How to learn with intentional mistakes: NoisyEnsembles to overcome poor tissue quality for deep learning in computational pathology.

There is a lot of recent interest in the field of computational pathology, as many algorithms are introduced to detect, for example, cancer lesions or molecular features. However, there is a large gap between artificial intelligence (AI) technology and practice, since only a small fraction of the applications is used in routine diagnostics. The main problems are the transferability of convolutional neural network (CNN) models to data from other sources and the identification of uncertain predictions. The role of tissue quality itself is also largely unknown. Here, we demonstrated that samples of the TCGA ovarian cancer (TCGA-OV) dataset from different tissue sources have different quality characteristics and that CNN performance is linked to this property. CNNs performed best on high-quality data. Quality control tools were partially able to identify low-quality tiles, but their use did not increase the performance of the trained CNNs. Furthermore, we trained NoisyEnsembles by introducing label noise during training. These NoisyEnsembles could improve CNN performance for low-quality, unknown datasets. Moreover, the performance increases as the ensemble become more consistent, suggesting that incorrect predictions could be discarded efficiently to avoid wrong diagnostic decisions.

Myofibroblastic CAF Density, Not Activated Stroma Index, Indicates Prognosis after Neoadjuvant Therapy of Pancreatic Carcinoma.

Neoadjuvant therapy (NT) for advanced PDAC is an emerging concept, affecting both stroma and tumor. The Activated Stroma Index (ASI; ratio of activated cancer-associated fibroblasts (CAF) to collagen deposition) is a prognostic marker in upfront resected pancreatic adenocarcinoma (PDAC). We assessed ASI and its prognostic relevance after NT. Tissue from resection specimens of n = 48 PDAC patients after neoadjuvant chemotherapy with FOLFIRINOX (FOL; n = 31), gemcitabine + nab-paclitaxel (GEM; 7) or combination treatment (COMB; 10) was compared with upfront resected matched controls (RES; 69). Activated CAFs were assessed by immunohistochemistry for α-SMA, and collagen was stained with aniline blue; the stained area was then determined by computational imaging analysis and ASI was calculated. In GEM, ASI was significantly higher and collagen deposition lower than in controls and FOL. The lowest quartile of ASI values had significantly longer overall survival (OS) in RES, whereas in FOL, the highest quartile had the best prognosis. After NT, OS was significantly improved in the α-SMA-high group; in RES, however, survival was independent of α-SMA. Reversed prognostic association of ASI thus points to the differing significance of stromal composition after FOL, while improved prognosis with high CAF abundance suggests a synergistic effect of myofibroblasts with chemotherapy. These divergences impede usability of ASI after NT.

Circulating 27-hydroxycholesterol and breast cancer tissue expression of CYP27A1, CYP7B1, LXR-ß, and ERß: results from the EPIC-Heidelberg cohort.

BACKGROUND: Experimental and epidemiological studies demonstrate a role for 27-hydroxycholesterol (27HC) in breast cancer development, though results are conflicting. Cholesterol 27-hydroxylase (CYP27A1) and oxysterol 7-alpha-hydroxylase (CYP7B1) regulate 27HC concentrations, while differential expression of the liver X receptor (LXR) and estrogen receptor beta (ERß) may impact the association between 27HC and breast cancer risk. METHODS: We evaluated correlates of tumor tissue expression of CYP27A1, CYP7B1, LXR-ß, and ERß and the association between circulating prediagnostic 27HC concentrations and breast cancer risk by marker expression in a nested case-control study within the European Prospective Investigation into Cancer and Nutrition (EPIC)-Heidelberg cohort including 287 breast cancer cases with tumor tissue available. Tumor protein expression was evaluated using immunohistochemistry, and serum 27HC concentrations quantified using liquid chromatography-mass spectrometry. Conditional logistic regression models were used to estimate odds ratios (ORs) and 95% confidence intervals (CIs). RESULTS: A higher proportion of CYP7B1-positive cases were progesterone receptor (PR)-positive, relative to CYP7B1-negative cases, whereas a higher proportion of ERß-positive cases were Bcl-2 low, relative to ERß-negative cases. No differences in tumor tissue marker positivity were observed by reproductive and lifestyle factors. We observed limited evidence of heterogeneity in associations between circulating 27HC and breast cancer risk by tumor tissue expression of CYP27A1, CYP7B1, LXR-ß, and ERß, with the exception of statistically significant heterogeneity by LXR-ß status in the subgroup of women perimenopausal at blood collection (p = 0.02). CONCLUSION: This exploratory study suggests limited associations between tumor marker status and epidemiologic or breast cancer characteristics. Furthermore, the association between circulating 27HC and breast cancer risk may not vary by tumor expression of CYP27A1, CYP7B1, LXR-ß, or ERß.

