The Journal of Pathology

Cover image for Vol. 240 Issue 1

Edited By: C Simon Herrington, Editor-in-Chief

Impact Factor: 7.381

ISI Journal Citation Reports © Ranking: 2015: 3/78 (Pathology); 18/213 (Oncology)

Online ISSN: 1096-9896

Associated Title(s): The Journal of Pathology: Clinical Research

Virtual Issues


Welcome to The Journal of Pathology's Virtual Issues page

List of issues

2016 Issue 1, August: Recent advances in breast cancer
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2013 Issue 3, August: Next generation sequencing in pathology
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2013 Issue 2, May: Progress in prostate cancer
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2013 Issue 1, February: The microenvironment and cancer
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2012 Issue 4, December: Animal Models of Disease
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2012 Issue 3, August: Getting your papers published: A view from The Journal of Pathology Editorial team
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2012 Issue 2, March: Stem cells, clonal expansion and cancer progression
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2012 Issue 1, February: Recent advances in our understanding of gynaecological pathology
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2011 Issue 4, November: Progress in our understanding of the pathobiology of hypoxia and angiogenesis
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2011 Issue 3, August: The molecular pathology of sarcomas
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2011 Issue 2, April: Recent advances in the molecular pathology of micro-RNAs
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2011 Issue 1, February: Neuropathology: Advances in technology and biology
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2010 Issue 4, December: Recent advances in the pathobiology of lymphoma
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2010 Issue 3, September: Recent advances in renal pathology
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2010 Issue 2, June: Recent advances in breast cancer
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2010 Issue 1, April: p53: Recent advances in our understanding of this key tumour suppressor
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The Journal of Pathology 2016 Virtual Issue Number 1, August

Recent advances in breast cancer
Compiled and annotated by Philip J Coates, Regional Centre for Applied Molecular Oncology, Masaryk Memorial Cancer Centre, Brno, Czech Republic; Reviews editor, The Journal of Pathology

Early changes/precursor lesions

Many cancers are known to develop from precursor lesions and the changes seen in such lesions therefore provide information into the early events that drive the neoplasia. Whilst epithelial atypia, ductal hyperplasia and ductal carcinoma in situ are well-recognized precursors for the common hormone receptor-positive forms of breast cancer, a precursor for triple-negative breast cancer (TNBC) is much less well established. Jorge Reis-Filho and colleagues at the Memorial Sloan Kettering Cancer Centre, with collaborators from Italy, Brazil, Japan, France and the UK have provided compelling evidence that microglandular adenosis (MGA) associated with TNBC is an advanced precursor for the synchronous cancer. Using massively parallel sequencing of microdissected tissues, they could show, for example, identical mutations in TP53 in the MGA and associated TNBC, and similar copy number alterations. They also found sub-clonal populations and clonal shifts during progression from MGA to TNBC. On the other hand, pure MGAs that were not found in association with TNBC lacked clonal non-synonymous mutations and had limited copy number alterations [1]. The biology and pathology of MGA and the clinical implications of the new findings of MGA as a non-obligatory precursor for TNBC are summarized in a linked Commentary by Tsang and Tse [2].

  1. Microglandular adenosis associated with triple-negative breast cancer is a neoplastic lesion of triple-negative phenotype harbouring TP53 somatic mutations.
    Elena Guerini-Rocco, Salvatore Piscuoglio, Charlotte KY Ng, Felipe C Geyer, Maria R De Filippo, Carey A Eberle, Muzaffar Akram, Nicola Fusco, Shu Ichihara, Rita A Sakr, Yasushi Yatabe, Anne Vincent-Salomon, Emad A Rakha, Ian O Ellis, Y Hannah Wen, Britta Weigelt, Stuart J Schnitt and Jorge S Reis-Filho.
    The Journal of Pathology 2016: 238: 677–688.

  1. Microglandular adenosis: a prime suspect in triple-negative breast cancer development.
    Julia YS Tsang and Gary MK Tse.
    The Journal of Pathology 2016; 239: 129–132

Breast Cancer Metastasis

Improved understanding of the processes of tumour progression, invasion and metastasis is of obvious importance for breast cancer. Here, I highlight three papers published recently in The Journal of Pathology that deal with each of these aspects. In the first of these, brain metastases of different cancer types were subjected to analysis of their genomic and transcriptomic landscapes. The data revealed i) new candidate genes that may drive brain metastasis, ii) the occurrence of HER2 status conversion and other actionable mutations allowing targeted therapy of the metastasis and iii) that many brain metastatic lesions may rely on locally produced growth factors for HER3, again with therapeutic implications [3]. In the second paper, massively parallel sequencing of phyllodes tumours identified a recurrent clonal hotspot mutation in the TERT promoter, with the frequency increasing stepwise from benign to borderline to malignant tumours, suggesting that TERT alterations drive the progression of phyllodes tumours. Importantly, TERT alterations are not seen in fibroadenomas and may therefore be used in differential diagnosis [4]. In the third article, the process of epithelial-mesenchymal-transition (EMT) as a mechanism of invasion and metastatic spread was studied in the histologically special type of breast cancer, invasive lobular cancer. This cancer is characterized by an invasive growth pattern with single cells or single files of cells that lose the epithelial marker E-cadherin. Surprisingly, given the prevalence of reports indicating that EMT is a major driving force for cancer invasion, only 2% of intralobular cancers showed early and coordinated changes of EMT markers. These data do not support a role for EMT in defining the phenotype of these cancers [5].

3. Integrated genomic and transcriptomic analysis of human brain metastases identifies alterations of potential clinical significance
Jodi M Saunus, Michael CJ Quinn, Ann-Marie Patch, John V Pearson, Peter J Bailey, Katia Nones, Amy E McCart Reed, David Miller, Peter J Wilson, Fares Al-Ejeh, Mythily Mariasegaram, Queenie Lau, Teresa Withers, Rosalind L Jeffree, Lynne E Reid, Leonard Da Silva, Admire Matsika, Colleen M Niland, Margaret C Cummings, Timothy JC Bruxner, Angelika N Christ, Ivon Harliwong, Senel Idrisoglu, Suzanne Manning, Craig Nourse, Ehsan Nourbakhsh, Shivangi Wani, Matthew J Anderson, J Lynn Fink, Oliver Holmes, Stephen Kazakoff, Conrad Leonard, Felicity Newell, Darrin Taylor, Nick Waddell, Scott Wood, Qinying Xu, Karin S Kassahn, Vairavan Narayanan, Nur Aishah Taib, Soo-Hwang Teo, Yock Ping Chow, kConFab, Parmjit S Jat, Sebastian Brandner, Adrienne M Flanagan, Kum Kum Khanna, Georgia Chenevix-Trench, Sean M Grimmond, Peter T Simpson, Nicola Waddell and Sunil R Lakhani.
The Journal of Pathology 2015; 237: 363–378.

