. 2017 Nov;134(5):705-714.

doi: 10.1007/s00401-017-1752-4. Epub 2017 Jul 21.

Immunohistochemical analysis of H3K27me3 demonstrates global reduction in group-A childhood posterior fossa ependymoma and is a powerful predictor of outcome

Pooja Panwalkar¹, Jonathan Clark¹, Vijay Ramaswamy^{2

3}, Debra Hawes⁴, Fusheng Yang⁴, Christopher Dunham^{5

6}, Stephen Yip⁶, Juliette Hukin⁷, Yilun Sun⁸, Matthew J Schipper⁸, Lukas Chavez⁹, Ashley Margol¹⁰, Melike Pekmezci¹¹, Chan Chung¹, Adam Banda¹, Jill M Bayliss¹, Sarah J Curry², Mariarita Santi¹², Fausto J Rodriguez¹³, Matija Snuderl¹⁴, Matthias A Karajannis¹⁵, Amanda M Saratsis^{16

17}, Craig M Horbinski¹⁸, Anne-Sophie Carret¹⁹, Beverly Wilson²⁰, Donna Johnston²¹, Lucie Lafay-Cousin²², Shayna Zelcer²³, David Eisenstat²⁴, Marianna Silva²⁵, Katrin Scheinemann^{26

27}, Nada Jabado^{28

29}, P Daniel McNeely³⁰, Marcel Kool^{9

31}, Stefan M Pfister^{9

31

32}, Michael D Taylor³³, Cynthia Hawkins³⁴, Andrey Korshunov³⁵, Alexander R Judkins³⁶, Sriram Venneti^{37

38}

Affiliations

¹ Department of Pathology, University of Michigan, Ann Arbor, MI, 48104, USA.
² Division of Haematology/Oncology, Hospital for Sick Children, University of Toronto, Toronto, ON, Canada.
³ Programme in Neuroscience and Mental Health, Hospital for Sick Children, University of Toronto, Toronto, ON, Canada.
⁴ Department of Pathology and Laboratory Medicine, Children's Hospital Los Angeles, Keck School of Medicine of University of Southern California, 4650 Sunset Boulevard, MS #43, Los Angeles, CA, 90027, USA.
⁵ Division of Anatomic Pathology, British Columbia Children's Hospital, 4500 Oak Street, Vancouver, BC, V6H 3N1, Canada.
⁶ Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, V6T1Z3, BC, Canada.
⁷ Divisions of Neurology and Hematology and Oncology, Children's and Women's Health Centre of B.C, University of British Columbia, Vancouver, BC, V6H 3N1, Canada.
⁸ Department of Biostatistics, University of Michigan, Ann Arbor, MI, USA.
⁹ Division of Pediatric Neurooncology, German Cancer Research Center (DKFZ), Heidelberg, Germany.
¹⁰ Department of Pediatrics, Children's Hospital Los Angeles, Keck School of Medicine University of Southern California, Los Angeles, CA, 90027, USA.
¹¹ Department of Pathology, University of California San Francisco, San Francisco, CA, USA.
¹² Department of Anatomic Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, Philadelphia, PA, 19104, USA.
¹³ Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
¹⁴ Department of Pathology, New York University, New York, NY, USA.
¹⁵ Department of Pediatrics, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA.