Obesity as risk factor for subtypes of breast cancer: results from a prospective cohort study.

BACKGROUND: Earlier epidemiological studies indicate that associations between obesity and breast cancer risk may not only depend on menopausal status and use of exogenous hormones, but might also differ by tumor subtype. Here, we evaluated whether obesity is differentially associated with the risk of breast tumor subtypes, as defined by 6 immunohistochemical markers (ER, PR, HER2, Ki67, Bcl-2 and p53, separately and combined), in the prospective EPIC-Germany Study (n = 27,012). METHODS: Formalin-fixed and paraffin-embedded (FFPE) tumor tissues of 657 incident breast cancer cases were used for histopathological analyses. Associations between BMI and breast cancer risk across subtypes were evaluated by multivariable Cox regression models stratified by menopausal status and hormone therapy (HT) use. RESULTS: Among postmenopausal non-users of HT, higher BMI was significantly associated with an increased risk of less aggressive, i.e. ER+, PR+, HER2-, Ki67low, Bcl-2+ and p53- tumors (HR per 5 kg/m2: 1.44 [1.10, 1.90], p = 0.009), but not with risk of more aggressive tumor subtypes. Among postmenopausal users of HT, BMI was significantly inversely associated with less aggressive tumors (HR per 5 kg/m2: 0.68 [0.50, 0.94], p = 0.018). Finally, among pre- and perimenopausal women, Cox regression models did not reveal significant linear associations between BMI and risk of any tumor subtype, although analyses by BMI tertiles showed a significantly lower risk of less aggressive tumors for women in the highest tertile (HR: 0.55 [0.33, 0.93]). CONCLUSION: Overall, our results suggest that obesity is related to risk of breast tumors with lower aggressiveness, a finding that requires replication in larger-scale analyses of pooled prospective data.

Cyclin K dependent regulation of Aurora B affects apoptosis and proliferation by induction of mitotic catastrophe in prostate cancer.

Cyclin K plays a critical role in transcriptional regulation as well as cell development. However, the role of Cyclin K in prostate cancer is unknown. Here, we describe the impact of Cyclin K on prostate cancer cells and examine the clinical relevance of Cyclin K as a biomarker for patients with prostate cancer. We show that Cyclin K depletion in prostate cancer cells induces apoptosis and inhibits proliferation accompanied by an accumulation of cells in the G2/M phase. Moreover, knockdown of Cyclin K causes mitotic catastrophe displayed by multinucleation and spindle multipolarity. Furthermore, we demonstrate a Cyclin K dependent regulation of the mitotic kinase Aurora B and provide evidence for an Aurora B dependent induction of mitotic catastrophe. In addition, we show that Cyclin K expression is associated with poor biochemical recurrence-free survival in patients with prostate cancer treated with an adjuvant therapy. In conclusion, targeting Cyclin K represents a novel, promising anti-cancer strategy to induce cell cycle arrest and apoptotic cell death through induction of mitotic catastrophe in prostate cancer cells. Moreover, our results indicate that Cyclin K is a putative predictive biomarker for clinical outcome and therapy response for patients with prostate cancer.

Spatial niche formation but not malignant progression is a driving force for intratumoural heterogeneity.