4. Massively parallel sequencing of phyllodes tumours of the breast reveals actionable mutations, and TERT promoter hotspot mutations and TERT gene amplification as likely drivers of progression.
Salvatore Piscuoglio, Charlotte KY Ng, Melissa Murray, Kathleen A Burke, Marcia Edelweiss, Felipe C Geyer, Gabriel S Macedo, Akiko Inagaki, Anastasios D Papanastasiou, Luciano G Martelotto, Caterina Marchio, Raymond S Lim, Rafael A Ioris, Pooja K Nahar, Ino De Bruijn, Lillian Smyth, Muzaffar Akram, Dara Ross, John H Petrini, Larry Norton, David B Solit, Jose Baselga, Edi Brogi, Marc Ladanyi, Britta Weigelt and Jorge S Reis-Filho.
The Journal of Pathology 2016; 238: 508–518.

5. An epithelial to mesenchymal transition programme does not usually drive the phenotype of invasive lobular carcinomas.
Amy E McCart Reed, Jamie R Kutasovic, Ana C Vargas, Janani Jayanthan, Amel Al-Murrani, Lynne E Reid, Rachael Chambers, Leonard Da Silva, Lewis Melville, Elizabeth Evans, Alan Porter, David Papadimos, Erik W Thompson, Sunil R Lakhani and Peter T Simpson.
The Journal of Pathology 2016; 238: 489–494.

In addition to informing our views of how cancers arise and progress, delineating the genomic landscapes of cancers has allowed improved classification schemes, added to diagnostic markers and provided new predictive markers and targets for therapy. This approach has been applied to pure and mixed acinic cell carcinomas, a rare form of TNBC. The analyses reveal that the genetics of these tumours is highly similar to the common forms of TNBC and that identical somatic mutations may be seen in the acinic cell and the high grade non-acinic components of mixed tumours, suggesting that acinic cell carcinomas may develop into aggressive TNBC [6]. In a similar study of another rare form of TNBC, adenoid cystic carcinoma, the data indicate a distinctly different mutational landscape and genomic structure from the more common forms of TNBC. Indeed, breast adenoid cystic carcinomas are more similar to their histopathological counterpart in the salivary gland than to TNBC. Whilst breast adenoid cystic carcinomas have a relatively simple genome, they also display intra-tumour heterogeneity at diagnosis, with implications for therapy. These data also highlight that tumours with similar morphological characteristics may arise from similar genetic changes irrespective of their site of origin [7].

Another use of genomic analysis is to compare the landscapes of animal models with the human disease, particularly where the model is being used for preclinical studies. One example is mouse models of inherited breast cancer due to inherited mutations in BRCA2. Exome sequencing of one such model found that, whilst certain tumour somatic mutations were shared between mouse and human breast cancers, many were not shared. Thus, although these models are useful, the differences in genetic landscapes need to be taken into account when extrapolating data from mouse to human [8]. Finally, within this section I draw attention to the intriguing concept that cancer genomic landscapes may provide a useful baseline for categorization of tumours. In a recent Perspective [9] a case is made for categorizing cancers into those with minimal, moderate or high genomic complexity, with consequences for i) clinical study design, ii) improved diagnostic accuracy, iii) stratification of patients for targeted therapies, iv) research study design and v) personalized treatment strategies.

6. The repertoire of somatic genetic alterations of acinic cell carcinomas of the breast: an exploratory, hypothesis-generating study.
Elena Guerini-Rocco, Zsolt Hodi, Salvatore Piscuoglio, Charlotte KY Ng, Emad A Rakha, Anne M Schultheis, Caterina Marchiò, Arnaud da Cruz Paula, Maria R De Filippo, Luciano G Martelotto, Leticia De Mattos-Arruda, Marcia Edelweiss, Achim A Jungbluth, Nicola Fusco, Larry Norton, Britta Weigelt, Ian O Ellis and Jorge S Reis-Filho.
The Journal of Pathology 2016; 237: 166–178.

7. Genomic landscape of adenoid cystic carcinoma of the breast.
Luciano G Martelotto, Maria R De Filippo, Charlotte KY Ng, Rachael Natrajan, Laetitia Fuhrmann, Joanna Cyrta, Salvatore Piscuoglio, Huei-Chi Wen, Raymond S Lim, Ronglai Shen, Anne M Schultheis, Y Hannah Wen, Marcia Edelweiss, Odette Mariani, Göran Stenman, Timothy A Chan, Pierre-Emmanuel Colombo, Larry Norton, Anne Vincent-Salomon, Jorge S Reis-Filho and Britta Weigelt.
The Journal of Pathology 2016; 237: 179–189.

8. Whole-exome DNA sequence analysis of Brca2- and Trp53-deficient mouse mammary gland tumours.
Jeffrey C Francis, Lorenzo Melchor, James Campbell, Howard Kendrick, Wenbin Wei, Javier Armisen-Garrido, Ioannis Assiotis, Lina Chen, Iwanka Kozarewa, Kerry Fenwick, Amanda Swain, Matthew J Smalley, Christopher J Lord and Alan Ashworth.
The Journal of Pathology 2015; 236: 186–200.

9. Categorization of cancer through genomic complexity could guide research and management strategies.
Hugo M Horlings, Adrienne M Flanagan and David G Huntsman.
The Journal of Pathology 2015; 236: 397–402.

