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Title: Let-7 miRNA-binding site polymorphism in the KRAS 3`UTR; colorectal
cancer screening population prevalence and influence on clinical outcome in
patients with metastatic colorectal cancer treated with 5-fluorouracil and
oxaliplatin +/- cetuximab
Authors:
Janne B Kjersem ([email protected])
Tone Ikdahl ([email protected])
Tormod Guren ([email protected])
Eva Skovlund ([email protected])
Halfdan Sorbye ([email protected])
Julian Hamfjord ([email protected])
Per Pfeiffer ([email protected])
Bengt Glimelius ([email protected])
Christian Kersten ([email protected])
Hiroko Solvang ([email protected])
Kjell M Tveit ([email protected])
Elin H Kure ([email protected])
Version: 4 Date: 15 October 2012
Author's response to reviews: see over
BMC Cancer
BioMed Central
236 Gray`s Inn Road
London WC1X 8HL
United Kingdom
Oslo 12th of October 2012
To the Editor,
Reply to reviewers and the editors comments for Manuscript 1442908651768315
– Let-7 miRNA-binding site polymorphism in the KRAS 3`UTR; colorectal cancer
screening population prevalence and influence on clinical outcome in patients
with metastatic colorectal cancer treated with 5-fluorouracil and oxaliplatin +/cetuximab
We are thankful to both reviewers and the editors for the comprehensive review of our
manuscript and helpful feedback. A revised manuscript has been prepared based on the
comments of the reviewers and the editorial board. Please find the comments addressed
individually below:
Reviewer 1
Although these are interesting data, the authors should be aware that the mode of
presentation is not reasonable as the results represent negative findings (which
are also important to report). Thus the authors have to write what they actually
found: No significant differences for the groups.
We recognize that the mode of the presentation of our negative findings were
misleading and have therefore made several changes throughout the manuscript.
They are addressed below.
“Numerically increased PFS and OS were found in patients with the LCS6 variant
allele (8.5 versus 7.8 months, P=0.16 and 23.5 versus 19.5 months, P=0.31,
respectively).” This message is rather misleading – as the study did not reveal
significant differences (P=0.16, P=0.31) – so the authors have to state this in a
proper meaning.
Under Results in the Abstract the sentence: “Numerically increased PFS and OS
were found in patients with the LCS6 variant allele (8.5 versus 7.8 months,
P=0.16 and 23.5 versus 19.5 months, P=0.31, respectively)” has been replaced by
the following sentence on page 2, lines 57-62: “No statistically significant
differences were demonstrated in the NORDIC-VII cohort even if numerically
increased progression-free survival (PFS) and overall survival (OS) were found in
patients with the LCS6 variant allele (8.5 (95% CI: 7.3-9.7 months) versus 7.8
months (95% CI: 7.4-8.3 months), P=0.16 and 23.5 (95% CI: 21.6-25.4 months)
versus 19.5 months (95% CI: 17.8-21.2 months), P=0.31, respectively).”
“Addition of cetuximab improved response rate more in variant carriers than in
wild-type carriers (from 35% to 57% versus 44% to 47%. Interaction P=0.16)”.
Again there was no significant difference (P=0.16). The authors have to state these
“negative findings” more clearly throughout the whole manuscript before the
paper should be considered for acceptance.
Under Results in the Abstract the sentence: “Addition of cetuximab improved
response rate more in variant carriers than in wild type carriers (from 35% to
57% versus 44% to 47%. Interaction P=0.16)” has been replaced by the following
sentence on page 2, lines 62-65: “Addition of cetuximab seemed to improve
response rate more in variant carriers than in wild-type carriers (from 35% to
57% versus 44% to 47%), however the difference was not statistically significant
(interaction P = 0.16).”
Under Conclusions in the Abstract the sentence: “The LCS6 variant allele may be a
prognostic factor and predict response to cetuximab in mCRC patients treated
with 5-fluorouracil and oxaliplatin.” has been changed to the following sentence
on page 2, lines 67-69: “No statistically significant effect of the LCS6 variant
allele on response rate, PFS or OS was found in mCRC patients treated with 1st
line 5-fluorouracil-oxaliplatin +/- cetuximab.”
The sentence: ”The LCS6 variant did not appear as a risk factor for CRC” under
Conclusions in the Abstract has been with replaced with ”The LCS6 variant allele
does not seem to be a risk factor for development of colorectal polyps or CRC.” on
page 2, lines 66-67.
