Thank you for the reviewers´ useful comments. We have now discussed them within our
group and address them in the following. All passages which were changed since the
previous version were flagged using a grey background.
Preliminary note 1: Number of patients
Preoperative neoadjuvant chemotherapy (NAC) in the management of advanced ovarian
cancer has been heavily contested and still remains an experimental therapy with only
few trials highlighting its role in the last years, as mentioned in our report. Although NAC
intervention provides an excellent opportunity for evaluating in situ the effects of
cytotoxic therapy on the tumor microenvironment, this area of research has not received
adequate attention in the past. To the best of our knowledge, only two reports have thus
far addressed this issue: Miller et al. (J Clin Pathol 2008) compared the
immunophenotype of 16 postoperative ovarian carcinomas with that of untreated tumors,
and Sassen et al. (Human Pathology 2007) systematically evaluated histopathologic
features of tumor regression in advanced-stage ovarian cancer treated with neoadjuvant
chemotherapy (n = 49) and in a control group treated with primary surgery (n = 35).
Admittedly, the small sample size of our trial does not endorse definite conclusions, and
the impact of MVD on patient outcome can only be estimated. However, extending the
cohort to a statistical satisfactory quantity would mean recruiting four times more
patients for evaluation, which in the face of the experimental character of this therapy
and that pre- and posttreatment specimens should be collected at the same institution to
ensure reproducibility of sampling is difficult to accomplish within a manageable time
frame. In sum, we see our report as a hypothesis generating study.
Preliminary note 2:
We provided an additional figure with mean pre- and posttreatment values of MVD.
We used a well described and standardized method for the measurement of microvessel
density. This method was first introduced by Weidner (Tumor angiogenesis and
metastasis--correlation in invasive breast carcinoma. N Engl J Med. 1991 Jan
3;324(1):1-8. 1991). Many other studies used this “hotspot” method for measuring
microvessel density in the past years. As stated in the report: Quantification of
Angiogenesis in Solid Human Tumours: an International Consensus on the Methodology
and Criteria of Evaluation (P.B. Vermeulen, European Journal of Cancer Vol. 32A, pp.
2474-2484, 1996) we provide an experienced pathologist for this analysis. We cannot
provide data of inter- or intraobserver variablility of this method within this study.
Reviewer 1:
Reviewer's report:
Major Compulsory Revision: The authors report that ovarian cancer intratumor
microvessel density (MVD) is increased following exposure to cytotoxic
chemotherapy, yet there is no effect on survival or on the ability to surgically
cytoreduce the tumor following the neaadjuvant chemotherapy. One could
conclude that the measurement of MVD may be irrelevant to the successful
treatment of women with advanced stage ovarian cancer rather than the authors
reported hypothesis that one needs to administer taxane chemotherapy on a
more frequent basis to achieve more effective anti-angiogenic activity. Perhaps
the experience they refer to to support the hypothesis (Ref 32)in which all
patients received the same dose of carboplatin but those who received weekly
paclitaxel at 80 mg/m2 did better because the total amount of chemotherapy
received by the patients treated weekly was much higher than the control group
who received the paclitaxel 180mg/m2 every 3 weeks. Could the authors please
comment of this alternative explanation for the efficacy of the dose dense
chemotherapy effect.
This is an important issue which we have now addressed in the manuscript.
A phase 3 trial (JGOG3016) with 637 advanced ovarian cancer patients enrolled
compared a dosedense weekly paclitaxel regimen (Carboplatin AUC 6 q21d and
Paclitaxel 80mg/m2 d1, 8, 15, x 6-9) with standard treatment (Carboplatin AUC 6 and
Paclitaxel 180mg/m2 q21 x 6-9) after cytoreductive surgery [41]. This study
demonstrated a significant improvement of PFS in the arm with weekly taxane (28 vs.17
months; p=0.02) and a significant improvement in the overall survival rate after 3-years (
72 vs. 65 %; p=0.03) Increased doses of paclitaxel of 200 mg/m2, 225 mg/m² or 250
mg/m² given every 3 weeks have not shown a benefit in survival rates compared to
standard dosage (175 mg/m2) [43-45]. The authors concluded that higher survival rates
without improved response rates in this study might be attributed to an additional
antiangiogenic effect of the weekly application of paclitaxel.