Breast Cancer Heterogeneity and Multiple Versus Single Tumours

Multifocal breast cancer is seen relatively frequently, but is largely ignored in clinical practice where treatment decisions are made based on phenotypic characterization of the largest lesion. To investigate whether different lesions share a common ancestry or not, mutations were identified in multiple lesions from a series of patients with multifocal breast cancer, selected for lesions having the same grade, ER and HER2 status. About one third of patients contained lesions with distinct, non-shared (private) somatic mutations in different lesions. This genomic inter-lesion heterogeneity suggests that these tumours will respond differently to specific molecular targeted therapies, and so deeper characterization of multifocal lesions is warranted [10]. An accompanying commentary summarizes these findings in the contexts of early clonal divergence and the rules governing breast cancer evolution, with implications for the clinical assessment of breast cancers and their driver alterations to guide appropriate patient management [11]. Intra-tumour heterogeneity of breast and other cancers is increasingly recognised through genomic studies. An alternative approach using the novel technology of imaging mass spectrometry to identify proteomic signatures was recently reported [12]. This exciting study showed that proteomic heterogeneity can be identified in breast and gastric cancers and reveals microscopically indistinct tumour sub-populations with impact on clinical outcome. In breast cancer, several subpopulations were associated with the presence of loco-regional metastases. Alongside genomic studies, the application of such proteomic technologies is likely to enable further molecular characterization of cancer biology and the impact of tumour heterogeneity.

10.Uncovering the genomic heterogeneity of multifocal breast cancer.
Christine Desmedt, Debora Fumagalli, Elisabetta Pietri, Gabriele Zoppoli, David Brown, Serena Nik-Zainal, Gunes Gundem, Françoise Rothé, Samira Majjaj, Anna Garuti, Enrico Carminati, Sherene Loi, Thomas Van Brussel, Bram Boeckx, Marion Maetens, Laura Mudie, Delphine Vincent, Naima Kheddoumi, Luigi Serra, Ilaria Massa, Alberto Ballestrero, Dino Amadori, Roberto Salgado, Alexandre de Wind, Diether Lambrechts, Martine Piccart, Denis Larsimont, Peter J Campbell and Christos Sotiriou.
The Journal of Pathology 2015; 236: 457–466.

11. Breast cancer heterogeneity: parallel evolution or conscious uncoupling?
Rachael C Natrajan.
The Journal of Pathology 2015; 237: 1–3.

12. De novo discovery of phenotypic intratumour heterogeneity using imaging mass spectrometry.
Benjamin Balluff, Christian K Frese, Stefan K Maier, Cédrik Schöne, Bernhard Kuster, Manfred Schmitt, Michaela Aubele, Heinz Höfler, André M Deelder, Albert JR Heck, Pancras CW Hogendoorn, Johannes Morreau, AF Maarten Altelaar, Axel Walch and Liam A McDonnell.
The Journal of Pathology 2015; 235: 3–13.

Novel Pathways with Prognostic and/or Therapeutic Implications

Much of the drive behind current cancer research is to identify the pathways involved and the specific abnormalities that cause cancer, with the aim of identifying new therapeutic or prognostic markers. In one such study, the RECQL4 gene was shown to be amplified and the protein over-expressed in aggressive ER-negative breast cancers. Depletion of RECQL4 led to increased chemosensitivity, suggesting that RECQL4 is both a prognostic marker and potential therapeutic target [13]. In another study, SF3B1 mutations were seen in 1.8% of unselected breast cancers, with higher frequencies in papillary and mucinous carcinomas of the breast. SF3B1 is involved in mRNA splicing, and tumours with mutant SF3B1 showed differentially spliced mRNAs and a long non-coding RNA. Importantly, cells with mutant SF3B1 were sensitive to spliceostatin A, an inhibitor of the SF3b splicing complex [14]. In an independent study, alternative splicing of the DMP1 gene was observed in about 30% of breast cancers, with an increased proportion of the DMP1 mRNA. DMP1 was sufficient to induce mammary gland hyperplasia and multifocal tumours in transgenic mice, and high levels of DMP1 associated with poor patient outcomes, again inferring that altered mRNA splicing is an important aspect of breast cancer with clinical implications [15]. Another study of therapeutic relevance was published recently in The Journal of Pathology: Clinical Research [16]. Here, the authors investigated the impact of Aurora kinase A in breast cancers from BRCA2 999del5 mutation carriers. Using multivariate analyses, nuclear Aurora kinase A was a prognostic marker for BRCA2 mutation carriers, independent of clinical parameters and adjuvant treatment. Thus, targeting this kinase for treatment is a rational approach for Aurora kinase A positive tumours in BRCA2 mutation carriers, and for sporadic tumours with high levels of Aurora kinase A [16]. A different approach of functional screening was employed to identify MCT4 as a regulator of breast cancer survival. The authors then investigated the effects of MCT4 on cell growth and metabolism. Importantly, they also showed that MCT4, a regulator of pH, lactate secretion and non-oxidative glucose metabolism, is expressed in HER2-positive breast cancers, making these a particular target for MCT4-based therapeutics [17].

13. RECQL4 helicase has oncogenic potential in sporadic breast cancers.
Arvind Arora, Devika Agarwal, Tarek MA Abdel-Fatah, Huiming Lu, Deborah L Croteau, Paul Moseley, Mohammed A Aleskandarany, Andrew R Green, Graham Ball, Emad A Rakha, Stephen YT Chan, Ian O Ellis, Lisa L Wang, Yongliang Zhao, Adayabalam S Balajee, Vilhelm A Bohr and Srinivasan Madhusudan.
The Journal of Pathology 2016; 238: 495–501.

14. SF3B1 mutations constitute a novel therapeutic target in breast cancer.
Sarah L Maguire, Andri Leonidou, Patty Wai, Caterina Marchiò, Charlotte KY Ng, Anna Sapino, Anne-Vincent Salomon, Jorge S Reis-Filho, Britta Weigelt and Rachael C Natrajan.
The Journal of Pathology 2015; 235: 571–580.

15. DMP1β, a splice isoform of the tumour suppressor DMP1 locus, induces proliferation and progression of breast cancer.Dejan Maglic, Daniel B Stovall, J. Mark Cline, Elizabeth A Fry, Ali Mallakin, Pankaj Taneja, David L Caudell, Mark C Willingham, Guangchao Sui and Kazushi Inoue.
The Journal of Pathology 2015; 236: 90–102.

16. Aurora A is a prognostic marker for breast cancer arising in BRCA2 mutation carriers.
Margret Aradottir, Sigridur T Reynisdottir, Olafur A Stefansson, Jon G Jonasson, Asgerdur Sverrisdottir, Laufey Tryggvadottir, Jorunn E Eyfjord and Sigridur K Bodvarsdottir.
The Journal of Pathology: Clinical Research 2015; 1: 33–40.

