (TNM). Data on tumor size, laterality and date of diagnosis, among other clinicopathological characteristics, were obtained through record linkage with the Netherlands Cancer Registry and the Pathologisch-Anatomisch Landelijk Geautomatiseerd Archief. Digital slides from the NLCS were reviewed by two urogenital pathologists according to the new ISUP grading and the 2022 WHO classification (5th edition). Immunohistochemistry staining for carbonic anhydrase IX was performed on cases with ambiguous morphology. A total of 373 cases of clear cell RCC (ccRCC), 61 cases of papillary RCC (pRCC), 13 cases of chromophobe RCC, 3 cases of collecting duct carcinoma and 4 cases of oncocytoma were identified. The subtyping showed no discrepancy with the previous diagnoses. A comparison of the WHO/ISUP grading to the original Fuhrman grading showed a similar grading in 245 (56.5%) cases of the total ccRCC and pRCC cases. The staging according to the novel TNM classification 8th edition showed a restaging in 286 cases (65.5%). Lymphovascular (microvascular) invasion (LVI) and tumor necrosis (TN) were present in 14.4% and 33.5% of the total number of cases, respectively. Furthermore, the presence of sarcomatoid differentiation in 5.1% and rhabdoid differentiation in 4.2% of the cases was observed. In conclusion, none of the newly accepted and emerging/provisional RCC entities were identified in the NLCS cases, which could be attributed to the high mean age (71.4 years) at diagnosis of the patients included in the present study. A restaging of the NLCS cases using the TNM 8th edition and regrading using ISUP grading was performed, which showed that it is possible to report on newer features, such as sarcomatoid differentiation and LVI, even in an old sample collection.

Efficient 3D light-sheet imaging of very large-scale optically cleared human brain and prostate tissue samples.

The ability to image human tissue samples in 3D, with both cellular resolution and a large field of view (FOV), can improve fundamental and clinical investigations. Here, we demonstrate the feasibility of light-sheet imaging of ~5 cm3 sized formalin fixed human brain and up to ~7 cm3 sized formalin fixed paraffin embedded (FFPE) prostate cancer samples, processed with the FFPE-MASH protocol. We present a light-sheet microscopy prototype, the cleared-tissue dual view Selective Plane Illumination Microscope (ct-dSPIM), capable of fast 3D high-resolution acquisitions of cm3 scale cleared tissue. We used mosaic scans for fast 3D overviews of entire tissue samples or higher resolution overviews of large ROIs with various speeds: (a) Mosaic 16 (16.4 µm isotropic resolution, ~1.7 h/cm3), (b) Mosaic 4 (4.1 µm isotropic resolution, ~ 5 h/cm3) and (c) Mosaic 0.5 (0.5 µm near isotropic resolution, ~15.8 h/cm3). We could visualise cortical layers and neurons around the border of human brain areas V1&V2, and could demonstrate suitable imaging quality for Gleason score grading in thick prostate cancer samples. We show that ct-dSPIM imaging is an excellent technique to quantitatively assess entire MASH prepared large-scale human tissue samples in 3D, with considerable future clinical potential.

Association between adjuvant therapy and survival in colorectal cancer patients according to metabolic Warburg-subtypes.

PURPOSE: Tumor location and tumor node metastasis (TNM) stage guide treatment decisions in colorectal cancer (CRC) patients. However, patients with the same disease stage do not benefit equally from adjuvant therapy. Hence, there remains an urgent clinical need to identify prognostic and/or predictive biomarker(s) to personalize treatment decisions. In this exploratory study, we investigated whether our previously defined metabolic Warburg-subtypes can predict which CRC patients might derive survival benefit from adjuvant therapy. METHODS: Information regarding treatment (surgery only: n = 1451; adjuvant radiotherapy: n = 82; or adjuvant chemotherapy: n = 260) and Warburg-subtype (Warburg-low: n = 485, -moderate: n = 641, or -high: n = 667) was available for 1793 CRC patients from the Netherlands Cohort Study (NLCS). Kaplan-Meier curves and Cox regression models were used to investigate survival benefit from adjuvant therapy compared to surgery-only for the different Warburg-subtypes. RESULTS: Patients with Warburg-moderate CRC (HRCRC-specific 0.64; 95% CI 0.47-0.86, HRoverall 0.61; 95% CI 0.47-0.80), and possibly Warburg-high CRC (HRCRC-specific 0.86; 95% CI 0.65-1.14, HRoverall 0.82; 95% CI 0.64-1.05), had survival benefit from adjuvant therapy. No survival benefit was observed for patients with Warburg-low CRC (HRCRC-specific 1.07; 95% CI 0.76-1.52, HRoverall 0.95; 95% CI 0.70-1.30). There was a significant interaction between Warburg-subtype and adjuvant therapy for CRC-specific survival (p = 0.049) and overall survival (p = 0.035). CONCLUSION: Our results suggest that Warburg-subtypes may predict survival benefit from adjuvant therapy in CRC patients. A survival benefit from adjuvant therapy was observed for patients with Warburg-moderate and possibly Warburg-high CRC, but not for patients with Warburg-low CRC. Future prospective studies are necessary to validate our findings.