Intratumoural heterogeneity (ITH) is a major cause of cancer-associated lethality. Extensive genomic ITH has previously been reported in clear cell renal cell carcinoma (ccRCC). Here we address the question whether ITH increases with malignant progression and can hence be exploited as a prognostic marker. Unexpectedly, precision quantitative image analysis reveals that the degree of functional ITH is virtually identical between primary ccRCCs of the lowest stage and advanced, metastatic tumours. Functional ITH was found to show a stage-independent topological pattern with peak proliferative and signalling activities almost exclusively in the tumour periphery. Exome sequencing of matching peripheral and central primary tumour specimens reveals various region-specific mutations. However, these mutations cannot directly explain the zonal pattern suggesting a role of microenvironmental factors in shaping functional ITH. In conclusion, our results indicate that ITH is an early and general characteristic of malignant growth rather than a consequence of malignant progression.

Dynamin-mediated Nephrin phosphorylation regulates glucose-stimulated insulin release in pancreatic beta cells.

We have previously demonstrated a role for Nephrin in glucose stimulated insulin release (GSIR). We now hypothesize that Nephrin phosphorylation is required for GSIR and that Dynamin influences Nephrin phosphorylation and function. MIN6-C3 Nephrin-deficient pancreatic beta cells and human islets were transfected with WT-Nephrin or with a mutant Nephrin in which the tyrosine residues responsible for SH2 domain binding were substituted with phenylalanine (3YF-Nephrin). GSIR and live images of Nephrin and vesicle trafficking were studied. Immunoprecipitation experiments and overexpression of WT-Dynamin or dominant negative Dynamin mutant (K44A-Dynamin) in WT-Nephrin, 3YF-Nephrin, or Nephrin siRNA-transfected cells were utilized to study Nephrin-Dynamin interaction. In contrast to WT-Nephrin or to single tyrosine mutants, 3YF-Nephrin did not positively affect GSIR and led to impaired cell-cell contacts and vesicle trafficking. K44A-Dynamin prevented the effect of Nephrin on GSIR in the absence of protein-protein interaction between Nephrin and Dynamin. Nephrin gene silencing abolished the positive effects of WT-Dynamin on GSIR. The effects of protamine sulfate and vanadate on Nephrin phosphorylation and GSIR were studied in MIN6 cells and human islets. WT-Nephrin phosphorylation after glucose occurred at Tyr-1176/1193 and resulted in improved GSIR. On the contrary, protamine sulfate-induced phosphorylation at Tyr-1176/1193/1217 was associated with Nephrin degradation and impaired GSIR. Vanadate, which prevented Nephrin dephosphorylation after glucose stimulation, improved GSIR in human islets and MIN6 cells. In conclusion, Dynamin-dependent Nephrin phosphorylation occurs in response to glucose and is necessary for Nephrin-mediated augmentation of GSIR. Pharmacological modulation of Nephrin phosphorylation may thus facilitate pancreatic beta cell function.

UVA-induced phenoxyl radical formation: A new cytotoxic principle in photodynamic therapy.

Psoralens are regularly used in therapy in combination with ultraviolet A light irradiation (PUVA) to treat skin diseases such as psoriasis, vitiligo, and mycosis fungoides. PUVA therapy is also used within the scope of extracorporeal photopheresis to treat a variety of diseases that have a suspected involvement of pathogenic T cells, including rejection of organ transplants, graft-vs-host disease, cutaneous T cell lymphoma, and autoimmune disorders. Because psoralens are the only photosensitizers used in PUVA therapies and are considered to be responsible for a number of side effects, the identification of alternative drugs is of practical interest. Here we investigated the impact of activated Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), a hydrophilic vitamin E analog lacking the phytyl tail, as an alternative photoactivatable agent with T cell cytotoxic properties. Despite the well-known antioxidative capacity of Trolox, we found that at low UVA doses and in the presence of supraphysiological concentration of nitrite, a natural constituent of human skin, this compound selectively enhances radical-mediated cytotoxicity toward T cells but not toward human skin fibroblasts, keratinocytes, or endothelial cells. The cytotoxic mechanism comprises a reaction of Trolox with photo-decomposition products of nitrite, which leads to increased Trolox phenoxyl radical formation, increased intracellular oxidative stress, and a consecutive induction of apoptosis and necrosis in fast proliferating T cells. Thus, the identified UVA/nitrite-induced phenoxyl radical formation provides an opportunity for a new cytotoxic photodynamic therapy.