17. Functional screening identifies MCT4 as a key regulator of breast cancer cell metabolism and survival.
Franziska Baenke, Sébastien Dubuis, Charlene Brault, Britta Weigelt, Beatrice Dankworth, Beatrice Griffiths, Ming Jiang, Alan Mackay, Becky Saunders, Bradley Spencer-Dene, Susana Ros, Gordon Stamp, Jorge S Reis-Filho, Michael Howell, Nicola Zamboni and Almut Schulze.
The Journal of Pathology 2015; 237: 152–165.

Studies from The Journal of Pathology: Clinical Research

The Journal of Pathology: Clinical Research is a sister journal to the Journal of Pathology; both are Journals of The Pathological Society of Great Britain and Ireland. As its name implies, the focus of The Journal of Pathology: Clinical Research is to publish studies that illuminate the clinical relevance of research into disease. This aim is exemplified by the following three studies. In the first, a multi-national, multi-centre study (52 authors with 36 author affiliations) tackled the issue of rapid, standardised scoring of tumour marker expression in samples from nine studies containing over 20,000 cores from 8,267 breast cancers. This comprehensive study demonstrated the usefulness of digital imaging and automated algorithms for rapid and robust scoring, but also identified substantial heterogeneity across tissue microarrays and false-positivity as a problem for some markers. The authors conclude that, although useful, continued optimisation and marker-specific quality controls are required to improve large-scale biomarker studies [18]. In the second study, a similarly large international group of investigators developed and assessed an automated protocol for Ki67 staining using material from 13 studies and including over 9,000 breast cancers. The authors found that automated Ki67 scoring performed well, with the expected correlations between Ki67 and clinical and pathological characteristics. However, variations according to TMA and study mean that rigorous pre- and post-analytical control procedures are required to ensure satisfactory results are obtained from multi-centre studies [19]. The third article reports the validation of the Nottingham Prognostic Index Plus (NPI+) tool for clinical decision making in breast cancer. The NPI+ is derived from expression of biomarkers and clinicopatholoical parameters to provide biological classes and prognostic groupings. The article validated the original findings for NPI+ in an independent series of 885 primary early stage breast cancers, confirming the prognostic value of this tool and indicating its potential for further validation in large randomised controlled trial material [20].

18. Performance of automated scoring of ER, PR, HER2, CK5/6 and EGFR in breast cancer tissue microarrays in the Breast Cancer Association Consortium.
William J Howat, Fiona M Blows, Elena Provenzano, Mark N Brook, Lorna Morris, Patrycja Gazinska, Nicola Johnson, Leigh-Anne McDuffus, Jodi Miller, Elinor J Sawyer, Sarah Pinder, Carolien H M van Deurzen, Louise Jones, Reijo Sironen, Daniel Visscher, Carlos Caldas, Frances Daley, Penny Coulson, Annegien Broeks, Joyce Sanders, Jelle Wesseling, Heli Nevanlinna, Rainer Fagerholm, Carl Blomqvist, Päivi Heikkilä, H Raza Ali, Sarah-Jane Dawson, Jonine Figueroa, Jolanta Lissowska, Louise Brinton, Arto Mannermaa, Vesa Kataja, Veli-Matti Kosma, Angela Cox, Ian W Brock, Simon S Cross, Malcolm W Reed, Fergus J Couch, Janet E Olson, Peter Devillee, Wilma E Mesker, Caroline M Seyaneve, Antoinette Hollestelle, Javier Benitez, Jose Ignacio Arias Perez, Primitiva Menéndez, Manjeet K Bolla, Douglas F Easton, Marjanka K Schmidt, Paul D Pharoah, Mark E Sherman and Montserrat García-Closas.
The Journal of Pathology: Clinical Research 2015; 1: 18–32.

19. High-throughput automated scoring of Ki67 in breast cancer tissue microarrays from the Breast Cancer Association Consortium.
Mustapha Abubakar, William J Howat, Frances Daley, Lila Zabaglo, Leigh-Anne McDuffus, Fiona Blows, Penny Coulson, H Raza Ali, Javier Benitez, Roger Milne, Herman Brenner, Christa Stegmaier, Arto Mannermaa, Jenny Chang-Claude, Anja Rudolph, Peter Sinn, Fergus J Couch, Rob A.E.M. Tollenaar, Peter Devilee, Jonine Figueroa, Mark E Sherman, Jolanta Lissowska, Stephen Hewitt, Diana Eccles, Maartje J Hooning, Antoinette Hollestelle, John WM Martens, Carolien HM van Deurzen, kConFab Investigators, Manjeet K Bolla, Qin Wang, Michael Jones, Minouk Schoemaker, Annegien Broeks, Flora E van Leeuwen, Laura Van't Veer, Anthony J Swerdlow, Nick Orr, Mitch Dowsett, Douglas Easton, Marjanka K Schmidt, Paul D Pharoah and Montserrat Garcia-Closas.
The Journal of Pathology: Clinical Research 2016; Version of Record online: 6 APR 2016 | DOI: 10.1002/cjp2.42

20. Nottingham Prognostic Index Plus: Validation of a clinical decision making tool in breast cancer in an independent series.
Andrew R Green, Daniele Soria, Jacqueline Stephen, Desmond G Powe, Christopher C Nolan, Ian Kunkler, Jeremy Thomas, Gillian R Kerr, Wilma Jack, David Cameron, Tammy Piper, Graham R Ball, Jonathan M Garibaldi, Emad A Rakha, John MS Bartlett and Ian O Ellis.
The Journal of Pathology: Clinical Research 2016; 2: 32–40.



The Journal of Pathology 2013 Virtual Issue Number 3, August


Next generation sequencing in pathology

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Compiled and annotated by David T. Bonthron, Senior Editor at the Journal of Pathology and Professor of Molecular Medicine, University of Leeds, St. James's University Hospital, Leeds, LS9 7TF, UK

"Next-generation sequencing" is one of those phrases that really should have quickly disappeared from use -- the massively parallel technologies to which it refers are now the staple diet of biomedical science. A new instrument bringing further incremental improvement lies around each corner. Hardly "next generation" - not anymore...