Results
Under the heading: “Predictive analyses for benefit of cetuximab treatment”, the
sentence: “Addition of cetuximab to FLOX improved response more in patients
with the LCS6 variant (35% in arm A versus 57% in arms B and C) than in wildtype carriers (44% in arm A versus 47% in arms B and C), but the difference did
not reach statistical significance (interaction P = 0.16, Figure 3).” has been
replaced by the following sentence on page 8, lines 246-249: “Addition of
cetuximab to FLOX seemed to improve treatment response more in patients with
the LCS6 variant (35% in arm A versus 57% in arms B and C) than in wild-type
carriers (44% in arm A versus 47% in arms B and C), but the difference was not
statistically significant (interaction P = 0.16, Figure 3).”
Discussion
The sentences: “Although not statistically significant, numerically increased
median PFS and OS were found in patients with the LCS6 variant allele when
compared to LCS6 wild-type carriers. This finding may indicate that mCRC
patients with the variant allele belong to a favorable prognostic group” have been
removed and replaced by the following sentence on page 9, lines 274-278:
“Numerically increased median PFS and OS were found in patients with the LCS6
variant allele when compared to LCS6 wild-type carriers, but the differences
were not statistically significant at the 5% level and we cannot conclude that
mCRC patients with the variant allele belong to a favorable prognostic group.”
The sentence: “The trend of numerically increased PFS and OS and response rate
was also observed independent of KRAS mutation status and in KRAS and BRAF
wild-type patients.” has been modified on page 9, lines 282-284 to: “The trend
of numerically increased PFS, OS and response rate was also observed
independent of KRAS mutation status and in KRAS and BRAF wild-type patients,
but none of the findings proved statistically significant.”
The following sentence has been added on page 9, lines 285-288: “Thus, any
potential predictive effect of LCS6 is likely to be too small to be demonstrated
with the patient sample available from the NORDIC-VII study.”
The following two sentences have been removed: “Our findings may indicate that
the LCS6 variant allele is favorable in respect of clinical outcome when cetuximab
is added on to Nordic FLOX in the first line treatment of mCRC patients.” and
“However, these results should be interpreted with caution.”
Conclusions
The sentence: “The LCS6 variant allele does not seem to be a risk factor for
development of colorectal polyps or CRC.” has been added on page 10, lines
328-329.
The sentences: “Our findings may indicate that mCRC patients with the LCS6
variant allele belong to a favorable prognostic group. Also, the LCS6 variant allele
may increase the probability of response when cetuximab is given in combination
with Nordic FLOX in the first-line treatment of mCRC patients” have been
replaced by the following sentences on page 10, lines 329-332: “No prognostic
effect of the LCS6 variant allele was demonstrated in the NORDIC-VII cohort. No
predictive effect of the LCS6 variant allele on response rate, PFS or OS when
cetuximab is given in combination with 5-fluorouracil-oxaliplatin in the first-line
treatment of mCRC patients could be proven.”
The authors should also include – at least in the discussion section of the
manuscript – some additional, more recently published studies with regard to
microRNAs and their potential as biomarkers/resistance mediators in EGFRinhibitor treated colorectal cancer patients. The meaning of these findings
should be carefully discussed in the context of their own results. These
citations should include:
1. Mekenkamp et al: “Beyond KRAS mutation status: influence of KRAS copy
number status and microRNAs on clinical outcome to cetuximab in metastatic
colorectal cancer patients.” BMC Cancer 2012.
2. Pichler et al: “Down-regulation of KRAS-interaction miRNA-143 predicts poor
prognosis but not response to EGFR-targeted agents in colorectal cancer.”
British Journal of Cancer 2012.
3. Ruzzo et al: “Role of KRAS let-7 LCS6 SNP in metastatic colorectal cancer
patients.” Annals of Oncology 2011.
4. Ragusa et al: “Specific alterations of microRNA transcriptome and global
network structure in colorectal carcinoma after cetuximab treatment.”
Molecular Cancer Therapeutics 2010.
We added the following sentence in the Background section on page 3, lines
100-102: “A number of miRNAs have been reported to be involved in CRC
development and KRAS regulation, and these may influence the effect of EGFRtargeted therapy [13,16,17].” (Mekenkamp et al, Pichler et al, and Ragusa et al)
Mekenkamp et al
We included the article by Mekenkamp et al in the Background section as we
found the article to be most relevant here. We added the following paragraph on
page 3, lines 87-94: “It was recently reported that copy number aberrations
(CNA) may provide additional information to mutation status and their use may
potentially further improve the selection of mCRC patients for EGFR targeted
therapy [13]. Mekenkamp et al demonstrated that KRAS copy number loss was
associated with good response in both KRAS wild-type and KRAS mutated mCRC
patients treated with a cetuximab-containing first-line regimen [13]. KRAS copy
number gains were associated with poor progression-free survival (PFS) in KRAS
wild-type mCRC patients given the same treatment [13].”
