Clare Verrill

Inhibiting WEE1 Selectively Kills Histone H3K36me3-Deficient Cancers by dNTP Starvation

Summary Histone H3K36 trimethylation (H3K36me3) is frequently lost in multiple cancer types, identifying it as an important therapeutic target. Here we identify a synthetic lethal interaction in which H3K36me3-deficient cancers are acutely sensitive to WEE1 inhibition. We show that RRM2, a ribonucleotide reductase subunit, is the target of this synthetic lethal interaction. RRM2 is regulated by two pathways here: first, H3K36me3 facilitates RRM2 expression through transcription initiation factor recruitment; second, WEE1 inhibition degrades RRM2 through untimely CDK activation. Therefore, WEE1 inhibition in H3K36me3-deficient cells results in RRM2 reduction, critical dNTP depletion, S-phase arrest, and apoptosis. Accordingly, this synthetic lethality is suppressed by increasing RRM2 expression or inhibiting RRM2 degradation. Finally, we demonstrate that WEE1 inhibitor AZD1775 regresses H3K36me3-deficient tumor xenografts.

Hepatocyte Expression of the Senescence Marker p21 Is Linked to Fibrosis and an Adverse Liver-Related Outcome in Alcohol-Related Liver Disease

Background and Aim Alcohol-related liver disease (ALD) remains a leading cause of liver-related morbidity and mortality. Age, fibrosis stage, MELD score and continued alcohol consumption predict outcome in everyday clinical practice. In previous studies increased hepatocyte nuclear area and hepatocyte expression of p21, both markers of senescence, were associated with increased fibrosis stage and a poor outcome in non-alcohol-related fatty liver disease, while increased hepatocyte nuclear area was related to liver dysfunction in ALD cirrhosis. This study, therefore, investigated the pattern of hepatocyte cell cycle phase distribution and hepatocyte p21 expression in relation to outcome in ALD. Methods Liver sections from two cohorts were studied. The first comprised 42 patients across the full spectrum of ALD. The second cohort comprised 77 patients with ALD cirrhosis. Immunohistochemistry assessed hepatocyte expression of cell cycle phase markers and p21. Regenerating liver (n=12) and “normal” liver sections (n=5) served as positive and negative controls, respectively. Results In the first cohort there was little cell cycle progression beyond G1/S phase and increased hepatocyte p21 expression (p<0.0001), which correlated independently with fibrosis stage (p=0.005) and an adverse liver-related outcome (p=0.03). In the second cohort, both hepatocyte p21 expression (p<0.001) and MELD score (p=0.006) were associated independently with an adverse liver-related outcome; this association was stronger with hepatocyte p21 expression (AUROC 0.74; p=0.0002) than with MELD score (AUROC 0.59; p=0.13). Further, hepatocyte p21 expression co-localised with increased hepatic stellate cell activation. Conclusions The findings are consistent with impaired cell cycle progression beyond the G1/S phase in ALD. The striking independent associations between increased hepatocyte p21 expression and both fibrosis stage and an adverse liver-related outcome in both cohorts suggests hepatocyte senescence plays an important role in ALD. Measuring hepatocyte p21 expression is simple and cheap and in this series was a useful measure of long-term prognosis in ALD.

Visualizing the origins of selfish de novo mutations in individual seminiferous tubules of human testes

Significance A major goal in genetics is to understand the processes that shape the frequency of new mutations, particularly those causing human disease. Here, we focus on specific mutations in the male germline that, although initially rare, confer a growth or survival advantage to the stem cell, leading to clonal expansion over time: a process similar to early tumor growth and currently described only in humans. Previous studies supporting this “selfish” selection quantified mutations in sperm or testis pieces using methods that destroyed their cellular origins. Here, we pinpoint and identify pathogenic mutations directly within individual seminiferous tubules, the structures that generate spermatozoa. This methodology provides unprecedented precision in documenting the spectrum and prevalence of selfish mutations in men’s testes. De novo point mutations arise predominantly in the male germline and increase in frequency with age, but it has not previously been possible to locate specific, identifiable mutations directly within the seminiferous tubules of human testes. Using microdissection of tubules exhibiting altered expression of the spermatogonial markers MAGEA4, FGFR3, and phospho-AKT, whole genome amplification, and DNA sequencing, we establish an in situ strategy for discovery and analysis of pathogenic de novo mutations. In 14 testes from men aged 39–90 y, we identified 11 distinct gain-of-function mutations in five genes (fibroblast growth factor receptors FGFR2 and FGFR3, tyrosine phosphatase PTPN11, and RAS oncogene homologs HRAS and KRAS) from 16 of 22 tubules analyzed; all mutations have known associations with severe diseases, ranging from congenital or perinatal lethal disorders to somatically acquired cancers. These results support proposed selfish selection of spermatogonial mutations affecting growth factor receptor-RAS signaling, highlight its prevalence in older men, and enable direct visualization of the microscopic anatomy of elongated mutant clones.