Maybe then, the persistence of this forward-looking jargon term tells us something about the impact of these technologies on the scientific process. Nowadays, in genetic and dependent disciplines, data acquisition is cheap and easy. Much more of the scientific challenge consists of analysis and interpretation, and because these depend heavily on computational methods, there has been a huge shift in the core skill-sets needed in biomedical research. Perhaps for the first time in the history of biology, many senior investigators find themselves lacking the scientific skills required to plan and direct research from an informed position. Like music or chess, the fundamentals underpinning modern bioinformatics are not best learnt in middle age. The real "next generation", then, is the human one, the young scientists driving the implementation of new methods of enquiry. They have assumed centre stage.

There is insufficient space here to attempt to summarize all the diverse ways in which NGS can be applied in biomedical research and diagnostics. It has notable capabilities, though, that deserve brief mention. It can be exquisitely sensitive; the resequencing of a known mutation target at high read depth can allow detection of mutations at levels of mosaicism far below the detection limits of conventional sequencing methods, and which would formerly have required laborious cloning methods. It is readily quantifiable; read numbers matching a given genomic or transcript target can thus be interpreted in terms of copy number or expression level. Finally, for diagnostic pathologists, the huge capacity of NGS can be harnessed for parallel analysis of multiple genes in multiple subjects, simultaneously broadening diagnostic coverage and reducing costs.

In this virtual issue, we highlight a range of studies that exploit the power of NGS in the study of human disease. Most of them focus on cancer, entirely unsurprising given its immense importance and largely genetic causation. Even a brief review of these impressive studies make it clear that our knowledge of cancer genomics already greatly exceeds our understanding. Should we question the value of cancer genomics, then? In their overview of this area, Taylor and Ladanyi [1] offer us many reminders of why we should resist becoming cynical. Genomic abberations that drive tumorigenesis, once they are tested out from the bewildering complexity of tumour genetics, have repeatedly led to the identification of key therapeutic targets.

 1 Clinical cancer genomics: how soon is now?
Taylor B.S. and Ladanyi M.et al.
The Journal of Pathology 2011; 223: 318-326
Tumour pathogenesis

Prostate cancer, more than most, has been illuminated by recent large-scale genomic analyses. Weier et al. [2] used discrepant mapping of the paired-end reads of individual genomic fragments to characterise the TMPRSS2-ERG translocation breakpoints in 25 of 83 prostate cancers, as well as less frequent translocations involving other genes. The clustering of these breakpoints at hotspots of androgen-induced chromosome breakage is consistent with models that invoke androgen-driven genome rearrangement as an important tumorigenic mechanism.

In a series of 6 prostate cancers lacking TMPRSS2-ERG fusions, Lapuk et al. [3] combined genome and transcriptome analysis to reveal a high frequency of novel fusion genes as well as characteristic patterns of gene expression. Strikingly, they were able to discern prostate-cancer-specific patterns even within histologically normal lymph node, suggesting the presence of undiagnosed occult metastatic disease.

Wu et al. [4] performed genome and transcriptome sequencing of an aggressive prostate cancer in a young subject, identifying 15 tumour-specific translocations that were common to primary and metastatic disease, but private and not reported in other prostate cancers. The identities of the genes involved in the rearrangements could be related directly to the intermediate gene expression patterns displayed by this unusual tumour, with both androgen-related and neuroendocrine characteristics.

 2 Nucleotide resolution analysis of TMPRSS2 and ERG rearrangements in prostate cancer
Weier C, Haffner MC, Mosbruger T, et al.
The Journal of Pathology 2013; 230: 174-183
 3 From sequence to molecular pathology, and a mechanism driving the neuroendocrine phenotype in prostate cancer
Lapuk A.V., Wu C., Wyatt A.W., et al.
The Journal of Pathology 2012; 227: 286-297.
 4

Integrated genome and transcriptome sequencing identifies a novel form of hybrid and aggressive prostate cancer
Wu C, Wyatt AW, Lapuk A.V., et al.
The Journal of Pathology 2012; 227: 53-61

Moving to breast cancer, Natrajan et al. [5] compared the genome sequences of ER+ and ER- tumours from germline BRCA1 mutation carriers, to try to resolve controversy about the relationship between BRCA1 mutation status and development of ER+ tumours. SImilar patterns of genetic aberrations (consistent with loss of homologous recombination DNA repair), together with loss of the wild-type BRCA1 allele, were seen in each case, arguing in favour of a true pathogenic status for the germ line BRCA1 mutation, even in ER+ tumours.

Mucosal melanoma is an uncommon tumour with a poor prognosis. Furney et al. [6] performed whole genome and whole exome sequencing to show that mucosal melanomas have far fewer (~70) non-synonymous exon mutations than sun-exposed cutaneous melanomas (~300-400). This, together with their four-fold higher rate of structural genomic aberrations, clearly indicates distinct genetic mechanisms driving the development of these two tumour types.

RNA-level analysis is also a powerful way to identify genomic aberrations that drive tumour development. Majewski et al. [7] used targeted enrichment of kinase transcripts to identify rearrangements fusing FGFR3 and ALK to other genes in non-small-cell lung cancer. Their findings underscore the extreme heterogeneity of genomic rearrangements in lung cancer, as well as intriguing overlaps with other tumour types such as bladder cancer, in which the same type of FGFR3 rearrangements and point mutations are seen.

 5 A whole-genome massively parallel sequencing analysis of BRCA1 mutant oestrogen receptor-negative and -positive breast cancers
Natrajan R., Mackay A., Lambros M.B., et al.
The Journal of Pathology 2012; 227: 29-41

 6 Genome sequencing of mucosal melanomas reveals that they are driven by distinct mechanisms from cutaneous melanoma
Furney S.J., Turajlic S., Stamp G., et al.
The Journal of Pathology 2013; 230: 261-269.

 7

Identification of recurrent FGFR3 fusion genes in lung cancer through kinome-centered RNA sequencing
Majewski I.J., Mittempergher L., Davidson N.M., et al.
The Journal of Pathology 2013; 230: 270–276

Tumour evolution

NGS also offers powerful ways to observe the processes of tumour evolution and response to treatment, by comparison of the genomic architecture and transcription patterns of primary and metastatic lesions at different times. Castellarin et al. [8] used whole exome sequencing to study somatic mutations in primary ovarian tumours and ascites fluid at recurrence. Most primary tumour mutations were still present at recurrence, indicating a failure of chemotherapy to eliminate many primary tumour clones. In contrast, Kasaian et al. [9] employed whole-genome and transcriptome sequencing to compare the genomes of a primary and recurrent parathyroid carcinoma (PTC). This revealed not only a novel spectrum of somatic point mutations, but tumour-specific rearrangements. Such intensive scrutiny of individual cases appears to be the best way to obtain the maximum amount of information on the pathogenesis of rare tumour types such as PTC.

