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The “European Hereditary Tumour Group” (former Mallorca
Group) hosts the Prospective Lynch Syndrome Database and the
Europ...
The Mallorca Group:
About 30 years ago, the International Collaborative Group on HNPCC (ICG-HNPCC)
and the Leeds Castle Po...
Among our interests are:
• How to determine pathogenicity of genetic variants
• Observe and report penetrance, expressivit...
JOINT PROJECT: Splicing analysis of MMR VUS
Medizinische Klinik und Poliklinik IV
Campus Innenstadt, Klinikum der Universi...
JOINT PROJECT: Splicing analysis of MMR VUS
European MMR Working Group
Two approaches:
1. Isolate RNA from blood to invest...
RNA from blood:
Two different protocols (Barcelona and Munich) compared
Concordant results (√) but Long-Range PCR (LR)poss...
Minigene splicing assay
Minigene
preparation
Analysis
of
minigene’s
transcripts
PCR
Transfection
human cells
RNA extractio...
Soukarieh et al., PLoS Genet. 2016
MLH1 exon 10 variants:
an unexpected high proportion of splicing mutations (77%)
MLH1 e...
Prospective Lynch Syndrome Database
• To escape from the selection biases in
retrospective studies
• To monitor effects of...
Segregation analysis is not appliccable
when phenocopies in the population and
penetrance < 50%
Pål Møller HVP 2016 UNESCO...
Power:
• 20,000 observation years needed for assumption-free methods
At 21,535 observation years filed
we analyzed the dat...
Unbiases input = unbiased output.
Once system established,
you may calculate on these
variables in any possible
way
– just...
Years
Cumulative incidence any cancer
path_MLH1
path_MSH2
path_MSH6
path_PMS2
Cumulative incidences first cancer in path_M...
MSH2
(GUT 2015)
Prospective Lynch Syndrome Database
Pål Møller HVP 2016 UNESCO
http://lscarisk.org
Open interactive web-site: Insert patient’s age, gender and genetic variant
and get his/her lifetime r...
Relative cumulative risk for subsequent cancer versus first
cancer in LS patients (GUT 2016 in press)
No difference
Pål Mø...
path_MLH1 path_MSH6path_MSH2
Years
Crude survival by genetic variant GUT 2016 in press
Prospective Lynch Syndrome Database...
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76 81
UGI_MLH1
UGI_MSH2
UGI_MSH6
UGI_PM...
European Hereditary Tumour Group (former Mallorca Group) hosts The Prospective Lynch Syndrome Database and the European MM...
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European Hereditary Tumour Group (former Mallorca Group) hosts The Prospective Lynch Syndrome Database and the European MMR cDNA Working Group and is open for all to join - Pål Møller

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The Mallorca group was formed by European members when InSiGHT was established 10 years ago to facilitate, coordinate and compile European research on inherited colorectal cancer syndromes and issue clinical guidelines (http://www.ncbi.nlm. nih.gov/pubmed/23408351). It is this year transformed into The European Hereditary Tumour Group (EHTG), open for world-wide membership (contact: Gabriela.Moeslein@helios-kliniken.de). The European MMR cDNA Working Group is focusing on in-vitro testing for determining pathogenicity of MMR gene variants (holinskifeder@ mgz-muenchen.de).

The Prospective Lynch Syndrome Database ( moller.pal@gmail. com) established 2013, is open for all to join. The goal is to migrate knowledge on Lynch Syndrome (LS) from educated guesses based on retrospective series invalidated by selection biases, to prospectively obtained empirical knowledge and revise clinical guidelines accordingly. Classification of mutations is made in collaboration with InSiGHT’s (http://insight-group.org/) Variant Interpretation Committee (VIC, contact: Maurizio.Genuardi@unicatt.it) and the LOVD database (http://chromium.lovd.nl/LOVD2/colon_cancer/), where all genetic variants are classified (http://www.ncbi.nlm.nih. gov/pubmed/24362816 ) and which is now recognized by HVP as the reference database for MMR mutations. Large numbers are needed to arrive at reliable results. The database currently includes ~3,200 LS patients prospectively observed for ~25,000 years. Results currently published (http://www.ncbi.nlm.nih.gov/pubmed/26657901) or in preparation include: Colonoscopy with removal of adenomas does not prevent colorectal cancer as assumed, but survival when cancer occurred was excellent. A 3 year interval was neither associated with lower incidence of colorectal cancer nor worse survival than shorter intervals between colonoscopies. Endometrial cancer had the highest lifetime incidence and had excellent survival. Ovarian cancer was seen early in life and most were cured. We now have a growing and aging cohort of LS patients who have survived both their first and subsequent cancers. Among these, path_MLH1 carriers have a high incidence of upper gastrointestinal cancers with serious prognosis, while path_MSH2 carriers have high incidence of urinary tract cancers with good prognosis but some get brain or gastric tumours with serious prognoses. Path_MSH6 carriers had as high incidence of endometrial cancer, but relatively low lifetime risk for colorectal cancer. Path_PMS2 carriers had, besides a few cases with endometrial and prostate cancers, no incidence of LS associated cancers. We have established the web-site http://lscarisk.org where risk for cancer according to age, genotype and gender may be calculated for any LS patient’s actual age which may be instrumental for genetic counselling.