Minor Essential Revisions:
Page 10 and Page 11 have the same 9 line statement beginning "It has been
hypothesized..." I would suggest deleting it from page 11.
We deleted the paragraph as suggested.
Discretionary Revisions:
Reference 32 is now published in a fulllmanuscript form.
We provided this reference as suggested.
Reviewer 2:
Reviewer's report:
- Major Compulsory Revisions
Introduction part:
1- The authors did not report the literature data in an objective way. The authors
are so turned to demonstrate the association of high MVD and tumor
aggressiveness in ovarian carcinoma, and consequently, forget many studies
which failed to show any association between MVD and survival (Ferrero et al
2004 for example). Indeed some studies (as Gadducci et al 2003) reported that
increased MVD predicted improved survival.
We provided a more detailed report of the available data.
Intratumor microvessel density (MVD) has been used to examine the role of
vascularization within the malignant process. High MVD is associated with parameters of
tumor aggressiveness such as greater incidence of metastases and decreased survival
[8] It was found to have independent prognostic significance when compared with
traditional prognostic markers by multivariate analysis in many types of cancer. While
the prognostic impact of high MVD In ovarian cancer patients was demonstrated in
numerous retrospective studies [9-14] other studies failed to prove a significant
association [15-17].
2- The introduction paragraph (“Anticancer chemotherapeutic agents […] tumor
vasculature [15]”) should be more appropriate in the discussion part. Indeed, the
hypothesis should be arguing more notably with more reference.
Thank you for this comment. We changed the introduction as suggested.
Anticancer chemotherapeutic agents are known to directly inhibit tumor cell proliferation.
In addition, chemotherapeutic drugs were reported to have antiangiogenic activity [22] At
the same time, current preclinical observations provide evidence that the secondary host
response to chemotherapy-associated endothelial damage can induce proangiogenic
effects. Specifically, paclitaxel and docetaxel have been shown to induce mobilization of
bone-marrow derived circulating progenitor cell (CEP) with subsequent homing to tumor
vasculature [23]
Patients and methods part:
3- The patient number (32) is too small in order to do a robust statistical study
We refer to our preliminary note 1.
4- In order to clarify the study, the authors should restrict their work to serous
tumors. What is the interest to include one endometrioid histological subtype
case?
5- In order to clarify the study, the authors should restrict their work to stage IIIC.
What is the interest to include two FIGO stage IV cases?
As noted in Ptients and methods, the study collective consists of patients treated within
this prospective phase II trial at our institution. Strict inclusion criteria for this study were
the availability of pre- and posttreatment samples. Further exclusion criteria were not
considered for this analysis. Post-hoc exclusions or subgroup analyses after
interpretation of the results were not done.
6- The paragraph which describes statistical analysis is misleading. For example,
the authors use both parametrics and nonparametrics tests without normality test
of distributions.
Thank you. Indeed, parametric tests were not performed.
In light of the increasing alpha value and a limited number of patients we do not consider
a normality test of distributions of benefit for this analysis.
Statistical analysis was performed using the software SAS 9.1.3 (SAS Institute Inc. Cary,
NC, USA) parametric and included non-parametric group comparison tests (MannWhitney and Wilcoxon-test) nonparametric (Spearman) correlation tests, and parametric
(Cox proportional) and nonparametric (Kaplan-Meier) survival analysis with p<0.05
considered significant
Results part
7- The authors did not report their results according to REMARK
recommendations (McShane et al 2005).
We don’t think that this trial is a tumor marker prognostic study. The primary aim was to
compare the microvessel density before and after chemotherapy. Therefore not all
Reporting recommendations for tumor marker prognostic studies (REMARK) were
followed, especially not those for prognostic considerations.
8- The number of vessels is the keystone in this study, but few details are
provided:
Quantitating blood vessels in hot spot areas implies several observer-dependent
steps, in particular in ovarian carcinomas which are heterogeneous tumor. Where
is the intra-reproducibility of data?
The authors indicate that “the number […] vessels was counted in 3 high power
fields”. Where is the standard deviation between these three areas for each
case?
We refer here to our preliminary note 2. A standard deviation for three values is not
usefull.
In the “Results section”, what is the meaning of the “253 specimens of 32
patients”. If many samples are used by patients, it is very important to give more
data concerning the quantification of vessels. How many samples are quantified
before and after treatment for each case?
