Author's response to reviews
Title: Detection of erbB2 copy number variations in plasma of patients with
esophageal carcinoma.
Authors:
Immacolata Andolfo ([email protected])
Giuseppe Petrosino ([email protected])
Loredana Vecchione ([email protected])
Pasqualino De Antonellis ([email protected])
Mario Capasso ([email protected])
Donatella Montanaro ([email protected])
Marica Gemei ([email protected])
Giancarlo Troncone ([email protected])
Achille Iolascon ([email protected])
Fortunato Ciardiello ([email protected])
Fernando De Vita ([email protected])
Massimo Zollo ([email protected])
Version: 2 Date: 10 January 2011
Author's response to reviews: see over
UNIVERSITA’ DEGLI STUDI DI NAPOLI FEDERICO II
DIP. DI BIOCHIMICA E BIOTECNOLOGIE MEDICHE
Editorial Office, BMC Cancer
Dear Editor, Professor Chae-Ok Yun,
Please find enclosed our revised manuscript (MS: 9958903345763859) entitled, “Detection of
erbB2 copy number variations in plasma of patients with esophageal carcinoma”, by Andolfo
et al., for consideration for publication as an Article in BMC Cancer.
The referees questions were point-by-point answered and all changes made in the text were
underlined and coloured in red.
Reviewer's report 1
Major Compulsory Revisions:
-
The mean erbB2 CN value (+/-SD) (and/or median values) for the 34 control samples should
be included in section 3.1. The only information about this is shown in figure 1, and the data
is so compressed that it is impossible to resolve these values.
The Referee’s point has been taken into consideration and the CN values for healthy control
subjects is 1, with a standard deviation of 0.16 (see section 3.1, pages 11 and 12 and
legend of figure 1A, page 28). To resolve these values a new chart in figure 1A was made
with a new scale (see new figure 1A).
-
In the last sentence of section 3.4 on page 15, it is stated that the CN values for the six CTC
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80131 Naples, Italy
e-mail: [email protected]
website: http://www.scienzebiotecnologiche.unina.it/Ricerca/Zollo.pdf
samples were similar to the CN values of the corresponding marched free DNA samples.
This statement is repeated on page 17 in the Discussions section. However, the data is
presented in a way that makes it impossible to evaluate this conclusion. The CN data on the
six CTC samples should be presented with the CN values of the matched plasma samples,
for example in a table.
The CN data from CTSs compared to DNA from plasma of the same patients are now
indicated in supplementary Table S3 and the CN of DNA from plasma is added in the chart
in Figure 4C (see section 3.4, page 15 and Figure 4C with its legend at page 29).
-
It would be very useful to include estimates of the DNA content in plasma, e.g., based on the
beta-actin measurements, of the controls and the patients. The authors conclude that a
majority of the DNA in the plasma of the patients must be tumor-derived (otherwise the
measured erbB2 CN increase could not be expected to be as high as it is). Consequently, the
overall DNA content in patient plasma should thus be higher than in the control samples. It
would be useful to include a supplementary table summarizing the underlying QPCR data
(the beta actin and erbB2 Ct values for all samples).
Following these points, we evaluated DNA content extracted from plasma of EC patients
and we found that it is higher than the one extracted from plasma of healthy subjects. We
reported here, the quantization of the DNA extracted to underline the differences of total
DNA content between patients and controls (mean value for patients 221ng/µl and for
controls 47 ng/µl, see table S4 and at page 17). We used for all the sample 500µl of plasma,
then the DNA extracted were dissolved in the same quantity of water. In the real time assay
the DNA content for both patients and controls were the same (100 ng). For this reason, the
β-actin values were almost similar for patients and controls (mean value for β- actin of
patients is 23.54 and 24.21 for controls), confirming that β- actin is a good normalized gene
in our analysis.
Furthermore, following referee point, we summarizing the QPCR data in supplementary
Table S2 with CT values of real time PCR for both erbB2 and β-actin genes in patients and
controls (see at page 12).
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Tel. + 39 081/3737875-Fax + 39 081 3737-711
e-mail: [email protected]
website: http://www.ceinge.unina.it/~zollo/zollo.webpage.htm
Dipartimento di Biochimica e Biotecnologie Mediche (DBBM) Via Sergio Pansini 5
80131 Naples, Italy
e-mail: [email protected]
website: http://www.scienzebiotecnologiche.unina.it/Ricerca/Zollo.pdf
Reviewer's report 2
Major:
-
Why did you chose to use a CN < 2 and CN >= 2 (i.e., included normal in with gains in
CN)? Did you look at if copy number was associated without the dichotomizing of the
variable, or look at gain, lose and normal?
The CN cut off was selected as 2 because the median of the CN distribution of the patients
is 2 as shown in figure 1A (a new chart in figure 1A was made with a new scale) and
described into its legend and also in section 3.1 at pages 11 and 12.