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European Hereditary Tumour Group (former Mallorca Group) hosts The Prospective Lynch Syndrome Database and the European MMR cDNA Working Group and is open for all to join - Pål Møller

  1. 1. The “European Hereditary Tumour Group” (former Mallorca Group) hosts the Prospective Lynch Syndrome Database and the European MMR cDNA Working Group and is open for all to join Pål Møller (moller.pal@gmail.com), Elke Holinski-Feder, Monika Morak, Toni Seppälä, Kirsi Pylvanainen, Jukka-Pekka Mecklin, Gabriel Capella, Ian Frayling, Julian Sampson, John Burn, Rolf Sijmons, Maurizio Genuardi, John-Paul Plazzer, Finaly Macrae, Gabriela Möslein on behalf of The Mallorca group (http://mallorca-group.org/ ) InSiGHT (http://insight-group.org/ ) The Prospective Lynch Syndrome Database (PLSD) The European MMR cDNA Working Group Pål Møller HVP 2016 UNESCO
  2. 2. The Mallorca Group: About 30 years ago, the International Collaborative Group on HNPCC (ICG-HNPCC) and the Leeds Castle Polyposis Group (LCPG) set out to find the genes causing inherited colorectal cancers due to non-polyposis and polyposis respectively. When the genes were found, we joined all activities on describing inherited intestinal cancers into one world-wide organisation:- The International Society for Gastrointestinal Hereditary Tumours InSiGHT (http://insight-group.org/ ) 11 years ago the European members of the former ICG-HNPCC and LCPG decided to meet annually to facilitate, coordinate and compile European research on inherited colorectal cancer syndromes and issue clinical guidelines: The Mallorca Group ( http://mallorca-group.org) Which this year was transformed into “The European Hereditary Tumour Group” (working title, name not finally decided) which is now open for all those interested in Europe and world-wide to join. Pål Møller HVP 2016 UNESCO
  3. 3. Among our interests are: • How to determine pathogenicity of genetic variants • Observe and report penetrance, expressivity and effects of interventions in carriers of pathogenic variants of the MMR genes • Translate the above into clinical guidelines for health care Most European countries have adapted the ‘Mallorca guidelines’ on health service for the various inherited cancer syndromes in question. This contribution describes the two research activities mentioned above. Pål Møller HVP 2016 UNESCO
  4. 4. JOINT PROJECT: Splicing analysis of MMR VUS Medizinische Klinik und Poliklinik IV Campus Innenstadt, Klinikum der Universität München, Germany Aims: - Assess quality of different cDNA analyses by comparison. - Improve uniformity of results interpretation. Provide an improved standardized protocol for cDNA analysis (SOP). European MMR Working Group See poster by Monika Morak , Annika Lindblom, Maurizio Genuardi, Gabriel Capella, Pål Møller, Alexandra Martins, Marta Pineda, Pascaline Gaildrat, Guido Plotz, Wenche Sjursen, Kristina Lagerstedt, Minna Nystrom, Niels de Wind, Robert Hofstra , Inge Bernstein, Lene Juel Rasmussen, Malcolm Dunlop, Niels Rahner, Brigitte Royer-Polka, Susan Mary Farrington, John Burn, Friedrik Wikman, Rolf Sijmons and Elke Holinski-Feder Pål Møller HVP 2016 UNESCO
  5. 5. JOINT PROJECT: Splicing analysis of MMR VUS European MMR Working Group Two approaches: 1. Isolate RNA from blood to investigate cDNA from a variant 2. DNA from blood to test a variant in an artificial cell- culture splicing assay (minigene) Why? The InSiGHT Variant Interpretation Committee identified the need for lab test to describe pathogenicity of the genetic variants (Nature Genetics 2014) Pål Møller HVP 2016 UNESCO