¹⁶ Department of Neurological Surgery, Northwestern University Feinberg School of Medicine, Chicago, IL, 60611, USA.
¹⁷ Division of Pediatric Neurosurgery, Ann & Robert H. Lurie Children's Hospital of Chicago, Chicago, IL, 60611, USA.
¹⁸ Department of Pathology and Neurosurgery, Northwestern University Feinberg School of Medicine, Chicago, IL, 60611, USA.
¹⁹ Division of Hematology-Oncology, Centre Hospitalier Universitaire Sainte-Justine, Université de Montréal, Montreal, QC, Canada.
²⁰ Division of Pediatric Hematology/Oncology, Stollery Children's Hospital, University of Alberta, Edmonton, AB, T2W3N2, Canada.
²¹ Division of Pediatric Hematology/Oncology, Department of Pediatrics, University of Ottawa, Ottawa, ON, Canada.
²² Division of Pediatric Hematology/Oncology, Alberta Children's Hospital, Calgary, AB, T3B6A8, Canada.
²³ Division of Pediatric Hematology/Oncology, London Health Sciences Center, Children's Hospital, London, ON, N6A5A5, Canada.
²⁴ Department of Pediatrics and Medical Genetics, University of Alberta, Edmonton, AB, Canada.
²⁵ Kingston General Hospital, Kingston, ON, Canada.
²⁶ Department of Pediatrics, McMaster University, Hamilton, ON, Canada.
²⁷ Division of Hematology/Oncology, University Children Hospital of Basel (UKBB) and University of Basel, Basel, Switzerland.
²⁸ Department of Pediatrics, McGill University, Montreal, QC, H3Z2Z3, Canada.
²⁹ Department of Human Genetics, McGill University, Montreal, QC, H3Z2Z3, Canada.
³⁰ Division of Neonatal Pediatrics, Department of Pediatrics, Dalhousie University, Halifax, NS, Canada.
³¹ German Cancer Consortium (DKTK), Heidelberg, Germany.
³² Department of Pediatric Oncology, Hematology and Immunology, University of Heidelberg, Heidelberg, Germany.
³³ Division of Neurosurgery, Arthur and Sonia Labatt Brain Tumor Research Centre, The Hospital for Sick Children, Toronto, ON, M5G 1X8, Canada.
³⁴ Pediatric Laboratory Medicine, Hospital for Sick Children, 555 University Avenue, Toronto, ON, M5G 1X8, Canada.
³⁵ Department of Neuropathology, German Cancer Research Center (DKFZ), University Hospital Heidelberg and CCU Neuropathology, Heidelberg, Germany.
³⁶ Department of Pathology and Laboratory Medicine, Children's Hospital Los Angeles, Keck School of Medicine of University of Southern California, 4650 Sunset Boulevard, MS #43, Los Angeles, CA, 90027, USA. ajudkins@chla.usc.edu.
³⁷ Department of Pathology, University of Michigan, Ann Arbor, MI, 48104, USA. svenneti@med.umich.edu.
³⁸ Pathology, University of Michigan Medical School, University of Michigan, 3520E MSRB 1, 1150 W. Medical Center Dr., Ann Arbor, MI, 41804, USA. svenneti@med.umich.edu.

