Author's response to reviews

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1 Author's response to reviews Title: EPHB2 germline variants in patients with colorectal cancer or hyperplastic polyposis Authors: Antti Kokko Paivi Laiho Rainer Lehtonen Sanna Korja Luis G Carvajal-Carmona (luis.carvajal@cancer.org.uk) Heikki Jarvinen (heikki.jarvinen@hus.fi) Jukka-Pekka Mecklin (jukka-pekka.mecklin@ksshp.fi) Charis Eng (engc@ccf.org) Johanna Schleutker (johanna.schleutker@uta.fi) Ian PM Tomlinson (ian.tomlinson@cancer.org.uk) Pia Vahteristo (pia.vahteristo@helsinki.fi) Lauri A Aaltonen (lauri.aaltonen@helsinki.fi) Version: 2 Date: 30 May 2006 see over Author's response to reviews:

2 U N I V E R S I T Y O F H E L S I N K I H A A R T M A N I N S T I T U T E D E P A R T M E N T O F M E D I C A L G E N E T I C S Professor Melissa Norton Medical Editor BMC Cancer Helsinki, 30 May 2006 Enclosed please find the revised manuscript entitled EPHB2 germline variants in patients with colorectal cancer or hyperplastic polyposis resubmitted for possible publication in the BMC Cancer. The manuscript has been formatted to conform to BMC cancer style and modified as suggested by the reviewers. We found the comments very helpful, and believe that the changes have improved the manuscript. The scientific criticism presented was very constructive and no major concerns were expressed. Below, the specific comments and suggestions presented by the reviewers are addressed point by point (in bold). We have fully addressed the issues raised. I look forward to hearing from you, Lauri A. Aaltonen, M.D., Ph.D. Research Professor of the Finnish Academy of Sciences Department of Medical Genetics Biomedicum Helsinki PO Box 63 (Haartmaninkatu 8) FIN University of Helsinki, Finland Phone: Fax: lauri.aaltonen@helsinki.fi

3 Reviewer #1 Barbara Burwinkel Reviewer's report: Minor Essential Revisions: In their manuscript entitled "EPHB2 germline variants in patients with colorectal cancer or hyperplastic polyposis" Kokko et al. report four EPHB2 missense alterations in patients with CRC or HPP. In general, this study has been well performed since the study design is rationale and sound. However, I would suggest improvements of the manuscript including a more explicit presentation. Main points: BACKGROUND section 1. Last sentence of the second paragraph: Give a short description of the known somatic and germline EPHB2 mutations (references 6-8). In which ethnic groups have they been detected? As suggested by the reviewer, we have added the following text to the manuscript (pages 3-4): Huusko et al. (2004) identified a nonsense mutation Q723X in a PC cell line, which led to the identification of other novel missense (R199H, A297S, D679N, T909M), nonsense (K1019X), frameshift and splice site mutations in prostate tumors obtained from patients in USA and Switzerland [7]. In a later study by Kittles et al. (2005) the nonsense mutation K1019X was found to increase risk for PC over two-fold in an African American hereditary PC sample set [8]. In 2005, Alazzouzi et al. observed frequent frameshift mutations on the A9 repeat of the EPHB2 gene in primary colorectal tumors in patients of mostly Caucasian origin (Finland, Germany, Spain and Japan) [6]. 2. The authors claim (in the second paragraph of the Discussion and first sentences of the Results/Conclusion sections, respectively) that they are the first to report previously unknown missense alterations in EPHB2. However, they refer to their submitted manuscript (Laiho et al.) in which they describe a EPHB2 germline mutation. The authors should comment on this and make their statements with caution. The submitted manuscript (Laiho et al., now in press) focused on expression profiling of two tumor types, and the EPHB2 gene, being differentially expressed between the two classes, was screened for somatic mutations in CRC samples. However, as during the screening a new potentially interesting germline missense alteration (R568W) was found, we decided to perform another study focusing on the germline variants, and report this finding there (the current study). Therefore, though we mention the study by Laiho et al. as background for the current study, no germline mutations are reported in the previous study.