  6. 6. RNA from blood: Two different protocols (Barcelona and Munich) compared Concordant results (√) but Long-Range PCR (LR)possibly more sensitive For details see poster. Pål Møller HVP 2016 UNESCO
  7. 7. Minigene splicing assay Minigene preparation Analysis of minigene’s transcripts PCR Transfection human cells RNA extraction & RT-PCR Electrophoresis Gel purification Sequencing Sequencing WT mut Cloning Tournier et al., Hum Mutat. 2008 Théry et al., Eur J Hum Genet. 2011 Gaildrat et al., J Med Genet. 2012 Gaildrat et al., Methods Mol Biol. 2010 Bonnet et al., J Med Genet. 2008 Gaildrat et al., J Med Genet. 2010 Vezain et al., Hum Mutat. 2010 Vezain et al., Hum Mutat. 2011 Di Giacomo et al., Hum Mutat. 2013 Thompson et al., Nature Genet 2014 Soukarieh et al., PLoS Genet 2016 de la Hoya et al., Hum Mol Genet 2016 Houdayer et al., Hum Mutat. 2012 alexandra.martins@univ-rouen.fr Inserm U1079, Rouen, FrancePål Møller HVP 2016 UNESCO
  8. 8. Soukarieh et al., PLoS Genet. 2016 MLH1 exon 10 variants: an unexpected high proportion of splicing mutations (77%) MLH1 exon 10MLH1 exon 10 pSPL3m No impact on splicing: 5 variants Increased exon inclusion: 4 variants Increased exon skipping: 13 variants alexandra.martins@univ-rouen.fr Inserm U1079, Rouen, France Variants (n= 22) Minigene splicing assay Pål Møller HVP 2016 UNESCO
  9. 9. Prospective Lynch Syndrome Database • To escape from the selection biases in retrospective studies • To monitor effects of interventions Why? Pål Møller HVP 2016 UNESCO
  10. 10. Segregation analysis is not appliccable when phenocopies in the population and penetrance < 50% Pål Møller HVP 2016 UNESCO HVP Paris UNESCO 2010
  11. 11. Power: • 20,000 observation years needed for assumption-free methods At 21,535 observation years filed we analyzed the data set Annual Incidence Rates in 5-year age cohorts for each gene and gender to determine penterance of MMR pathogenic variants Prospective Lynch Syndrome Database Pål Møller HVP 2016 UNESCO
  12. 12. Unbiases input = unbiased output. Once system established, you may calculate on these variables in any possible way – just put in the data and the results pop out. Prospective Lynch Syndrome Database Pål Møller HVP 2016 UNESCO
  13. 13. Years Cumulative incidence any cancer path_MLH1 path_MSH2 path_MSH6 path_PMS2 Cumulative incidences first cancer in path_MMR carriers (GUT 2015) Prospective Lynch Syndrome Database Pål Møller HVP 2016 UNESCO Colonoscopy with polypectomy does not protect against colon cancer as much as we hoped for Significant differences between the genes
  14. 14. MSH2 (GUT 2015) Prospective Lynch Syndrome Database Pål Møller HVP 2016 UNESCO
  15. 15. http://lscarisk.org Open interactive web-site: Insert patient’s age, gender and genetic variant and get his/her lifetime risk in a sec. Pål Møller HVP 2016 UNESCO
  16. 16. Relative cumulative risk for subsequent cancer versus first cancer in LS patients (GUT 2016 in press) No difference Pål Møller EHTG Mallorca 2016 Prospective Lynch Syndrome Database
  17. 17. path_MLH1 path_MSH6path_MSH2 Years Crude survival by genetic variant GUT 2016 in press Prospective Lynch Syndrome Database Pål Møller HVP 2016 UNESCO
  18. 18. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76 81 UGI_MLH1 UGI_MSH2 UGI_MSH6 UGI_PMS2 Cumulative incidence upper gastrointestinal cancer Survival Gastric duodenal bile duct pancreatic ca Candididates for phase2 trials immunotherapy? Vaccines? What now? (in preparation) Prospective Lynch Syndrome Database Pål Møller HVP 2016 UNESCO

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