Nuclear iASPP may facilitate prostate cancer progression

One of the major challenges in prostate cancer (PCa) research is the identification of key players that control the progression of primary cancers to invasive and metastatic disease. The majority of metastatic PCa express wild-type p53, whereas loss of p63 expression, a p53 family member, is a common event. Here we identify inhibitor of apoptosis-stimulating protein of p53 (iASPP), a common cellular regulator of p53 and p63, as an important player of PCa progression. Detailed analysis of the prostate epithelium of iASPP transgenic mice, iASPPΔ8/Δ8 mice, revealed that iASPP deficiency resulted in a reduction in the number of p63 expressing basal epithelial cells compared with that seen in wild-type mice. Nuclear and cytoplasmic iASPP expression was greater in PCa samples compared with benign epithelium. Importantly nuclear iASPP associated with p53 accumulation in vitro and in vivo. A pair of isogenic primary and metastatic PCa cell lines revealed that nuclear iASPP is enriched in the highly metastatic PCa cells. Nuclear iASPP is often detected in PCa cells located at the invasive leading edge in vivo. Increased iASPP expression associated with metastatic disease and PCa-specific death in a clinical cohort with long-term follow-up. These results suggest that iASPP function is required to maintain the expression of p63 in normal basal prostate epithelium, and nuclear iASPP may inactivate p53 function and facilitate PCa progression. Thus iASPP expression may act as a predictive marker of PCa progression.Cell Death and Disease (2014) 5, e1492; doi:10.1038/cddis.2014.456; published online 23 October 2014

Dsh homolog DVL3 mediates resistance to IGFIR inhibition by regulating IGF-RAS signaling

Drugs that inhibit insulin-like growth factor 1 (IGFI) receptor IGFIR were encouraging in early trials, but predictive biomarkers were lacking and the drugs provided insufficient benefit in unselected patients. In this study, we used genetic screening and downstream validation to identify the WNT pathway element DVL3 as a mediator of resistance to IGFIR inhibition. Sensitivity to IGFIR inhibition was enhanced specifically in vitro and in vivo by genetic or pharmacologic blockade of DVL3. In breast and prostate cancer cells, sensitization tracked with enhanced MEK-ERK activation and relied upon MEK activity and DVL3 expression. Mechanistic investigations showed that DVL3 is present in an adaptor complex that links IGFIR to RAS, which includes Shc, growth factor receptor-bound-2 (Grb2), son-of-sevenless (SOS), and the tumor suppressor DAB2. Dual DVL and DAB2 blockade synergized in activating ERKs and sensitizing cells to IGFIR inhibition, suggesting a nonredundant role for DVL3 in the Shc-Grb2-SOS complex. Clinically, tumors that responded to IGFIR inhibition contained relatively lower levels of DVL3 protein than resistant tumors, and DVL3 levels in tumors correlated inversely with progression-free survival in patients treated with IGFIR antibodies. Because IGFIR does not contain activating mutations analogous to EGFR variants associated with response to EGFR inhibitors, we suggest that IGF signaling achieves an equivalent integration at the postreceptor level through adaptor protein complexes, influencing cellular dependence on the IGF axis and identifying a patient population with potential to benefit from IGFIR inhibition.

Reporting and Staging of Testicular Germ Cell Tumors: The International Society of Urological Pathology (isup) Testicular Cancer Consultation Conference Recommendations

The International Society of Urological Pathology held a conference devoted to issues in testicular and penile pathology in Boston in March 2015, which included a presentation and discussion led by the testis microscopic features working group. This conference focused on controversies related to staging and reporting of testicular tumors and was preceded by an online survey of the International Society of Urological Pathology members. The survey results were used to initiate discussions, but decisions were made by expert consensus rather than voting. A number of recommendations emerged from the conference, including that lymphovascular invasion (LVI) should always be reported and no distinction need be made between lymphatic or blood invasion. If LVI is equivocal, then it should be regarded as negative to avoid triggering unnecessary therapy. LVI in the spermatic cord is considered as category pT2, not pT3, unless future studies provide contrary evidence. At the time of gross dissection, a block should be taken just superior to the epididymis to define the base of the spermatic cord, and direct invasion of tumor in this block indicates a category of pT3. Pagetoid involvement of the rete testis epithelium must be distinguished from rete testis stromal invasion, with only the latter being prognostically useful. Percentages of different tumor elements in mixed germ cell tumors should be reported. Although consensus was reached on many issues, there are still areas of practice that need further evidence on which to base firm recommendations.

UICC drops the ball in the 8th edition TNM staging of urological cancers

The publication of each successive edition of the TNM staging classification by the International Union Against Cancer (UICC) is widely anticipated, as the staging system is accepted and utilized internationally. Late in 2016 the eighth edition of the TNM classification (UICC 8)1 was released but unfortunately, and despite the passage of six years since the publication of the seventh edition of the staging classification, the new edition incorporates little new data. In addition to this there are a number of differences between the staging recommendations of UICC 8 and those recently published as part of the 8th edition of the American Joint Committee for Cancer (AJCC 8). This article is protected by copyright. All rights reserved.