We did not look if copy number was associated without the dichotomizing because the
number of patients here analysed is too small to be divided into three groups. However, by
dividing the patients in lose (CN<1), normal (2≥CN ≥1) and gain (CN ≥ 2), 6, 14 and 21
patients respectively, and by using Kaplan-Meier analyses we did not reach a statistical
significance but we saw the same tendency as for the survival analysis with cut-off 2, that it
is shown in figure 2A (this data is now shown in supplementary figure 1S C, in result section
at pages 11 and 12 and in discussion section at page 18).
-
How was the formula to determine CN determined? How did you handle non-integer
results? Is this formula specific to this study (data set)?
Following referee point, the formula for CN determination was the standard formula used to
calculate the copy number. Examples of the determination of CN by this formula were the
references here listed:
Ponchel F, et al. Real-time PCR based on SYBR-Green I fluorescence: an alternative to
the TaqMan assay for a relative quantification of gene rearrangements, gene
CEINGE Biotecnologie Avanzate s.c.a r.l.: Via Comunale Margherita 482 - 80145 Naples – ITALY
Tel. + 39 081/3737875-Fax + 39 081 3737-711
e-mail: [email protected]
website: http://www.ceinge.unina.it/~zollo/zollo.webpage.htm
Dipartimento di Biochimica e Biotecnologie Mediche (DBBM) Via Sergio Pansini 5
80131 Naples, Italy
e-mail: [email protected]
website: http://www.scienzebiotecnologiche.unina.it/Ricerca/Zollo.pdf
amplifications and micro gene deletions. BMC Biotechnol 2003, 3:18 (reference 31);
Dhanasekaran S, Doherty TM, Kenneth J; TB Trials Study Group. Comparison of
different standards for real-time PCR-based absolute quantification. J Immunol
Methods. 2010 Mar 31;354(1-2):34-9. Epub 2010 Jan 25. (reference 33, now added in
the reference list);
Godornes C, Leader BT, Molini BJ, Centurion-Lara A, Lukehart SA. Quantitation of
rabbit cytokine mRNA by real-time RT-PCR.. Cytokine. 2007 Apr;38(1):1-7. Epub 2007
May 22 (reference 34, now added in the reference list);
The formula takes into account 6.022×1023(molecules/mole) that is the Avogadro's number
and 660 Da that is the average weight of a single base pair. The non-integer results were
treated on the basis of the cut-off CN>2 and CN ≤2 and they were rounded off, particularly,
the values were rounded down if the decimal value was between 0 and 4; while they were
rounded up if the decimal value was between 5 and 9 (see section 2.4.2 at pages 8 and 9).
-
Need additional information on the subjects and survival variable. How was survival
measured? From time from diagnosis or entry into the study? All subjects from one clinic?
Was treatment the same for all subjects? Where all the subjects the same race (ie, any issue
with possible population stratification)?
Following these points by the Referee, the progression free survival time was measured
from the date of registration in the study to the date of progression or last follow-up visit.
The patients derived all from one clinic and all plasma samples from the patients with EC
included in the present study were collected before surgical tumor resection and before
chemotherapy treatment. Altogether, these data are now written in material and methods
section at page 11, section 2.8.
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Tel. + 39 081/3737875-Fax + 39 081 3737-711
e-mail: [email protected]
website: http://www.ceinge.unina.it/~zollo/zollo.webpage.htm
Dipartimento di Biochimica e Biotecnologie Mediche (DBBM) Via Sergio Pansini 5
80131 Naples, Italy
e-mail: [email protected]
website: http://www.scienzebiotecnologiche.unina.it/Ricerca/Zollo.pdf
Minor:
• On page 9, please give standard error for slopes as needed to determine if the
slopes are similar or not. Also, can you test that the slopes are different and
report p-value.
Following these points by the Referee, now at page 8 the standard errors for the slopes were
added. Slopes β-actin: -3.122; -2.772; -3.215, standard deviation is 0.234 and standard
error is 0.135; slopes erbB2: -3.263; -3.232; -3.360, standard deviation is 0,066 and
standard error 0.039. A slope of -3.3 +/- 10% reflects an efficiency of 100% +/- 10% of the
PCR reaction as demonstrated by Joshua S Yuan et al., (Statistical analysis of real-time
PCR data. BMC Bioinformatics. 2006, 7:85, now added as reference 32 in the reference
list). For this reason, the slopes for the two genes are similar and the p values = 0,150.
These data are now showed in section 2.4.2 at page 8, and two tables showing the values of
all points of the calibration curves are added in figure 1S.
Following our responses to the concerns we hope that you will find our manuscript suitable for
publication on BMC Cancer.
I am looking forward to hearing from you soon,
Prof. Massimo Zollo,
for and on behalf of all of the authors.
CEINGE Biotecnologie Avanzate s.c.a r.l.: Via Comunale Margherita 482 - 80145 Naples – ITALY
Tel. + 39 081/3737875-Fax + 39 081 3737-711
e-mail: [email protected]
website: http://www.ceinge.unina.it/~zollo/zollo.webpage.htm
Dipartimento di Biochimica e Biotecnologie Mediche (DBBM) Via Sergio Pansini 5
80131 Naples, Italy
e-mail: [email protected]
website: http://www.scienzebiotecnologiche.unina.it/Ricerca/Zollo.pdf