Pichler et al
We also included the article by Pichler et al in the Background section as we
found the article to be most relevant here. We added the following paragraph on
page 3, lines 102-107: “An important miRNA in CRC seems to be miR-143,
which, has been described to be down regulated in CRC [18] and to inhibit the
translation of KRAS mRNA, thereby altering RAS signaling and inhibiting tumor
cell growth [19]. Pichler et al found that low miR-143 expression was an
independent negative prognostic factor for cancer specific survival in mCRC, and
they reported a decreased PFS in KRAS wild-type mCRC patients treated with
EGFR-targeted agents [16].”
Ruzzo et al
The article by Ruzzo et al has been cited in the Discussion section on pages 9 and
10, lines 300-304: “The conflicting results in these studies suggest that the
chemotherapy backbone may play a role, and that the LCS6 variant allele has
different predictive values in mCRC patients treated with cetuximab alone or in
combination with 5-fluorouracil-oxaliplatin than in patients treated with
cetuximab in combination with irinotecan [41,42].”
Ragusa et al
We added the following paragraph in the Discussion section on page 10, lines
306-317: “Ragusa et al demonstrated that cetuximab treatment induced miRNA
transcriptome changes in drug-sensitive and drug-resistant CRC cell lines [17].
The set of differentially expressed miRNAs in the two cell lines (one sensitive and
the other resistant) was almost entirely not overlapping. These data suggest that
different responses to cetuximab are associated with different sets of miRNAs
and thereby different molecular signaling. Interestingly, 67% of the differentially
expressed miRNAs were involved in cancer, including CRC, whereas 19 miRNA
targets had previously been reported to be involved in the cetuximab pathway
and CRC. Based on their result, they suggest down regulation of let-7b and let-7e
and the up regulation of miR-17* to be associated with cetuximab resistance [17].
Although these miRNAs were generated from cell studies, they illustrate that
miRNAs may be promising predictive markers of cetuximab response to be
further studied in mCRC patients.”
Reviewer 2
The authors should delve more into the different results from the prior
studies, one used irinotecan in combination with cetuximab, and actually
Zhang had the same results of a separate cohort using this same
combination, showing that LCS6 patients receiving cetuximab plus
irinotecan did worse than those getting irinotecan alone. Therefore, it
should not be assumed that this is due to sample size. In contrast it could
really be due to chemotherapy backbone. This should be more directly
discussed. The Zhang study that provided cetuximab delivered it alone.
Your study is the first reporting on FLOX, and suggests that this may be a
promising agent for these patients, as they appear to do better. This should
be discussed.
The paragraph “Our results are consistent with the results of Zhang et al who
studied the association of the LCS6 variant allele and clinical outcome in KRAS
wild-type mCRC patients enrolled in a study of salvage cetuximab monotherapy
[24]. Contrary to our results, a smaller Italian study [23] on mCRC patients who
underwent salvage cetuximab – irinotecan therapy, demonstrated that patients
with the variant allele had shorter PFS and OS compared to wild-type. Possible
explanations for conflicting results could be small sample sizes, different
chemotherapy backbone, prior treatment and line of therapy.” has been replaced
by the following paragraph on pages 9-10, lines 292-304:
“Zhang et al demonstrated that of 67 KRAS wild-type mCRC patients, there was a
higher response rate and a trend of longer PFS and OS in patients with LCS6
variant allele (N=12) compared to patients with LCS6 wild-type (N=55) when
treated with cetuximab monotherapy [34]. Contrary, another study on 121 BRAF
wild-type mCRC patients who underwent salvage cetuximab – irinotecan therapy,
of which 58 were KRAS mutated, reported that patients with LCS6 variant allele
(N=34) had shorter PFS and OS compared to LCS6 wild-type (N=87) [33]. Similar
results were reported by Winder et al who found mCRC patients with mutant
KRAS and LCS6 variant allele to have shorter PFS when treated with irinotecan
and cetuximab [35]. The conflicting results in these studies suggest that the
chemotherapy backbone may play a role, and that the LCS6 variant allele have
different predictive values in mCRC patients treated with cetuximab alone or in
combination with 5-fluorouracil-oxaliplatin than in patients treated with
cetuximab in combination with irinotecan [41,42].”
We have added Table 3: “Progression-free survival and overall survival by LCS6
genotype and treatment” under “Predictive analyses for benefit of cetuximab
treatment” in the Results section.