Osteoclast-like giant cells in hepatocellular carcinoma case description and review of the literature.

BACKGROUND: The presence of osteoclast-like giant cells (OGC) in hepatocellular carcinoma (HCC) is rare and literature on this topic is scarce. In this article, we report on a case of a 77-year-old male patient with HCC with OGC and provide an overview of the current literature. METHODS: We conducted a systematic search to find all available literature on OGC in HCC. The electronic databases PubMed, Web of Science, Embase and CENTRAL were searched from inception until October 2020. RESULTS: Thirteen articles on this topic were identified and were included in this review. Data on 14 patients were available, described in twelve case reports, one patient in a patient series and the present case. Median age of included patients was 68 years. Two patients underwent neoadjuvant therapy prior to surgery. Of the 14 cases, eight tumours with OGC arose in a cirrhotic liver. Oncological outcome in this series was unfavourable, even after surgical resection, with a median disease-free survival of 75 d. CONCLUSIONS: The presence of OGC in HCC is rare. Current literature is scarce, and suggests an unfavourable outcome in regard to overall survival of HCC.

Association between mutational subgroups, Warburg-subtypes, and survival in patients with colorectal cancer.

BACKGROUND: Previous research suggests that Warburg-subtypes are related to potentially important survival differences in colorectal cancer (CRC) patients. In the present study, we investigated whether mutational subgroups based on somatic mutations in RAS, BRAF, PIK3CA, and MET, which are known to promote the Warburg-effect, as well as mismatch repair (MMR) status, hold prognostic value in CRC. In addition, we investigated whether Warburg-subtypes provide additional prognostic information, independent of known prognostic factors like TNM stage. METHODS: CRC patients (n = 2344) from the prospective Netherlands Cohort Study (NLCS) were classified into eight mutually exclusive mutational subgroups, based on observed mutations in RAS, BRAF, PIK3CA, and MET, and MMR status: All-wild-type + MMRproficient , KRASmut  + MMRproficient , KRASmut  + PIK3CAmut  + MMRproficient , PIK3CAmut  + MMRproficient , BRAFmut  + MMRproficient , BRAFmut  + MMRdeficient , other + MMRproficient , and other + MMRdeficient . Kaplan-Meier curves and Cox regression models were used to investigate associations between mutational subgroups and survival, as well as associations between our previously established Warburg-subtypes and survival within these mutational subgroups. RESULTS: Compared to patients with all-wild-type + MMRproficient CRC, patients with KRASmut  + MMRproficient , KRASmut  + PIK3CAmut  + MMRproficient , BRAFmut  + MMRproficient , or other + MMRproficient CRC had a statistically significant worse survival (HRCRC-specific ranged from 1.29 to 1.88). In contrast, patients with other + MMRdeficient CRC had the most favorable survival (HRCRC-specific 0.48). No statistically significant survival differences were observed for the Warburg-subtypes within mutational subgroups. CONCLUSION: Our results highlight the prognostic potential of mutational subgroups in CRC. Warburg-subtypes did not provide additional prognostic information within these mutational subgroups. Future larger-scale prospective studies are necessary to validate our findings and to examine the potential clinical utility of CRC subtyping based on mutational subgroups.

Human polyomaviruses JCPyV and MCPyV in urothelial cell carcinoma: a single institution experience.