4 METHODS section Patient material 1. Have the CRC patients been tested for mutations in high-penetrance genes? The Finnish CRC patients have been tested for predisposing mutations in MLH1 and MSH2 (Aaltonen et al., 1998; Salovaara et al., 2000) and three founder mutations in MYH (Enholm et al., 2003; Alhopuro et al., 2005). The UK hyperplastic polyposis patients have been tested for MLH1, MSH2 and two MYH mutations (unpublished data). The US hyperplastic polyposis patients have been tested for PTEN, BMPR1A, STK11, SMAD4, ENG, BRAF, MYH, and BHD as described by Sweet et al. (2005). References: Aaltonen et al. N Engl J Med May; 338(21): Alhopuro et al. Hum Mutat Oct; 26(4):393. Enholm et al. Am J Pathol Sep; 163(3): Salovaara et al. J Clin Oncol Oct; 18(19):3456. Sweet et al. JAMA Nov; 294(19): The authors should add mean ages of onset and age ranges for patient groups 1) and 2) (Methods, lines 3-4) and the further analyzed cancer patients and controls (Methods, paragraph 2). We have added the data for the patient groups and further analyzed cancer patients (pages 4 and 5), as suggested. Unfortunately, for the healthy controls this information was not available. The Finnish control samples were obtained from anonymous healthy blood donors from The Finnish Red Cross Blood Service. The UK controls were a mixture of healthy middle-aged and old individuals from various sources without detailed information related to age. 3. The authors selected the 63 CRC patients out of a population-based series 1042 Finnish CRC patients. There were only 63 CRC patients with either personal or family history of PC or were there additional selection criteria? As the reviewer has commented, in filtering of the series of 1042 Finnish CRC patients there were only 63 CRC patients with either personal or family history of PC in the first degree relatives. Family history of second degree relatives (if available) would have undoubtedly increased the numbers substantially. Mutational analysis 4. Did the authors sequence all exons of the EPHB2 gene? Did they sequence the exonintron boundaries, and if so, how many intronic nucleotides can be claimed to be mutation-

5 free (e.g. all EPHB2 exons or all coding exons were sequenced including at least 20 nucleotides intronic sequence at the exon-intron boundaries each )? All EPHB2 exons, except exon 1, were sequenced with primers designed to flank at least 70 intronic nucletides on both sides of the exons. Exon 1 has a long CpG-rich promoter area, and despite multiple efforts to design primers we could not sequence the region. To our knowledge, no commercial primers are available, and no other research group has been able to screen this region either. The following remark was added to the text at page 6: All coding exons with at least 70 nucleotides intronic sequence at the exon-intron boundaries, except exon 1, were sequenced. RESULTS section 1. One should mention that the four germline mutations were heterozygous (Results section, line 1). We have added this information at page 6, as suggested: Altogether four heterozygous germline missense changes 2. Initial (Results section, line 2) should be specified (e.g. the initial ). The wording has been clarified in the manuscript accordingly, at page 6: in the initial EPHB2 mutation screening of 101 samples The authors should mention the varying and low genotyping rates ; e.g. sequencing data of only 87% (139 out of 159) of familial CRC patients and 85% (239 out of 282) of controls were obtained etc.. The genotyping rates have been expressed in more detail, as requested by the reviewer. At pages 6 and 7: in 139 of 159 familial CRC patients (data from 87% of cases) or in 239 of 282 healthy population-matched controls (data from 85% of cases) in 145 familial CRC patients (data from 91% of cases) 4. The authors may discuss a putative protective effect of R80H (0.9% in cases vs. 3.2% in controls). We appreciate this comment, which is an intriguing remark. However, in our analyses the comparison did not reach statistical significance ( 2 p-value = 0.39, Yate s correction). Due to the rarity of the polymorphism and few numbers of observations we feel hesitant to include this putative protective effect into the text as a result, but can of course discuss this point in general, if considered a prerequisite for publication. DISCUSSION section