 8 Clonal evolution of high-grade serous ovarian carcinoma from primary to recurrent disease
Castellarin M., Milne K., Zeng T., et al.
The Journal of Pathology 2013; 229: 515-524
 9 Complete genomic landscape of a recurring sporadic parathyroid carcinoma
Kasaian K., Wiseman S.M., Thiessen N., et al.
The Journal of Pathology 2013; 230: 249–260.

Tumour classification

With its ability to analyse multiple genes simultaneously, NGS has the potential in a diagnostic setting to complement or even replace older methods of disease classification, such as those based on histology or immunohistochemistry. The classification of gynaecological tumour is the subject of much attention using these new methods. McConechy et al. [10] demonstrate characteristic differences between subtypes of endometrial carcinoma, in their profiles of mutations within a panel of nine genes.Subsequent identification of outlying cases with apparently discrepant mutational and morphological classification enabled the reclassification of a number of cases that had borderline histology

Jones et al. [11] performed whole exome sequencing of enriched tumour cell populations from low-grade serous ovarian cancers, finding that very few somatic point mutations occur in this tumour type, aside from those in wither BRAF or KRAS. Despite the low mutational yield of this study, it carries the highly significant message that the KRAS-BRAF-MEK-MAPK pathway should be investigated as a therapeutic target in this subgroup of ovarian cancers. McBride et al. [12] characterised genomic rearrangements in ovarian cancers using paired-end genomic sequencing. A subset of high-grade serous tumours displayed a high frequency of tandem duplications, a molecular phenotype shared with triple-negative breast cancers.

 10 Use of mutation profiles to refine the classification of endometrial carcinomas
McConechy M.K., Ding J., Cheang M.C.U., et al.
The Journal of Pathology 2012; 228: 20-30
 11 Low-grade serous carcinomas of the ovary contain very few point mutations
Jones S., Wang T.L., Kurman R.J., et al.
The Journal of Pathology 2012; 226: 413-420.

 12

Tandem duplication of chromosomal segments is common in ovarian and breast cancer genomes
McBride D.J., Etemadmoghadam D. Cooke S.L., et al.
The Journal of Pathology 2012; 227: 446-455

NGS in the elucidation of gene regulatory networks

ChIP-seq (chromatin immunoprecipitation followed by NGS) is a powerful method for directly defining the genomic targets of transcriptional regulators. By determining which DNA sequences are enriched in chromatin immunoprecipitates, target genes and consensus DNA binding sites can be elucidated. Nelson et al. [13] used a combination of transcriptional profiling and ChIP-seq to show that the hallmark chordoma oncogene T (brachyury) up-regulates a network of genes including a large subset of those involved in cell cycle control.

 13 An integrated functional genomics approach identifies the regulatory network directed by brachyury (T) in chordoma
Nelson A.C., Pillay N., Henderson S., et al. The Journal of Pathology 2012; 228: 274-285
Non-invasive diagnosis

Human plasma normally contains minute quantities of highly fragmented cell-free DNA. In pregnancy, a proportion of this is fetal, and in cancer patients, some is tumour-derived. Lo and Chiu [14] review the diverse ways in which NGS technology now allows access to this DNA for diagnostic purposes, permitting applications as different as prenatal diagnosis of chromosomal abnormalities and monitoring of remission samples for the re-appearance of a tumour-specific genetic marker.

 14 Plasma nucleic acid analysis by massively parallel sequencing: pathological insights and diagnostic implications
Lo Y.M. and Chiu R.W.K.
The Journal of Pathology 2011; 225: 318-323
Infection, epidemiology and disease history

Finally to infection, still untoppled as the biggest killer of all. Compared to the challenges of analyzing the large repetitive genomes that vertebrates are endowed with, NGS has made the task of de novo sequencing and assembly of microbial genomes trivial. Viruses, too, can be sub-classified and their evolution followed much more precisely on the basis of their sequences than by immunological criteria. The advent of NGS has also completely changed what is possible in the field of pathogen detection. Xiao et al. [15] provide a powerful demonstration of how they could reassemble the 1918 influenza pandemic virus in a few days, using formalin-fixed tissue nearly a century old. Remarkably, in this RNA-based sequencing study, they were also even able to deduce aspects of the pattern of host defence, based on gene expression analysis.

The memory of disease battles, long since fought and lost, lives on in a multitude of preserved tissue samples around the world.The new lessons that modern sequencing technology are empowering us to learn are remarkable indeed. The "next generation" -- of humans, that is, not sequencing machines -- will benefit from biomedical knowledge on a scale unimaginable evena short while ago.

 15 High-throughput RNA sequencing of a formalin-fixed, paraffin-embedded autopsy lung tissue sample from the 1918 influenza pandemic
Xiao Y.L., Kash J.C., Beres S.B., et al.
The Journal of Pathology 2013; 229: 535-545

Questions

The following questions can be answered by reading, and reflecting upon, the above annotation and the papers that are cited within it. Within the Royal College of Pathologists Continuing Professional Development (CPD) scheme, CPD points may be earned by writing reflective notes on the papers in this Virtual Issue and the questions are designed to act as a focus for this activity. To do this, you may wish to use the Royal College of Pathologists' reflective notes form.

 Question 1 What are the differences between a human genome, an "exome" and a "transcriptome"? What are their relative sizes? For what purposes might each of these be the analytical target of choice?

 Question 2 Large-scale genomic abnormalities (copy-number variations and translocations) are common and diverse in tumours. What type of NGS experimental designs would be required to detect (a) copy number variations at high resolutions and (b) balanced translocations?

 Question 3 What are the key advantages offered by NGS methods in the analysis of archival pathological material? WHich other genomic analysis methods cannot be applied to such material, and why?