Objective: Urothelial cell carcinoma (UCC) is the most common type of urinary bladder. JCPyV and BKPyV have been detected in the urine and tissue of urothelial cell carcinomas (UCC) in immunocompetent patients. Here, we investigated the presence of several HPyVs in UCC samples using diverse molecular techniques to study the prevalence of HPyVs in UCC. Methods: A large single-institution database of urine cytology specimens (UCS; n = 22.867 UCS) has previously been searched for decoy cells (n = 30), suggesting polyomavirus infection. The available urine sediments and formalin-fixed paraffin-embedded (FFPE) tissue samples of UCC patients were tested for the presence of JCPyV-LTAg expression by immunohistochemistry (IHC) labeled with SV40-LTAg antibody (clone: PAb416) and subsequent PCR followed by sequencing. In addition, the presence of the oncogenic Merkel cell polyomavirus (MCPyV) and the presence of human polyomavirus 6 (HPyV6) and 7 (HPyV7) DNA were tested with DNA PCR or IHC. Results: Of the 30 patients harboring decoy cells, 14 were diagnosed with UCC of the urinary bladder (14/30; 46.6%) before presenting with decoy cells in the urine. The SV40-LTAg IHC was positive in all 14 UCC urine sediments and negative in the FFPE tissues. JCPyV-DNA was identified in all five available UCS and in three FFPE samples of UCC (three of 14; 21.4%). Two UCC cases were positive for MCPyV-DNA (two of 14; 14.3%), and one of them showed protein expression by IHC (one of 14; 7.1%). All specimens were HPyV6 and HPyV7 negative. Conclusion: Our findings show the presence of JCPyV in the urine and UCC of immunocompetent patients. Moreover, MCPyV was detected in two UCC cases. In total, five UCC cases showed the presence of either JCPyV or MCPyV. The evidence here supports the hypothesis that these viruses might sporadically be associated with UCC. Further studies are needed to confirm the relevance of JCPyV or MCPyV as a possible risk factor for UCC development.

Energy balance-related factors in childhood and adolescence and risk of colorectal cancer based on KRAS, PIK3CA, and BRAF mutations and MMR status.

KRAS mutations (KRASmut ), PIK3CAmut , BRAFmut , and deficient DNA mismatch repair (dMMR) have been associated with the Warburg effect. We previously reported differential associations between early-life energy balance-related factors (height, energy restriction, body mass index [BMI]) and colorectal cancer (CRC) subtypes based on the Warburg effect. We now investigated associations of early-life energy balance-related factors and the risk of CRC subgroups based on mutation and MMR status. Data from the Netherlands Cohort Study was used. KRASmut , PIK3CAmut, BRAFmut, and MMR status were available for 2349 CRC cases, and complete covariate data for 1934 cases and 3911 subcohort members. Multivariable-adjusted Cox regression was used to estimate associations of height, energy restriction proxies (exposure to Dutch Hunger Winter, Second World War, Economic Depression), and early adult BMI (age 20 years) with risk of CRC based on individual molecular features and combinations thereof (all-wild-type+MMR-proficient [pMMR]; any-mutation/dMMR). Height was positively associated with any-mutation/dMMR CRC but not all-wild-type+pMMR CRC, with the exception of rectal cancer in men, and with heterogeneity in associations observed for colon cancer in men (p-heterogeneity = 0.049) and rectal cancer in women (p-heterogeneity = 0.014). Results on early-life energy restriction proxies in relation to the risk of CRC subgroups did not show clear patterns. Early adult BMI was positively, but not significantly, associated with KRASmut colon cancer in men and with BRAFmut and dMMR colon cancer in women. Our results suggest a role of KRASmut , PIK3CAmut , BRAFmut , and dMMR in the etiological pathway between height and CRC risk. KRASmut might potentially play a role in associations of early adult BMI with colon cancer risk in men, and BRAFmut and dMMR in women.

Cutaneous metastases of internal malignancies: a single-institution experience.

