Endometriosis and Isthmocele: Common or Rare?

Higher cesarean section rates and better ultrasound diagnostics have led to a more frequent diagnosis of isthmocele, a cesarean scar defect. Sometimes, endometriosis is found in the isthmocele, but simultaneous extrauterine endometriosis and endometriosis in the isthmocele have not yet been reported. Additionally, the surgical technique to repair the isthmocele is the subject of ongoing controversy. The aim of this study is to analyze a possible correlation between uterine scar (isthmocele) endometriosis and extrauterine endometriosis and to investigate the outcome of laparoscopic isthmocele resection in the rendezvous technique. In this single-center retrospective study, we included 83 women of reproductive age with symptomatic isthmocele undergoing laparoscopic isthmocele repair in rendezvous technique from 2004 to 2020 at the University of Bern. We collected data on patient and surgical characteristics as well as on postoperative outcomes (symptoms, further pregnancy, and pregnancy outcomes) retrospectively. We analyzed and compared these data for patients with and without endometriosis. Endometriosis was diagnosed during surgery in 22 out of 83 operated patients (26.5%). Diagnosis of isthmocele endometriosis (n = 9, 11%) was significantly higher in patients with extrauterine endometriosis (n = 6, p = 0.004). While the duration of surgery was significantly longer for patients with endometriosis (p = 0.006), the groups did not differ with regard to blood loss or complications. In addition, both groups showed similar indications for isthmocele repair (infertility, abnormal uterine bleeding, or dysmenorrhea). Surgery significantly improved abnormal uterine bleeding (χ2 p < 0.001), dysmenorrhea (χ2, p = 0.03), and infertility (χ2, p < 0.001). Regardless of the presence of endometriosis, 25 of 40 (63%) infertile patients became pregnant after surgery. In one out of eight pregnancies, however, we observed scar complications during pregnancy such as uterine scar pregnancy (n = 3), uterine scar dehiscence (n = 3), and placenta previa (n = 1). Endometriosis is a non-negligible intraoperative finding in patients with symptomatic isthmocele. The laparoscopic approach in the rendezvous technique is safe and effective. Therefore, this method should be recommended, especially in women with secondary infertility, and preoperatively simultaneous endometriosis resection should be discussed with the patient. In follow-up, postoperative pregnancies have to be monitored with care

Real-life data from standardized preanalytical coding (SPREC) in tissue biobanking and its dual use for sample characterization and process optimization.

The standardized preanalytical code (SPREC) aggregates warm ischemia (WIT), cold ischemia (CIT), and fixation times (FIT) in a precise format. Despite its growing importance underpinned by the European in vitro diagnostics regulation or broad preanalytical programs by the National Institutes of Health, little is known about its empirical occurrence in biobanked surgical specimen. In several steps, the Tissue Bank Bern achieved a fully informative SPREC code with insights from 10,555 CIT, 4,740 WIT, and 3,121 FIT values. During process optimization according to LEAN six sigma principles, we identified a dual role of the SPREC code as a sample characteristic and a traceable process parameter. With this preanalytical study, we outlined real-life data in a variety of organs with specific differences in WIT, CIT, and FIT values. Furthermore, our FIT data indicate the potential to adapt the SPREC fixation toward concrete paraffin-embedding time points and to extend its categories beyond 72 h due to weekend delays. Additionally, we identified dependencies of preanalytical variables from workload, daytime, and clinics that were actionable with LEAN process management. Thus, streamlined biobanking workflows during the day were significantly resilient to workload peaks, diminishing the turnaround times of native tissue processing (i.e. CIT) from 74.6 to 46.1 min under heavily stressed conditions. In conclusion, there are surgery-specific preanalytics that are surgico-pathologically limited even under process optimization, which might affect biomarker transfer from one entity to another. Beyond sample characteristics, SPREC coding is highly beneficial for tissue banks and Institutes of Pathology to track WIT, CIT, and FIT for process optimization and monitoring measurements

Brown bowel syndrome: A rare complication in diseases associated with long-standing malabsorption