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The Journal of Pathology 2013 Virtual Issue Number 2, May


Progress in prostate cancer

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Compiled and annotated by Daniel M. Berney, Department of Molecular Pathology, Barts Cancer Institute, Queen Mary University of London, UK

Prostate cancer remains a major challenge for both diagnostic and experimental pathologists. It is one of the commonest cancers, yet we know less about its pathogenesis than most other common malignancies. It is a significant cause or mortality and morbidity in the Western world, yet most cases, especially those detected by screening, do not progress and patients usually die with their prostate cancer rather than from it. The search for biomarkers associated with prostate cancer aggressive behaviour has been accelerated by investigation of prostate cancer pathogenesis and genetics. The discovery of potential clinical targets has also accelerated in the past few years and the treatment options for both early and advanced prostate cancer seems to change on a yearly basis. The Journal of Pathology has been in the forefront of this search, with many papers exploring mechanistic and more clinically associated areas of the disease.

The genetics of prostate cancer

The most striking genetic finding in prostate cancer has been the high frequency of TMPRSS2-ERG rearrangements that occur in 50% of prostate cancers. Weier et al. [1] investigated the genomic architecture of these rearrangements using next generation sequencing to examine rearrangement breakpoints in the fusion gene in 83 primary prostate cancers. They show that the rearrangement breakpoints show strong clustering in specific intronic regions of TMPRSS2 and ERG and 88% occurred at or near regions of microhomology or involved insertions of one or more base pairs showing the specific nature of these re-arrangements. A number of studies have shown the existence of genetic regions which predispose for prostate cancer. Helfand et al. [2] demonstrate that chromosomal regions associated with prostate cancer susceptibility are preferentially localized to lamin B-deficient microdomains (LDMDs). LDMD frequency is correlated with prostate cancer cell line aggessiveness and increased cell motility. Furthermore, LDMDs were observed in human prostate cancer tissue and their frequency was correlated with increased Gleason grade. Nuclear morphological changes are a key feature of neoplasia and it perhaps extraordinary that we understand so little of the mechanisms that determine this.

The ability to perform increasingly detailed genetic analysis of cancers is demonstrated by Lapuk et al. [3]. They performed massive parallel sequencing, performing deep RNA and shallow DNA sequencing in 6 primary tumours. They massively expanded on the number of novel fusion genes known, linked fusion gene aetiology and gene expression profiles and showed the utility of fusion genes for molecular pathology by implicating the RE1-Silencing Transcription factor (REST) in the development of neuroendocrine prostatic cancers. A second paper from the same group [4] used next-generation sequencing on a single aggressive prostate adenocarcinoma using primary and metastatic tissues. They identified the area of primary cancer most likely to give rise to the metastasis and hypothesized that the amplification and over-expression of the stem cell gene MSI2 may have contributed to a hybrid luminal-neuroendocrine tumour of aggressive type. Lehmusvaara et al. [5] examined the molecular mechanisms of hormonal therapy by comparing the effects genetic effects of therapy with a control group in 28 men where frozen specimens were used for gene expression profiling for all known protein-coding genes. They show the significantly different effects of an anti-androgen and a GnRH agonist on gene expression. Also TMPRSS2-ERG fusion seems to bring many proliferation-related genes under androgen regulation.

 1 Nucleotide resolution analysis of TMPRSS2 and ERG rearrangements in prostate cancer
Weier C, Haffner MC, Mosbruger T, et al. The Journal of Pathology 2013; Accepted manuscript online. DOI:10.1002/path.4186
The Journal of Pathology 2013; Accepted manuscript online. DOI:10.1002/path.4186

 2 Chromosomal regions associated with prostate cancer risk localize to lamin B-deficient microdomains and exhibit reduced gene transcription
Helfand BT, Wang Y, Pfleghaar K, et al.
The Journal of Pathology 2012; 226: 735–745.

 3

From sequence to molecular pathology, and a mechanism driving the neuroendocrine phenotype in prostate cancer
Lapuk AV, Wu C, Wyatt AW, et al.
The Journal of Pathology 2012; 227: 286–297

 4 Integrated genome and transcriptome sequencing identifies a novel form of hybrid and aggressive prostate cancer
Wu C, Wyatt AW, Lapuk AV, et al.
The Journal of Pathology 2012; 227: 53–61

 5 Chemical castration and anti-androgens induce differential gene expression in prostate cancer
Lehmusvaara S, Erkkilä T, Urbanucci A, et al.
The Journal of Pathology 2012; 227:336–345.

In vitro studies

Metallothioneins (MT) are a group of metal binding proteins thought to play a role in the detoxification of heavy metals. Han et al. [6] show that MT1h, a metallothienin, demonstrates, in culture and xenografts, that MT1h demonstrates tumor suppressor activity that is dependent on activation of histone methylation. Prostatic squamous metaplasia is seen in response to oestrogen and oestrogen receptor Chen et al. [7] used a mouse model that has selectively lost ERα in either stromal or epithelial prostate cells to determine the requirements of ERα for oestrogen-stimulated prostate proliferation. They suggest that epithelial ERα is required for oestrogen-mediated proliferative response and could be an appropriate target for preventing aberrant oestrogen signalling in the prostate. The PMEPA1 gene has been shown to suppress the androgen receptor (AR) and TGFβ signalling pathways and is abnormally expressed in prostate tumours. Liu et al. [8] demonstrate that inhibition of PMEPA1 suppresses AR-negative cell lines through up-regulating p21 transcription. PMEPA1 may promote AR-negative prostate cancer cell proliferation through p21 and this may be a potential target for future therapies.

 6 Metallothionein 1h tumor suppressor activity in prostate cancer is mediated by euchromatin methyltransferase
Han Y-C, Zheng Z-L, Zuo Z-H, et al.
The Journal of Pathology 2013; Accepted manuscript online. DOI: 10.1002/path.4169

 7 Loss of epithelial oestrogen receptor α inhibits oestrogen-stimulated prostate proliferation and squamous metaplasia via in vivo tissue selective knockout models
Chen M, Yeh C-R, Chang H-C, et al.
The Journal of Pathology 2012; 226:17–27.

 8

PMEPA1 promotes androgen receptor-negative prostate cell proliferation through suppressing 3 the Smad3/4–c-Myc–p21 signaling pathway
Liu R, Zhou Z, Huang J and Chen C.
The Journal of Pathology 2011; 223:683–694.