Deep learning based tissue analysis predicts outcome in colorectal cancer

Image-based machine learning and deep learning in particular has recently shown expert-level accuracy in medical image classification. In this study, we combine convolutional and recurrent architectures to train a deep network to predict colorectal cancer outcome based on images of tumour tissue samples. The novelty of our approach is that we directly predict patient outcome, without any intermediate tissue classification. We evaluate a set of digitized haematoxylin-eosin-stained tumour tissue microarray (TMA) samples from 420 colorectal cancer patients with clinicopathological and outcome data available. The results show that deep learning-based outcome prediction with only small tissue areas as input outperforms (hazard ratio 2.3; CI 95% 1.79–3.03; AUC 0.69) visual histological assessment performed by human experts on both TMA spot (HR 1.67; CI 95% 1.28–2.19; AUC 0.58) and whole-slide level (HR 1.65; CI 95% 1.30–2.15; AUC 0.57) in the stratification into low- and high-risk patients. Our results suggest that state-of-the-art deep learning techniques can extract more prognostic information from the tissue morphology of colorectal cancer than an experienced human observer.

Discrepancy rates in liver biopsy reporting

Medical liver biopsy reporting is challenging, and maintaining competency with small case numbers is potentially difficult. This study evaluates the discrepancies identified in cases referred to a specialist centre between the specialist reports and those of the referring general departments. Fifty consecutive recently referred cases were selected, and original and final reports were compared. Discrepancies were classified as per the Royal College of Pathologists guidelines and scored for potential clinical impact. The overall rate of discrepancy was 38% with most of these due to differences in interpretation of morphology. Seventy per cent of these discrepancies were judged to have major clinical impact (26% of all referred cases). This study highlights the need for robust systems of quality control of liver biopsies in a general setting.

The genomic diversity of prostate cancer: our Achilles heel explored.

In this month’s issue of European Urology, Lindberg and colleagues explored the monoclonality of a case of metastatic prostate cancer (PCa) using exome sequencing of lymph node metastases and matched primary PCa tissue [1]. This was undertaken as a follow-up to their recently published exome-sequencing analysis of a much larger number of samples [2]. The authors elegantly demonstrated that none of the somatic mutations identified within the lymph nodemetastases from an individual patient matched those originally identified in the initial primary cancer sample. Consequently, the analysis was expanded to include multiple distinct areas of laser-microdissected primary PCa samples from the same patient. Intriguingly, this revealed that the metastatic clones most closely matched a seeding focus of primary intraductal PCa, a histologic variant known to be associated with poor outcome [3]. This new evidence supports the notion that metastases arise as a consequence of the development of a lethal clone of cells within the primary tumour [4,5] andmay not always be a late event [6]. This latest study provides new insight into themolecular heterogeneity of PCa and emphasises the importance of thorough tissue sampling, along with detailed genetic analysis of high-quality specimens, to better understand the linear cancer evolution and to design precision-driven therapeutic approaches to improve patient outcomes [1]. The interesting conundrum in the work described by Lindberg and colleagues is that the tumour had metastasised from a clone growingwithin ducts rather than clonal areas showing locally invasive behaviour, such as foci of extraprostatic extension or seminal vesicle invasion, where one might postulate metastasis would arise. There is indeed increasing interest in the prognostic significance of intraductal carcinoma as well as cribriform invasive lesions with elements of Gleason pattern 4 [7]. Molecularly, histologically, and clinically, PCa is a very heterogeneous disease, and many patients harbour several independent foci of malignancy within the prostate [8]. The past decade has seen an increased focus of research activity to identify germline mutations and single nucleotide polymorphisms associated with clinically aggressive disease. Despite some success in identifying such lesions, this approach has not hitherto delivered any clinical benefit. It is likely that the characterisation of somatic mutations within individual PCa cases will yield a tangible clinical benefit in terms of risk stratification of disease or prediction of outcome from treatment with targeted therapies. Further research is needed in this area before this approach can be incorporated reliably into standard urologic practice. Significant advances are being made in understanding the molecular drivers of castration-resistant PCa [9], providing greater hope that novel agents can be developed to treat men with this lethal form of disease. Despite these advances, the best hopes for cure of men with localised PCa, without overtreatment, involve precision medicine with accurate profiling of each individual malignancy, thorough molecular evaluation to understand its potential aggressiveness and behaviour, and subsequent optimisation of clinical management based on risk calculation. The genomic diversity of PCa is emerging as our Achilles heel in managing the disease, and our definition of significant versus insignificant PCa is increasingly being recognised as inadequate [10]. There is a large reservoir of latent PCawithin the general population of adult men, and a EU RO P E AN URO LOGY 6 7 ( 2 0 1 5 ) 8 2 3 – 8 2 4