AIMS: Cutaneous metastases of internal malignancies occur in 1-10% of cancer patients. The diagnosis can sometimes be challenging, especially in cases with an unknown primary cancer. MATERIALS AND METHODS: A retrospective case review was performed including all cases of skin metastases from primary internal malignancies diagnosed at the Department of Pathology at the Maastricht University Medical Centre+ from 2007 to 2021. The clinicopathological data were collected and immunohistochemical and molecular diagnostic tests were performed to confirm the primary origin of the metastases. RESULTS: We identified 152 cases (71 female; 31 male patients) of cutaneous metastases of internal malignancies. 28 patients (20 women and 8 men) were diagnosed with multiple cutaneous metastases. Among the female patients, the most common primary tumour was breast cancer (50% of the cases), followed by lung (13.6%), gynaecological (7.3%), and gastrointestinal origin (7.3%). Among the male patients, the most common primary sites were gastrointestinal and lung origin (altogether, 50% of the cases). In 19 patients, the cutaneous metastasis was the first presentation of a clinically silent internal malignancy (18.6%), of which most (78.9%) represented metastatic lung carcinomas. Finally, metastasizing patterns were different across tumour types and gender. CONCLUSION: Breast, lung, gastrointestinal, and gynaecologic cancers are the most common primary tumours demonstrating skin metastases. Infrequently, cutaneous metastases can be the first clinically visual manifestation of an underlying not yet diagnosed internal malignancy; therefore, occasional broad immunohistochemical profiling, molecular clonal analysis, and a continuous high level of awareness are necessary for a precise diagnosis of cutaneous metastases of internal malignancies.

Energy balance-related factors and risk of colorectal cancer based on KRAS, PIK3CA, and BRAF mutations and MMR status.

INTRODUCTION: KRAS mutations (KRASmut), PIK3CAmut, BRAFmut, and mismatch repair deficiency (dMMR) have been associated with the Warburg-effect. We previously observed differential associations between energy balance-related factors (BMI, clothing-size, physical activity) and colorectal cancer (CRC) subtypes based on the Warburg-effect. We now investigated whether associations between energy balance-related factors and risk of CRC differ between subgroups based on mutation and MMR status. METHODS: Information on molecular features was available for 2349 incident CRC cases within the Netherlands Cohort Study (NLCS), with complete covariate data available for 1934 cases and 3911 subcohort members. Multivariable-adjusted Cox-regression was used to estimate associations of energy balance-related factors with risk of CRC based on individual molecular features (KRASmut; PIK3CAmut; BRAFmut; dMMR) and combinations thereof (all-wild-type + MMR-proficient (pMMR); any-mutation/dMMR). RESULTS: In men, BMI and clothing-size were positively associated with risk of colon, but not rectal cancer, regardless of molecular features subgroups; the strongest associations were observed for PIK3CAmut colon cancer. In women, however, BMI and clothing-size were only associated with risk of KRASmut colon cancer (p-heterogeneityKRASmut versus all-wild-type+pMMR = 0.008). Inverse associations of non-occupational physical activity with risk of colon cancer were strongest for any-mutation/dMMR tumors in men and women, and specifically for PIK3CAmut tumors in women. Occupational physical activity was inversely associated with both combination subgroups of colon cancer in men. CONCLUSION: In men, associations did not vary according to molecular features. In women, a role of KRAS mutations in the etiological pathway between adiposity and colon cancer is suggested, and of PIK3CA mutations between physical activity and colon cancer.

Energy balance-related factors in childhood and adolescence and risk of colorectal cancer expressing different levels of proteins involved in the Warburg-effect.

Early-life (childhood to adolescence) energy balance-related factors (height, energy restriction, BMI) have been associated with adult colorectal cancer (CRC) risk. Warburg-effect activation via PI3K/Akt-signaling might explain this link. We investigated whether early-life energy balance-related factors were associated with risk of Warburg-subtypes in CRC. We used immunohistochemistry for six proteins involved in the Warburg-effect (LDHA, GLUT1, MCT4, PKM2, P53, and PTEN) on tissue microarrays of 2399 incident CRC cases from the prospective Netherlands Cohort Study (NLCS). Expression levels of all proteins were combined into a pathway-based sum score and categorized into three Warburg-subtypes (Warburg-low/-moderate/-high). Multivariable Cox-regression analyses were used to estimate associations of height, energy restriction proxies (exposure to Dutch Hunger Winter; Second World War [WWII]; Economic Depression) and adolescent BMI with Warburg-subtypes in CRC. Height was positively associated with colon cancer in men, regardless of Warburg-subtypes, and with Warburg-low colon and Warburg-moderate rectal cancer in women. Energy restriction during the Dutch Hunger Winter was inversely associated with colon cancer in men, regardless of Warburg-subtypes. In women, energy restriction during the Hunger Winter and WWII was inversely associated with Warburg-low colon cancer, whereas energy restriction during the Economic Depression was positively associated with Warburg-high colon cancer. Adolescent BMI was positively associated with Warburg-high colon cancer in men, and Warburg-moderate rectal cancer in women. In conclusion, the Warburg-effect seems to be involved in associations of adolescent BMI with colon cancer in men, and of energy restriction during the Economic Depression with colon cancer in women. Further research is needed to validate these results.