The following sentence has been added on page 10, lines 319-325: “We have
only investigated one miRNA binding site polymorphism in this study,
representing a small piece in a large puzzle of polymorphisms in the miRNA
pathway. Future research on miRNA pathway polymorphisms as potential
prognostic and/or predictive markers in mCRC should ideally include an
integrated approach using bioinformatical tools combined with biological data to
get a comprehensive understanding of the role and functions of miRNA
polymorphisms and cetuximab response.”
The repeated comment that the relationship with ovarian cancer is not possible is
incorrect. See two follow up publications explaining the issues with the metaanalysis (Ratner, Oncogene and Pilarski, PLOS One). The conclusion is that this
marker is important in ovarian cancer, predicts poor outcome and response to
platinum chemotherapy (which is relevant for your publication), and is a risk for
high risk families. This must be corrected as your statements are incorrect.
The LCS6 variant allele and reported cancer risks are now only referred to in the
Background section as we did not find it necessary to refer to this twice. The
following sentences in the Discussion section have therefore been removed: “The
variant allele is associated with increased risk for NSCLC in moderate smokers
[19] and triple negative breast cancer in premenopausal women [20]. The
reported association between the variant allele and increased risk of ovarian
cancer [21] was subsequently excluded by a meta-analysis [22].”
The following paragraph in the Background section: “The LCS6 variant allele is
associated triple-negative breast cancer [20] and non-small cell lung cancer
(NSCLC) in moderate smokers [19]. The LCS6 variant allele was reported to be
associated with increased risk of ovarian cancer [21]; however a subsequent
meta-analysis excluded the possibility of an association between the variant
allele and risk of ovarian cancer or of familial ovarian cancer [22].” has been
replaced by the following paragraph on page 4, lines 119-131: “The LCS6
variant allele is associated with increased risk of non-small cell lung cancer
(NSCLC) in moderate smokers [24], triple-negative breast cancer in
premenopausal women [25], and ovarian cancer in BRCA negative women from
hereditary breast and ovarian cancer syndrome families [26]. Moreover, the LCS6
variant allele is enriched in BRCA negative double primary breast and ovarian
cancer patients [27]. One study failed to find an association between the LCS6
variant allele and sporadic or familial ovarian cancer risk [28]. A recent study
confirms the importance of the LCS6 variant allele in postmenopausal ovarian
cancer patients and demonstrates that it is a biomarker of poor outcome in this
disease, probably due to platinum resistance [29]. The LCS6 variant allele is also
associated with reduced survival in oral cancers [30]. On the contrary, Smits et al
found that early-stage CRC patients with the LCS6 variant allele had better
outcome [31], whereas Ryan et al recently reported the LCS6 variant allele to be
associated with reduced risk of mortality in late-stage CRC [32].”
Editorial board
Ethical Approval - Research involving human subjects (including human material
or human data) that is reported in the manuscript must have been performed
with the approval of an appropriate ethics committee. Research carried out on
humans must be in compliance with the Helsinki Declaration
(http://www.wma.net/en/30publications/10policies/b3/index.html).
A statement to this effect must appear in the Methods section of the manuscript,
including the name of the body, which gave approval, with a reference number
where appropriate.
Under the heading “NORDIC-VII” in the Materials and Methods section the
following sentence has been added on page 5, lines 164-166: “The study was
approved by the national ethics committees and governmental authorities in
each country and was conducted in accordance with the Declaration of Helsinki.”
Under the heading “KAM cohort” in the Materials and Methods section the
following sentence has been added on page 6, lines 174-176: “The project was
approved by the Ethics Committee REK South-East, Norway (Regional komite for
medisinsk og helsefaglig forskningsetikk Sør-Øst).”
Consent - Please state in the Methods section whether written informed consent
for participation in the study was obtained from participants or, where
participants are children, a parent or guardian.
Under the heading “NORDIC-VII” in the Materials and Method section the
following sentence has been added on page 5, line 166: “All patients provided
written informed consent.”
Under the heading “KAM cohort” in the Materials and Method section the
following sentence has been added on page 6, lines 173-174: “All participants in
the study provided written informed consent.”
We hope our revised manuscript is acceptable for publication and look forward to
hearing from you.
Kind regards,
Elin Kure
Group Leader Molecular profiling of GI cancers
Department of Genetics, Institute for Cancer Research
Oslo University Hospital HF
Radiumhospitalet, Postboks 4953 Nydalen
0424 Oslo, Norway
Office Phone: +47 22 78 13 77
Mobile Phone: +47 909 155 78
E-mail: [email protected]