PMID: 28733933
PMCID: PMC5647236
DOI: 10.1007/s00401-017-1752-4

Immunohistochemical analysis of H3K27me3 demonstrates global reduction in group-A childhood posterior fossa ependymoma and is a powerful predictor of outcome

Pooja Panwalkar et al. Acta Neuropathol. 2017 Nov.

. 2017 Nov;134(5):705-714.

doi: 10.1007/s00401-017-1752-4. Epub 2017 Jul 21.

Authors

Affiliations

¹ Department of Pathology, University of Michigan, Ann Arbor, MI, 48104, USA.
² Division of Haematology/Oncology, Hospital for Sick Children, University of Toronto, Toronto, ON, Canada.
³ Programme in Neuroscience and Mental Health, Hospital for Sick Children, University of Toronto, Toronto, ON, Canada.
⁴ Department of Pathology and Laboratory Medicine, Children's Hospital Los Angeles, Keck School of Medicine of University of Southern California, 4650 Sunset Boulevard, MS #43, Los Angeles, CA, 90027, USA.
⁵ Division of Anatomic Pathology, British Columbia Children's Hospital, 4500 Oak Street, Vancouver, BC, V6H 3N1, Canada.
⁶ Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, V6T1Z3, BC, Canada.
⁷ Divisions of Neurology and Hematology and Oncology, Children's and Women's Health Centre of B.C, University of British Columbia, Vancouver, BC, V6H 3N1, Canada.
⁸ Department of Biostatistics, University of Michigan, Ann Arbor, MI, USA.
⁹ Division of Pediatric Neurooncology, German Cancer Research Center (DKFZ), Heidelberg, Germany.
¹⁰ Department of Pediatrics, Children's Hospital Los Angeles, Keck School of Medicine University of Southern California, Los Angeles, CA, 90027, USA.
¹¹ Department of Pathology, University of California San Francisco, San Francisco, CA, USA.
¹² Department of Anatomic Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, Philadelphia, PA, 19104, USA.
¹³ Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
¹⁴ Department of Pathology, New York University, New York, NY, USA.
¹⁵ Department of Pediatrics, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA.
¹⁶ Department of Neurological Surgery, Northwestern University Feinberg School of Medicine, Chicago, IL, 60611, USA.
¹⁷ Division of Pediatric Neurosurgery, Ann & Robert H. Lurie Children's Hospital of Chicago, Chicago, IL, 60611, USA.
¹⁸ Department of Pathology and Neurosurgery, Northwestern University Feinberg School of Medicine, Chicago, IL, 60611, USA.
¹⁹ Division of Hematology-Oncology, Centre Hospitalier Universitaire Sainte-Justine, Université de Montréal, Montreal, QC, Canada.
²⁰ Division of Pediatric Hematology/Oncology, Stollery Children's Hospital, University of Alberta, Edmonton, AB, T2W3N2, Canada.
²¹ Division of Pediatric Hematology/Oncology, Department of Pediatrics, University of Ottawa, Ottawa, ON, Canada.
²² Division of Pediatric Hematology/Oncology, Alberta Children's Hospital, Calgary, AB, T3B6A8, Canada.
²³ Division of Pediatric Hematology/Oncology, London Health Sciences Center, Children's Hospital, London, ON, N6A5A5, Canada.
²⁴ Department of Pediatrics and Medical Genetics, University of Alberta, Edmonton, AB, Canada.
²⁵ Kingston General Hospital, Kingston, ON, Canada.
²⁶ Department of Pediatrics, McMaster University, Hamilton, ON, Canada.
²⁷ Division of Hematology/Oncology, University Children Hospital of Basel (UKBB) and University of Basel, Basel, Switzerland.
²⁸ Department of Pediatrics, McGill University, Montreal, QC, H3Z2Z3, Canada.
²⁹ Department of Human Genetics, McGill University, Montreal, QC, H3Z2Z3, Canada.
³⁰ Division of Neonatal Pediatrics, Department of Pediatrics, Dalhousie University, Halifax, NS, Canada.
³¹ German Cancer Consortium (DKTK), Heidelberg, Germany.
³² Department of Pediatric Oncology, Hematology and Immunology, University of Heidelberg, Heidelberg, Germany.
³³ Division of Neurosurgery, Arthur and Sonia Labatt Brain Tumor Research Centre, The Hospital for Sick Children, Toronto, ON, M5G 1X8, Canada.
³⁴ Pediatric Laboratory Medicine, Hospital for Sick Children, 555 University Avenue, Toronto, ON, M5G 1X8, Canada.
³⁵ Department of Neuropathology, German Cancer Research Center (DKFZ), University Hospital Heidelberg and CCU Neuropathology, Heidelberg, Germany.
³⁶ Department of Pathology and Laboratory Medicine, Children's Hospital Los Angeles, Keck School of Medicine of University of Southern California, 4650 Sunset Boulevard, MS #43, Los Angeles, CA, 90027, USA. ajudkins@chla.usc.edu.
³⁷ Department of Pathology, University of Michigan, Ann Arbor, MI, 48104, USA. svenneti@med.umich.edu.
³⁸ Pathology, University of Michigan Medical School, University of Michigan, 3520E MSRB 1, 1150 W. Medical Center Dr., Ann Arbor, MI, 41804, USA. svenneti@med.umich.edu.