Expression of proteins associated with the Warburg-effect and survival in colorectal cancer.

Previous research has suggested that the expression of proteins related to the Warburg effect may have prognostic value in colorectal cancer (CRC), but results remain inconsistent. Our objective was to investigate the relationship between Warburg-subtypes and patient survival in a large population-based series of CRC patients. In the present study, we investigated the expression of six proteins related to the Warburg effect (LDHA, GLUT1, MCT4, PKM2, p53, PTEN) by immunohistochemistry on tissue microarrays (TMAs) from 2,399 incident CRC patients from the prospective Netherlands Cohort Study. Expression levels of the six proteins were combined into a pathway-based sum-score and patients were categorised into three Warburg-subtypes (low/moderate/high). The associations between Warburg-subtypes and CRC-specific and overall survival were investigated using Kaplan-Meier curves and Cox regression models. CRC patients were classified as Warburg-low (n = 695, 29.0%), Warburg-moderate (n = 858, 35.8%) or Warburg-high (n = 841, 35.1%). Patients with Warburg-high CRC had the poorest CRC-specific [hazard ratio (HR) 1.17; 95% CI 1.00-1.38] and overall survival (HR 1.19; 95% CI 1.05-1.35), independent of known prognostic factors. In stratified analyses, this was particularly true for patients with tumour-node-metastasis (TNM) stage III CRC (HRCRC-specific 1.45; 95% CI 1.10-1.92 and HRoverall 1.47; 95% CI 1.15-1.87), and cancers located in the rectum (HRoverall 1.56; 95% CI 1.15-2.13). To our knowledge, this is the first study to identify the prognostic value of immunohistochemistry-based Warburg-subtypes in CRC. Our data suggest that Warburg-subtypes are related to potentially important differences in CRC survival. Further research is required to validate our findings and to investigate the potential clinical utility of these Warburg-subtypes in CRC.

Energy Balance-Related Factors and Risk of Colorectal Cancer Expressing Different Levels of Proteins Involved in the Warburg Effect.

BACKGROUND: Energy balance-related factors [body mass index (BMI), waist circumference, physical activity] have been associated with colorectal cancer risk. Warburg effect activation via PI3K/Akt signaling is one of the proposed mechanisms. We investigated whether energy balance-related factors were associated with risk of Warburg subtypes in colorectal cancer. METHODS: We investigated this using immunohistochemistry for six proteins involved in the Warburg effect (LDHA, GLUT1, MCT4, PKM2, P53, PTEN) on tissue microarrays of 2,399 incident colorectal cancer cases from the prospective Netherlands Cohort Study (ntotal = 120,852; nsubcohort = 5,000; aged 55-69 in 1986; 20.3 years follow-up). Data analyses included 3,911 subcohort members and 1,972 colorectal cancer cases with complete covariate data. Expression levels of all proteins were combined into a pathway-based sum score and categorized into three "Warburg subtypes" (Warburg-low/moderate/high). Multivariable Cox regression analyses were used to estimate associations of BMI, clothing size (waist circumference proxy), and physical activity with Warburg subtypes in colorectal cancer. RESULTS: BMI and clothing size were positively associated with Warburg-moderate and Warburg-high colon cancer risk in men (Pheterogeneity = 0.192). In women, clothing size was positively associated with Warburg-low and Warburg-high colon cancer (Pheterogeneity = 0.005). Nonoccupational physical activity was inversely associated with Warburg-low and Warburg-moderate colon cancer in women (Pheterogeneity = 0.045), but positively associated with Warburg-high rectal cancer in men (Pheterogeneity = 0.089). CONCLUSIONS: The Warburg effect might be involved in associations between adiposity and colon cancer risk, though additional mechanisms could be at play in women as well. The inverse association between physical activity and colon cancer might be explained by mechanisms other than the Warburg effect. IMPACT: Further research is needed to reproduce these results and investigate possible additional mechanisms.