BACKGROUND/AIMS Longtime chronic malabsorption may among other things cause a lack of liposoluble vitamins. Vitamin E deficiency can lead to formation of lipofuscin aggregates. Its deficiency is also associated with an increased lipofuscinosis of the bowel, i.e. brown bowel syndrome. METHODS Systematic research via Medline on brown bowel syndrome, lipofuscinosis, and vitamin E deficiency was performed. We combined our own clinical experience and a review of the literature for this paper. Its goal is to inform about the possible consequences of severe malabsorption and brown bowel syndrome. RESULTS Systematic data about the occurrence of severe malabsorption and brown bowel syndrome are rare. Only about 27 scientific reports can be found on this subject. Brown bowel syndrome is found mostly in conjunction with vitamin E deficiency and lipofuscinosis of the bowel. The clinical findings are caused by both malabsorption and lipofuscinosis. Case reports show a therapeutic effect of vitamin E. CONCLUSION Vitamin deficiency caused by longtime chronic malabsorption can lead to the development of brown bowel syndrome, which is seen as the expression of lipofuscinosis of the bowel, and can cause further clinical disorders. Patients with malabsorption should therefore be monitored regarding their vitamin E levels

Pathological processing of sentinel lymph nodes in endometrial carcinoma - routine aspects of grossing, ultra-staging, and surgico-pathological parameters in a series of 833 lymph nodes.

Sentinel lymph nodes are widely accepted in the treatment of endometrial carcinoma. Whereas surgical aspects are well studied, the pathological work-up in terms of grossing, frozen section, and the so-called ultra-staging is still a matter of debate. This results in conflicting national or center-based recommendations. In a series of consecutive 833 sentinel lymph nodes from 206 patients in endometrial carcinomas, we compared three different grossing techniques and the use of frozen section in terms of anatomy, detection rates, and survival. In total, 42 macro-metastases, 6 micro-metastases, and 25 nodes with isolated tumor cells were found. Lymph nodes affected at least with micro-metastasis were about 0.5cm enlarged. Detection rates in lamellation technique increased with a step of 5.9% to 8.3% in comparison to bi-valved or complete embedding. The lamellation technique presented with a slight beneficial prognosis in pN0 subgroup (OS, p=0.05), which besides size effects might be attributed to trimming loss. In frozen section, this effect was less pronounced than expected (OS, p=0.56). Ultra-staging only revealed additional micro-metastases and isolated tumor cells. Exclusively, macro-metastases showed poor survival (p<0.001). In multivariate analysis, T-stage, subtype, and lympho-vascular invasion status outperformed this staging parameter significantly. Grossing of sentinel lymph nodes is the most essential step with evidence to prefer lamellation in 2 mm steps. Step sectioning should consider widely spaced protocols to exclude macro-metastases. Frozen sections might add value to the intra-operative assessment of endometrial carcinoma in selected cases. The excellent biological behavior of cases with isolated tumor cells might question the routine application of pan-cytokeratin as ultra-staging method

Closing the loop – the role of pathologists in digital and computational pathology research

An increasing number of manuscripts related to digital and computational pathology are being submitted to The Journal of Pathology: Clinical Research as part of the continuous evolution from digital imaging and algorithm-based digital pathology to computational pathology and artificial intelligence. However, despite these technological advances, tissue analysis still relies heavily on pathologists’ annotations. There are three crucial elements to the pathologist’s role during annotation tasks: granularity, time constraints, and responsibility for the interpretation of computational results. Granularity involves detailed annotations, including case level, regional, and cellular features; and integration of attributions from different sources. Time constraints due to pathologistshortages have led to the development of techniques to expedite annotation tasks from cell-level attributions up to so-called unsupervised learning. The impact of pathologists may seem diminished, but their role is crucial inproviding ground truth and connecting pathological knowledge generation with computational advancements. Measures to display results back to pathologists and reflections about correctly applied diagnostic criteria are mandatory to maintain fidelity during human–machine interactions. Collaboration and iterative processes, such as human-in-the-loop machine learning are key for continuous improvement, ensuring the pathologist’s involvement in evaluating computational results and closing the loop for clinical applicability. The journal is interested particularly in the clinical diagnostic application of computational pathology and invites submissions that address the issues raised in this editoria

Thymoquinone-induced conformational changes of PAK1 interrupt prosurvival MEK-ERK signaling in colorectal cancer