6 1. The authors comment on their subsequent analysis of I361V and R568W in the extensive set of Finnish patients, but they miss to describe their additional analysis of D861N in UK patients. I would suggest an additional paragraph on that issue. As the reviewer has pointed out, the UK patients were indeed analyzed further as well. This has been added to the manuscript at page 9: The UK variant D861N was not observed in additional 40 HPP patients or in 200 healthy UK controls, suggesting as well a possible role for the variant in colorectal tumor susceptibility. 2. The sentence Overall, EPHB2... (Page 9, first line) is confusing. Either delete it or comment on the total number of analyzed samples. This sentence has been modified to depict the information more clearly, as the reviewer has suggested. The total number of samples analyzed for the EPHB2 gene is 101, as all the additional screenings were performed only for the exons with detected alterations. At page 9: Overall, germline variants that may be associated with the disease phenotype were seen in 3/101 (3.0%) of patients analyzed for the EPHB2 gene. 3. The authors may estimate the effects of the detected variants using the PolyPhen tool ( The detected variants have been characterized using PolyPhen and SIFT prediction software, as suggested. Two of the variant (R80H, R568W) were predicted as probably damaging, and the remaining two as benign (I361V, D861N) by PolyPhen, while SIFT predicted I361V to be tolerated and all the others as nontolerated (data not included into the manuscript but can be added, if desired). However, in our experience (as in this study) these prediction software have not produced very reliable results, and we would be cautious to add these predictions. ABSTRACT section The Abstract sections should be adapted to the corresponding parts of the manuscript: ABSTRACT/Results: 1. The authors may avoid the expression four previously unknown as a manuscript including one missense mutation is submitted. Please see our response to the reviewer s question at cover letter pages 1 and 2. (Main points, background section, comment #2). The manuscript by Laiho et al. (now in press) does not mention any missense mutations. 2. They should write..which may be associated.. Revised as suggested, at page 2: which may be associated with the disease phenotype.

7 ABSTRACT/Conclusions: 1. As in the CONCLUSION section, the authors should be more cautious and suggest a limited role of the EPHB2 variants. This has been modified to the text, as proposed, at page 2: The results suggest a limited role for these EPHB2 variants in colon tumor predisposition. Minor points: 1. References within the text should be placed in front of a full stop. We have revised the references to conform to the current style, as suggested. 2. Discussion, line 11: Write In our recent study.... The text has been modified as suggested. 3. Since it is very complex, I would suggest a clearer presentation of Table 2 (e.g. a better optical separation of the different studies). We have revised Table 2, as proposed, to depict the results more clearly to the readers. 4. For data mining reasons, nucleotide changes should be presented as follows: 239G>A etc. (Table 2 and text). The nucleotide change markings have been modified as suggested. What next?: Accept after minor essential revisions Level of interest: An article of importance in its field Quality of written English: Acceptable Statistical review: No Declaration of competing interests: I declare that I have no competing interests

8 Reviewer #2 Maurizio Genuardi Reviewer's report: General The manuscript reports the results of constitutional mutation analysis in the EPHB2 gene in a series of familial colorectal cancer/hyperplastic polyposis patients. EPHB2 was investigated as a candidate predisposing gene due to its tumor suppressor activity in colorectal cancer. Overall, 4 variants were identified in the patient series investigated. These are all missense changes, whose pathogenicity cannot be demonstrated based on the results of mutation analysis. One of the variants was also detected in a control population, and was therefore classified as a low-frequency polymorphic allele. Overall, the results presented by the authors are worth publication, since this is the first report of EPHB2 mutation analysis in familial colorectal cancer. Although the results are essentially negative, it is worth bringing them to the attention of other groups interested in this field. In addition, the finding of a limited number of missense changes in this series is worth further investigation, and could mean that the gene may have a limited role in colorectal cancer susceptibility. However, this is quite hard to demonstrate, and, as the authors correctly state, final proof would likely stem from functional assays Major Compulsory Revisions (that the author must respond to before a decision on publication can be reached) Minor Essential Revisions (such as missing labels on figures, or the wrong use of a term, which the author can be trusted to correct) Discretionary Revisions (which the author can choose to ignore) Since the authors refer to the structural domains of the ephrin receptor B2 protein, it would be worthwhile to analyze the degree of conservation across species of the residues where the mutations are located and the (presumptive) degree of tolerance of the aminoacid changes, using available programs. Although the results of such analyses would not certainly solve the issue of pathogenicity/non pathogenicity of these variants, this additional piece of work should be easy to perform and could represent a valuable piece of information for the readers. The altered residues in EPHB2 peptide sequences have been analyzed for conservation across species, and were found to be identically conserved down to fish. These result have been added to the text, as suggested, at page 8: All three alterations are located in amino acids showing conservation across species from mouse, rat, chicken and cow down to fish, and were not observed in more than 200 healthy population-matched controls. On page 9, the sentence "Furthermore, similar pattern..." should be re-written: "Furthermore, a pattern of inactivation similar to EPHB2 has been observed..." The text has been revised, as proposed by the reviewer.

9 What next?: Accept after discretionary revisions Level of interest: An article whose findings are important to those with closely related research interests Quality of written English: Acceptable Statistical review: No Declaration of competing interests: I declare that I have no competing interests