Stem cells in the prostate

Until recently, our knowledge of stem cells in the prostate have been rudimentary. Two recent papers in Journal of Pathology have illuminated this area. Knowledge of stem cells is vital to understand cancer pathogenesis and also potentially for future clinical drug targets to the progenitor cells. Blackwood et al. [9] use mitochondrial DNA (mtDNA) mutations to map stem cell fate. The clonal relationships within the human prostate epithelial cell layers were explored by this method. They suggests that individual acini are typically generated from multiple stem cells. Most interestingly they show a common clonal origin for basal, luminal and neuroendocrine cells. Similar results were also reported by Gaisa et al. [10] who also investigated PIN and malignant human prostates Again, cells deficient for a mitochondrial enzyme, cytochrome c oxidase (CCO) were identified in frozen tissue samples using dual colour enzyme histochemistry. They also demonstrate that the normal, atrophic, hypertrophic and atypical (PIN) epithelium of human prostate contains stem cell-derived clonal units that actively replenish the epithelium during ageing. These deficient areas usually included the basal compartment indicating the basal layer as the location of the stem cell. Importantly, single clonal units comprised both PIN and invasive cancer, conforming PIN as the pre-invasive lesion for prostate cancer.

 9 In situ lineage tracking of human prostatic epithelial stem cell fate reveals a common clonal origin for basal and luminal cells
Blackwood JK, Williamson SC, Greaves LC, et al.
The Journal of Pathology 2011; 225:181–188.

 10 Clonal architecture of human prostatic epithelium in benign and malignant conditions
Gaisa NT, Graham TA, McDonald SAC, et al.
The Journal of Pathology 2011; 225: 172–180.

These are really important studies and further highlight the crucial importance of in situ studies of clonal architecture and stem cell hierarchies [11,12].

 11 Stem cell identification—in vivo lineage analysis versus in vitro isolation and clonal expansion
Wright NA.
The Journal of Pathology 2012; 227:255–266.

 12 The living-tissue microscope: the importance of studying stem cells in their natural, undisturbed microenvironment
Spradling A.
The Journal of Pathology 2011; 225: 161–162.

Future therapies for metastatic carcinoma

Options for metastatic prostate cancer are limited. Androgen deprivation remains the standard of care, though most cases show escape in time and secondary therapies are only temporary. Therefore the search for new targeted therapies for hormone resistant metastatic cancer is of vital clinical importance. Akfirat et al. [13] show that survival mechanisms differ between visceral and bone metastases, suggesting therapies of the future might depend on the pattern of metastasis or a multi-drug may be appropriate in those with both visceral and bone metastases. Methods of targeting bone metastasis are investigated by Caley et al. [14] who show that the collagen receptor Endo180 participates in collagen deposition by primary human osteoblasts during de novo osteoid formation and this was suppressed by co-culture with prostate tumour cells. Immunohistochemical analysis of core biopsies from bone metastasis revealed higher levels of Endo180 expression in tumour cell foci than cells in the surrounding stroma providing a rationale for targeting collagen remodelling by Endo180 in bone metastases of prostate cancer. Recent interest has focused on the potential of targeting metabolic pathways that may be altered during prostate tumorigenesis and progression. Flavin et al. [15] have reviewed in detail metabolic changes in prostate cancer and in particular the role of fatty acid and cholesterol secretion. Over-expression of the enzyme fatty acid synthase FASN has been suggested as a diagnostic marker in prostate cancers and several small molecule inhibitors of FASN have now been described or are in development.

Overexpression of the pro-survival protein heme oxygenase-1 (HO-1) and loss of the pro-apoptotic tumour suppressor PTEN are common events in prostate cancer (PCA). Li et al. [16] assessed these proteins in men with localized and castration-resistant prostate cancer (CRPC). The combined status of both markers correlated with disease progression. In a preclinical model, inhibition of HO-1 in PTEN-deficient PC3M cell lines and their matched cells where PTEN is restored strongly reduced cell growth and lung metastasis in xenografts. The cooperation between epithelial HO-1 expression and PTEN deletions could lead to the discovery of novel therapeutic modalities. Fibroblast growth factors (FGFs) have oncogenic roles in many cancers including prostate cancer. Rococa et al. show [17] pentraxin-3 (PTX3) acts as a natural FGF antagonist and show in murine and human cultured prostate cancer cells that it has an anti-mitogenic and anti-angiogenic effects and shows decreased expression in clinical prostate cancer samples. It may be another route to target metastatic disease.

 13 Tumor Cell Survival Mechanisms in Lethal Metastatic Prostate Cancer Differ Between Bone and Soft Tissue Metastases
Akfirat C, Zhang X, Ventura A, et al.
The Journal of Pathology 2013; Accepted manuscript online. DOI: 10.1002/path.4180

 14 TGFβ1–Endo180-dependent collagen deposition is dysregulated at the tumour–stromal interface in bone metastasis
Caley MP, Kogianni G, Adamarek A, et al.
The Journal of Pathology 2012; 226:775–783, Corrected by: Corrigendum: 2013; 229: e4.

 15 Metabolic alterations and targeted therapies in prostate cancer
Flavin R, Zadra G and Loda M.
The Journal of Pathology 2011; 223:284–295.

 16

PTEN deletion and heme oxygenase-1 overexpression cooperate in prostate cancer progression and are associated with adverse clinical outcome
Li Y, Su J, DingZhang X, et al.
The Journal of Pathology 2011; 224: 90–100.

 17

Long Pentraxin-3 As An Epithelial-Stromal Fibroblast Growth Factor-Targeting Inhibitor In Prostate Cancer
Ronca R, Alessi P, Coltrini D, et al.
The Journal of Pathology 2013; Accepted manuscript online. DOI: 10.1002/path.4181

Questions

The following questions can be answered by reading and reflecting upon the above annotation and the papers that are cited within it. Within the Royal College of Pathologists Continuing Professional Development (CPD) scheme, CPD points may be earned by writing reflective notes on the papers in this Virtual Issue and the questions are designed to act as a focus for this activity. To do this, you may wish to use the Royal College of Pathologists' reflective notes form.

 Question 1 What molecular abnormalities and in particular translocations have been described in prostate cancer?

 Question 2 Outline the stem cell structure of the prostate. What are the advantages of in situ methods for analyzing clonal architecture?

 Question 3 What are the possible metabolic changes seen in prostate cancer cells?


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