Background Thymoquinone (TQ) was shown to reduce tumor growth in several cancer models both in vitro and in vivo. So far only a few targets of TQ, including protein kinases have been identified. Considering that kinases are promising candidates for targeted anticancer therapy, we studied the complex kinase network regulated by TQ. Methods Novel kinase targets influenced by TQ were revealed by in silico analysis of peptide array data obtained from TQ-treated HCT116wt cells. Western blotting and kinase activity assays were used to determine changes in kinase expression patterns in colorectal cancer cells (HCT116wt, DLD-1, HT29). To study the viability/apoptotic effects of combining the PAK1 inhibitor IPA-3 and TQ, crystal violet assay and AnnexinV/PI staining were employed. Interactions between PAK1 and ERK1/2 were investigated by co-immunoprecipitation and modeled by docking studies. Transfection with different PAK1 mutants unraveled the role of TQ-induced changes in PAK1 phosphorylation and TQ´s effects on PAK1 scaffold function. Results Of the 104 proteins identified, 50 were upregulated ≥2 fold by TQ and included molecules in the AKT-MEK-ERK1/2 pathway. Oncogenic PAK1 emerged as an interesting TQ target. Time-dependent changes in two PAK1 phosphorylation sites generated a specific kinase profile with early increase in pPAKThr212 followed by late increase in pPAKThr423. TQ induced an increase of pERK1/2 and triggered the early formation of an ERK1/2-PAK1 complex. Modeling confirmed that TQ binds in the vicinity of Thr212 accompanied by conformational changes in ERK2-PAK1 binding. Transfecting the cells with the non-phosphorylatable mutant T212A revealed an increase of pPAKThr423 and enhanced apoptosis. Likewise, an increase in apoptosis was observed in cells transfected with both the kinase-dead K299R mutant and PAK1 siRNA. Using structural modeling we suggest that TQ interferes also with the kinase domain consequently disturbing its interaction with pPAKThr423, finally inhibiting MEK-ERK1/2 signaling and disrupting its prosurvival function. pERK1/2 loss was also validated in vivo. Conclusions Our study shows for the first time that the small molecule TQ directly binds to PAK1 changing its conformation and scaffold function. Because TQ affects the central RAF/MEK/ERK1/2 pathway, the combination of TQ with targeted therapies is worth considering for future anticancer treatments

Spatial distribution of CD3- and CD8-positive lymphocytes as pretest for POLE wild-type in molecular subgroups of endometrial carcinoma.

INTRODUCTION Over the years, the molecular classification of endometrial carcinoma has evolved significantly. Both POLEmut and MMRdef cases share tumor biological similarities like high tumor mutational burden and induce strong lymphatic reactions. While therefore use case scenarios for pretesting with tumor-infiltrating lymphocytes to replace molecular analysis did not show promising results, such testing may be warranted in cases where an inverse prediction, such as that of POLEwt, is being considered. For that reason we used a spatial digital pathology method to quantitatively examine CD3+ and CD8+ immune infiltrates in comparison to conventional histopathological parameters, prognostics and as potential pretest before molecular analysis. METHODS We applied a four-color multiplex immunofluorescence assay for pan-cytokeratin, CD3, CD8, and DAPI on 252 endometrial carcinomas as testing and compared it to further 213 cases as validation cohort from a similar multiplexing assay. We quantitatively assessed immune infiltrates in microscopic distances within the carcinoma, in a close distance of 50 microns, and in more distant areas. RESULTS Regarding prognostics, high CD3+ and CD8+ densities in intra-tumoral and close subregions pointed toward a favorable outcome. However, TCGA subtyping outperforms prognostication of CD3 and CD8 based parameters. Different CD3+ and CD8+ densities were significantly associated with the TCGA subgroups, but not consistently for histopathological parameter. In the testing cohort, intra-tumoral densities of less than 50 intra-tumoral CD8+ cells/mm2 were the most suitable parameter to assume a POLEwt, irrespective of an MMRdef, NSMP or p53abn background. An application to the validation cohort corroborates these findings with an overall sensitivity of 95.5%. DISCUSSION Molecular confirmation of POLEmut cases remains the gold standard. Even if CD3+ and CD8+ cell densities appeared less prognostic than TCGA, low intra-tumoral CD8+ values predict a POLE wild-type at substantial percentage rates, but not vice versa. This inverse correlation might be useful to increase pretest probabilities in consecutive POLE testing. Molecular subtyping is currently not conducted in one-third of cases deemed low-risk based on conventional clinical and histopathological parameters. However, this percentage could potentially be increased to two-thirds by excluding sequencing of predicted POLE wild-type cases, which could be determined through precise quantification of intra-tumoral CD8+ cells