PMID: 28733933
PMCID: PMC5647236
DOI: 10.1007/s00401-017-1752-4

Abstract

Posterior fossa ependymomas (EPN_PF) in children comprise two morphologically identical, but biologically distinct tumor entities. Group-A (EPN_PFA) tumors have a poor prognosis and require intensive therapy. In contrast, group-B tumors (EPN_PFB) exhibit excellent prognosis and the current consensus opinion recommends future clinical trials to test the possibility of treatment de-escalation in these patients. Therefore, distinguishing these two tumor subtypes is critical. EPN_PFA and EPN_PFB can be distinguished based on DNA methylation signatures, but these assays are not routinely available. We have previously shown that a subset of poorly prognostic childhood EPN_PF exhibits global reduction in H3K27me3. Therefore, we set out to determine whether a simple immunohistochemical assay for H3K27me3 could be used to segregate EPN_PFA from EPN_PFB tumors. We assembled a cohort of 230 childhood ependymomas and H3K27me3 immunohistochemistry was assessed as positive or negative in a blinded manner. H3K27me3 staining results were compared with DNA methylation-based subgroup information available in 112 samples [EPN_PFA (n = 72) and EPN_PFB tumors (n = 40)]. H3K27me3 staining was globally reduced in EPN_PFA tumors and immunohistochemistry showed 99% sensitivity and 100% specificity in segregating EPN_PFA from EPN_PFB tumors. Moreover, H3K27me3 immunostaining was sufficient to delineate patients with worse prognosis in two independent, non-overlapping cohorts (n = 133 and n = 97). In conclusion, immunohistochemical evaluation of H3K27me3 global reduction is an economic, easily available and readily adaptable method for defining high-risk EPN_PFA from low-risk posterior fossa EPN_PFB tumors to inform prognosis and to enable the design of future clinical trials.

Keywords: Childhood ependymoma; Epigenetics; H3K27me3; Molecular subgrouping.

PubMed Disclaimer

Conflict of interest statement

Declaration of interests: The authors declare no competing interests

Figures

**Figure. 1. H3K27me3 immunohistochemistry segregates EPN_PFA from EPN_PF B tumor**
a. Representative images of H3K27me3 immunostaining in grade II and grade III EPN_PFA and EPN_PFB. Arrowheads (black) indicate cells within the microenvironment of tumor including endothelial cells that stained positive for H3K27me3 and were used as internal positive control. Scale bars represents 200 µM b. Percent distribution of H3K27me3 negative (Red) and H3K27me3 positive (Blue) cases in predefined EPN_PFA and EPN_PFB tumors. Numbers in parentheses indicate the number of tumors studied. c. Quantification of H3K27me3 immunostaining in EPN_PFA and EPN_PFB. Each point represents a single tumor. Error bars represent mean +/− S.D. d. Percent sensitivity, specificity, positive predictive value, negative predictive value, false positive rate, false negative rate and false discovery rate for detection of childhood posterior fossa EPN_PFA vs. EPN_PFB molecular subgroups using H3K27me3 immunostaining.

**Figure. 2. Global loss of H3K27me3 immunostaining relates with younger age and poor prognosis in two independent cohorts**
a. Box and whisker plot of age distribution of H3K27me3 negative and H3K27me3 positive tumors in cohort 1. **b–c.** Progression free survival (PFS, B) or overall survival (OS, C) of EPN stratified by H3K27me3 staining in cohort 1. d. Box and whisker plot of age distribution of H3K27me3 negative and H3K27me3 positive tumors in cohort 2. **e–f.** Progression free survival (PFS, E) or overall survival (OS, F) of EPN stratified by H3K27me3 staining in cohort 2. The numbers below the X-axis indicates the number of persons at risk at each time point. g. Percentage of H3K27me3 negative and H3K27me3 positive tumors (Y-axis) plotted against corresponding patient age (X-axis). **h–i.** Progression free survival (PFS, H) or overall survival (OS, I) of EPN stratified by H3K27me3 staining in combined cohorts 1 and 2. For a and d, data are plotted as median with interquartile range with 95% CI and each point represents a single tumor. Data was analyzed using unpaired, two-tailed Mann Whitney test. For survival curves, the numbers below the X-axis indicates the number of persons at risk at each time point. Data was analyzed using the Log-Rank test.

See this image and copyright information in PMC

Comment in

Neuro-oncology: A new approach to ependymoma subtyping.
Wood H. Wood H. Nat Rev Neurol. 2017 Sep;13(9):512-513. doi: 10.1038/nrneurol.2017.114. Epub 2017 Aug 4. Nat Rev Neurol. 2017. PMID: 28776599 No abstract available.

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Immunohistochemical analysis of H3K27me3 demonstrates global reduction in group-A childhood posterior fossa ependymoma and is a powerful predictor of outcome

Affiliations

Immunohistochemical analysis of H3K27me3 demonstrates global reduction in group-A childhood posterior fossa ependymoma and is a powerful predictor of outcome

Authors

Affiliations

Abstract

Conflict of interest statement

Figures

Comment in

References

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Medical