Histopathologic and clinical comparison of recurrent and non-recurrent urethral stricture disease treated by reconstructive surgery.

BACKGROUND: Urethral stricture is a relatively frequent problem often requiring multiple surgical interventions. The objective of this study was to compare the clinicopathologic features of urethral resections from patients who underwent open end-to-end anastomotic urethroplasty and later recurred compared to those who did not. METHODS: A retrospective review of the pathology files identified 36 consecutive patients who underwent urethroplasty. The histopathological analysis included evaluation of the inflammatory infiltrate based on the predominant (>50%) cell type: lymphocyte-rich, neutrophil-rich, plasma cell-rich, and mixed; length and thickness of the fibrous plaque; and the cellularity of the fibrous plaque: cellular (>40 stroma nuclei/HPF) or paucicellular (<40 stroma nuclei/high power field). RESULTS: Ten (28%) patients recurred, and 26 (72%) did not. There was no significant difference between recurrent and non-recurrent cases in age, race, comorbidities, location of the stricture, and etiology. All patients with recurrent strictures showed dense paucicellular fibrotic plaques (10/10; 100%), while this was seen in 14/26 (53.8%) non-recurrent cases (P=0.01). Only one patient with cellular fibrosis showed recurrence during follow-up. The log-rank test shows that time to recurrence is significantly shorter in patients with paucicellular fibrosis compared to those with cellular fibrosis (P=0.036). The inflammation consisted of a mixed population of CD3(+) T-lymphocytes, CD20(+) B-lymphocytes, and CD68(+) histiocytes, and there was no difference in the composition of the inflammation between groups. All cases with plasma cell-rich infiltrate showed normal IgG4:IgG. CONCLUSIONS: Our study supports reporting cellularity of the fibrous plaque as a potential predictor of outcome in patients undergoing reconstructive urethroplasty. Patients with paucicellular fibrosis are at increased risk of recurrence.

Validity and Reproducibility of Immunohistochemical Scoring by Trained Non-Pathologists on Tissue Microarrays.

BACKGROUND: Scoring of immunohistochemistry (IHC) staining is often done by non-pathologists, especially in large-scale tissue microarray (TMA)-based studies. Studies on the validity and reproducibility of scoring results from non-pathologists are limited. Therefore, our main aim was to assess interobserver agreement between trained non-pathologists and an experienced histopathologist for three IHC markers with different subcellular localization (nucleus/membrane/cytoplasm). METHODS: Three non-pathologists were trained in recognizing adenocarcinoma and IHC scoring by a senior histopathologist. Kappa statistics were used to analyze interobserver and intraobserver agreement for 6,249 TMA cores from a colorectal cancer series. RESULTS: Interobserver agreement between non-pathologists (independently scored) and the histopathologist was "substantial" for nuclear and membranous IHC markers (κrange = 0.67-0.75 and κrange = 0.61-0.69, respectively), and "moderate" for the cytoplasmic IHC marker (κrange = 0.43-0.57). Scores of the three non-pathologists were also combined into a "combination score" (if at least two non-pathologists independently assigned the same score to a core, this was the combination score). This increased agreement with the pathologist (κnuclear = 0.74; κmembranous = 0.73; κcytopasmic = 0.57). Interobserver agreement between non-pathologists was "substantial" (κnuclear = 0.78; κmembranous = 0.72; κcytopasmic = 0.61). Intraobserver agreement of non-pathologists was "substantial" to "almost perfect" (κnuclear,range = 0.83-0.87; κmembranous,range = 0.75-0.82; κcytopasmic = 0.69). Overall, agreement was lowest for the cytoplasmic IHC marker. CONCLUSIONS: This study shows that adequately trained non-pathologists are able to generate reproducible IHC scoring results, that are similar to those of an experienced histopathologist. A combination score of at least two non-pathologists yielded optimal results. IMPACT: Non-pathologists can generate reproducible IHC results after appropriate training, making analyses of large-scale molecular pathological epidemiology studies feasible within an acceptable time frame.