Clinicopathological and molecular characterisation of “multiple classifier” endometrial carcinomas

Endometrial carcinoma (EC) molecular classification based on four molecular subclasses identified in The Cancer Genome Atlas (TCGA) has gained relevance in recent years due to its prognostic utility and potential to predict benefit from adjuvant treatment. While most ECs can be classified based on a single classifier (POLE exonuclease domain mutations - POLEmut, MMR deficiency - MMRd, p53 abnormal - p53abn), a small but clinically relevant group of tumours harbour more than one molecular classifying feature and are referred to as 'multiple-classifier' ECs. We aimed to describe the clinicopathological and molecular features of multiple-classifier ECs with abnormal p53 (p53abn). Within a cohort of 3518 molecularly profiled ECs, 107 (3%) tumours displayed p53abn in addition to another classifier(s), including 64 with MMRd (MMRd-p53abn), 31 with POLEmut (POLEmut-p53abn), and 12 with all three aberrations (MMRd-POLEmut-p53abn). MMRd-p53abn ECs and POLEmut-p53abn ECs were mostly grade 3 endometrioid ECs, early stage, and frequently showed morphological features characteristic of MMRd or POLEmut ECs. 18/28 (60%) MMRd-p53abn ECs and 7/15 (46.7%) POLEmut-p53abn ECs showed subclonal p53 overexpression, suggesting that TP53 mutation was a secondary event acquired during tumour progression. Hierarchical clustering of TCGA ECs by single nucleotide variant (SNV) type and somatic copy number alterations (SCNAs) revealed that MMRd-p53abn tumours mostly clustered with single-classifier MMRd tumours (20/23) rather than single-classifier p53abn tumours (3/23), while POLEmut-p53abn tumours mostly clustered with single-classifier POLEmut tumours (12/13) and seldom with single-classifier p53abn tumours (1/13) (both p ≤ 0.001, chi-squared test). Finally, the clinical outcome of patients with MMRd-p53abn and POLEmut-p53abn ECs [stage I 5-year recurrence-free survival (RFS) of 92.2% and 94.1%, respectively] was significantly different from single-classifier p53abn EC (stage I RFS 70.8%, p = 0.024 and p = 0.050, respectively). Our results support the classification of MMRd-p53abn EC as MMRd and POLEmut-p53abn EC as POLEmut. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland

IL-9 and its receptor are predominantly involved in the pathogenesis of UC

Objective: Several pathogenic roles attributed over the past two decades to either T helper (Th)1 or Th2 cells are increasingly becoming associated with interleukin (IL)-17 and most recently IL-9 signalling. However, the implication of IL-9 in IBD has not been addressed so far. Design: We investigated the expression of IL-9 and IL-9R by using peripheral blood, biopsies and surgical samples. We addressed the functional role of IL-9 signalling by analysis of downstream effector proteins. Using Caco-2 cell monolayers we followed the effect of IL-9 on wound healing. Results: IL-9 mRNA expression was significantly increased in inflamed samples from patients with UC as compared with controls. CD3+ T cells were major IL-9-expressing cells and some polymorphonuclear leucocytes (PMN) also expressed IL-9. IL-9 was co-localised with the key Th9 transcription factors interferon regulatory factor 4 and PU.1. Systemically, IL-9 was abundantly produced by activated peripheral blood lymphocytes, whereas its receptor was overexpressed on gut resident and circulating PMN. IL-9 stimulation of the latter induced IL-8 production in a dose-dependent manner and rendered PMN resistant to apoptosis suggesting a functional role for IL-9R signalling in the propagation of gut inflammation. Furthermore, IL-9R was overexpressed on gut epithelial cells and IL-9 induced STAT5 activation in these cells. Moreover, IL-9 inhibited the growth of Caco-2 epithelial cell monolayers in wound healing experiments. Conclusions: Our results provide evidence that IL-9 is predominantly involved in the pathogenesis of UC suggesting that targeting IL-9 might become a therapeutic option for patients with UC