We agree that this is misunderstanding (32 patients x2(pre-and post) x 4 Markers =256
specimens)
For each marker, pre- and posttreatment specimens of 32 patients were analyzed. In
three cases the pretreatment specimens showed severe artefacts, which prevented their
use for the designated analyses due to poor quality. Pre- and posttreatment values were
excluded pairwise in these cases.
Discussion part
9- The discussion is very confused. Two paragraphs were duplicated in two
different places.
We deleted the paragraph as suggested.
- Minor Essential Revisions
10- In figure 1, where are the error bars?
We refer here to our preliminary note 2
Reviewer 3:
Reviewer's report:
The authors present in a well written and clearly constructed manuscript results
of microvessel density in ovarian cancer tissue before and after chemotherapy.
They conclude that carboplatin/docetaxel lead to an increase of MVD after
two-three courses given in a neo-adjuvant scheme.
1) Major Compulsory Revisions
Was any erythropoietin stimulating agent (ESA) given during the chemotherapy
as treatment of therapy induced anemia?
We addressed the use of ESA as suggested.
laparotomy were assessable for the evaluation of MVD before and after treatment.
None of the patients received erythropoetin stimulating agents before cytoreductive
surgery. The protocol was approved by the institutional review board. All patients gave
written informed consent.
2) Minor Essential Revisions
-The authors state that carbo/paclitaxel is the standard chemotherapy in ovarian
cancer patients. Why did they use carbo/docetaxel?
The patients were treated within a phase II trial and the protocol was designed in 2002;
at this time, phase II trials showed a different toxicity profile compared to Paclitaxel and
the role of Docetaxel in the treatment of advanced ovarian cancer was still under
investigation (Results of a phase III Trial with Carboplatin Paclitaxel vs. Carboplatin
Docetaxel SCOTROC Trial were published 2004 by Vasey et al.)
(We stated:
Although about 80% of advanced ovarian cancer patients respond well to standard
management - primary cytoreductive surgery followed by platinum/taxane-based
chemotherapy - the majority of patients develop recurrent disease and die of progressive
disease)
-How do you explain that if there is an increase in MVD there is no increase in
surgical problems during interval debulking suergery (IDS), in contrary as shown
in the study by Vergote et al, morbidity decreases during IDS.
We don’t know any data concerning MVD and surgical outcome. We don’t know whether
differences in the microvessel environment could be noticed during surgery or whether
they could influence perioperative morbidity.
3) Discretionary Revisions
Page 3: Results of phase III study demonstrate similar results …. Add also the
difference between the two groups. Now it seems as there was no difference in
outcome between the neo-adjuvant and the primary debulked groups.
Thank you for this important comment.
Results of a recently reported phase 3 study with 704 patients enrolled and a treatment
schedule with 3 cycles carboplatin/paclitaxel preoperatively demonstrated that
neoadjuvant chemotherapy produces similar PFS and OS rates compared to standard
primary cytoreductive surgery and provides a significantly lower perioperative morbidity
and mortality in the group treated with neoadjuvant chemotherapy [5].
Reviewer's report
Reviewer 4: Reviewer's report:
The manuscript is a descriptive report on the density of small vessels in ovarian
tumors under chemotherapy. It is well written. However it has some major
problems:
1. The authors state that their findings were unexpected since the chemotherapy
was supposed to decrease and not increase the number of small vessels. They
try to explain it by the hypothesis of a long period between the treatments. This
hypothesis is discussed in details without any support from the results.
The results of this study showed an increase in MVD during neoadjuvant chemotherapy.
The pathogenesis of these findings is not clear. A possible explanation is the long
treatment interval. However, this explanation needs to be evaluated within a prospective
study.
2. The method of counting the vessels is prone to bias and one could expect to
have some digital imaging analysis or at least an itra- and inter- observer
variation analysis.
We refer here to our preliminary note 2
3. The discussion is too long and has for example duplication of paragraphs (the
JGOG study).
We deleted the paragraph as suggested
4. Figure 1 does not add anything to the manuscript and Figure 2 does not show
the post slides for CD34 and D2-40 (known to be unchanged). Moreover the
structure of the tumor seems to be different in the pre compared to the post.
We provided pictures of all markers before and after treatment in figure 2. Changes in
tumor specimens after neoadjuvant chemotherapy are discussed by Sassen et al.
(Human Pathology 2007)