Impact of adenylyltransferase GlnE on nitrogen starvation response in Corynebacterium glutamicum.

Adenylyltransferases regulate glutamine synthetase activity in enterobacteria and actinomycetes such as Streptomyces coelicolor, Mycobacterium tuberculosis and Corynebacterium glutamicum. In this study the effects of a mutation of the glnE gene, coding for adenylyltransferase, on transcriptome and metabolome profiles of C. glutamicum was investigated. As expected, the glnE deletion led to a loss of activity regulation of glutamine synthetase. Astonishingly, additionally the glnE mutation caused a nitrogen limitation response on the transcript level as well. Interestingly, induction of the nitrogen starvation response in the mutant strain was unusually weak and GlnK was present in adenylylated form even without nitrogen starvation. The results obtained might hint to a moonlighting function of adenylyltransferase and might be explained by protein interaction of adenylyltransferase and an unknown interaction partner of the nitrogen regulatory network.

Protein p0071, an armadillo plaque protein of adherens junctions, is predominantly expressed in distal renal tubules.

Protein p0071 is a member of the p120-subfamily of armadillo proteins and is well known as a junctional plaque component involved in cell-cell adhesion, especially in adherens junctions. By systematic immunohistochemical analysis of mouse and human kidney tissues, p0071 was prominently detected in distinct kidney tubules. Upon double-labeling immunolocalization experiments with segment-specific markers, p0071 was predominantly localized in distal straight and convoluted tubules and to a lesser extent in proximal tubules, in the ascending thin limb of loop of Henle and in the collecting ducts. In capillaries of the kidney, p0071 co-localized with VE-cadherin an endothelium-specific cadherin. Protein p0071 was also detected in both, renal cell carcinomas derived from distal tubules and in maturing nephrons of early mouse developmental stages. Immunoblotting of total extracts of cultured cells of renal origin showed that p0071 was detected in all human and murine cells analyzed. Upon immunolocalization, p0071 was observed in adherens junctions but also in distinct cytoplasmic structures at the cell periphery of cultured cells. Possible structural and functional roles of p0071 are suggested by its preferential occurrence in distinct tubule segments, and its potential use as a cytodiagnostic cell type marker in renal pathology is discussed.

Differential expression pattern of protein ARVCF in nephron segments of human and mouse kidney.

The protein ARVCF is a member of the p120 subfamily of armadillo proteins whose members have been described to occur in junction-bound and non-junction-bound forms. Studies on ARVCF were constrained because the endogenous protein was difficult to detect with the available reagents. We have generated novel monoclonal and polyclonal antibodies usable for biochemical and localization studies. By systematic immunohistochemical analysis of various tissues protein ARVCF is prominently detected in mouse, bovine and human kidney. Using antibodies against specific markers of nephron segments protein ARVCF is localized in proximal tubules according to double label immunofluorescence. Besides its occurrence in proximal tubules of adult kidney and in renal cell carcinoma derived from proximal tubules ARVCF is also detected in maturing nephrons in early mouse developmental stages such as, for example, 15 days of gestation (E15). Immunoblotting of total extracts of cultured cells of renal origin showed that ARVCF is detected in all human and murine cultured cells analyzed. Upon immunolocalization ARVCF is mostly detected in the cytoplasm occurring in a fine granular form. This prominent cytoplasmic localization of ARVCF in cultured cells and its occurrence in proximal tubules implies an involvement of ARVCF in specific functional processes of proximal tubules of kidney.

Dissection of ammonium uptake systems in Corynebacterium glutamicum: mechanism of action and energetics of AmtA and AmtB.

Corynebacterium glutamicum has two different Amt-type proteins. While AmtB has a low substrate affinity and is not saturable up to 3 mM methylammonium, AmtA has a high substrate affinity and mediates saturable, membrane potential-dependent transport, resulting in a high steady-state accumulation of methylammonium, even in the absence of metabolic trapping.

A genomic view of sugar transport in Mycobacterium smegmatis and Mycobacterium tuberculosis.

We present a comprehensive analysis of carbohydrate uptake systems of the soil bacterium Mycobacterium smegmatis and the human pathogen Mycobacterium tuberculosis. Our results show that M. smegmatis has 28 putative carbohydrate transporters. The majority of sugar transport systems (19/28) in M. smegmatis belong to the ATP-binding cassette (ABC) transporter family. In contrast to previous reports, we identified genes encoding all components of the phosphotransferase system (PTS), including permeases for fructose, glucose, and dihydroxyacetone, in M. smegmatis. It is anticipated that the PTS of M. smegmatis plays an important role in the global control of carbon metabolism similar to those of other bacteria. M. smegmatis further possesses one putative glycerol facilitator of the major intrinsic protein family, four sugar permeases of the major facilitator superfamily, one of which was assigned as a glucose transporter, and one galactose permease of the sodium solute superfamily. Our predictions were validated by gene expression, growth, and sugar transport analyses. Strikingly, we detected only five sugar permeases in the slow-growing species M. tuberculosis, two of which occur in M. smegmatis. Genes for a PTS are missing in M. tuberculosis. Our analysis thus brings the diversity of carbohydrate uptake systems of fast- and a slow-growing mycobacteria to light, which reflects the lifestyles of M. smegmatis and M. tuberculosis in their natural habitats, the soil and the human body, respectively.

Nitrogen metabolism and nitrogen control in corynebacteria: variations of a common theme.

The published genome sequences of Corynebacterium diphtheriae, Corynebacterium efficiens, Corynebacterium glutamicum and Corynebacterium jeikeium were screened for genes encoding central components of nitrogen source uptake, nitrogen assimilation and nitrogen control systems. Interestingly, the soil-living species C. efficiens and C. glutamicum exhibit a broader spectrum of genes for nitrogen transport and metabolism than the pathogenic species C. diphtheriae and C. jeikeium. The latter are characterized by gene decay and loss of functions like urea metabolism and nitrogen-dependent transcription control. The global regulator of nitrogen regulation AmtR and its DNA-binding motif are conserved in C. diphtheriae, C. efficiens and C. glutamicum, while in C. jeikeium, an AmtR-encoding gene as well as putative AmtR-binding motifs are missing.

Ammonium toxicity in bacteria.

Although an excellent nitrogen source for most bacteria, ammonium was-in analogy to plant and animal systems-assumed be detrimental to bacteria when present in high concentrations. In this study, we examined the effect of molar ammonium concentrations on different model bacteria, namely, Corynebacterium glutamicum, Escherichia coli, and Bacillus subtilis. The studied bacteria are highly resistant to ammonium. When growth was impaired upon addition of molar (NH4)2SO4 concentrations, this was not caused by an ammonium-specific effect but was due to an enhanced osmolarity or increased ionic strength of the medium. Therefore, it was concluded that ammonium is not detrimental to C. glutamicum and other bacteria even when present in molar concentrations.

DNA microarray analysis of the nitrogen starvation response of Corynebacterium glutamicum.

Nitrogen is an essential component of nearly all of the complex macromolecules in a bacterial cell, e.g. proteins, nucleic acids, and cell wall components. Accordingly, most prokaryotes have developed elaborate control mechanisms to provide an optimal supply of nitrogen for cellular metabolism and to cope with situations of nitrogen limitation. In this communication, a global analysis of the Corynebacterium glutamicum nitrogen starvation response by transcriptional profiling using DNA microarrays is presented. Our results show that C. glutamicum reacts to nitrogen starvation with a rearrangement of the cellular transport capacity, changes in metabolic pathways concerning nitrogen assimilation and amino acid biosynthesis, and a decreased capacity for protein synthesis.