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. 2020 Mar 31;15(3):e0230553. doi: 10.1371/journal.pone.0230553

Signal changes in T2-weighted MRI of liver metastases under bevacizumab—A practical imaging biomarker?

Johannes Thüring 1,*, Christiane Katharina Kuhl 1, Alexandra Barabasch 1, Lea Hitpass 1, Maike Bode 1, Nina Bünting 1, Philipp Bruners 1, Nils Andreas Krämer 1
Editor: Michael C Burger2
PMCID: PMC7108712  PMID: 32231380

Abstract

Objective

The purpose of this study was to investigate signal changes in T2-weighted magnetic resonance imaging of liver metastases under treatment with and without bevacizumab-containing chemotherapy and to compare these signal changes to tumor contrast enhancement.

Materials and methods

Retrospective analysis of 44 patients, aged 36–84 years, who underwent liver magnetic resonance imaging including T2-weighted and dynamic contrast enhancement sequences. Patients received bevacizumab-containing (n = 22) or conventional cytotoxic chemotherapy (n = 22). Magnetic resonance imaging was obtained at baseline and at three follow-ups (on average 3, 6 and 9 months after initial treatment). Three independent readers rated the T2 signal intensity and the relative contrast enhancement of the metastases on a 5-point scale.

Results

T2 signal intensity of metastases treated with bevacizumab showed a significant (p<0.001) decrease in T2 signal intensity after initial treatment and exhibit compared to conventionally treated metastases significantly (p<0.001 for each follow-up) hypointense (bevacizumab: 0.70 ± 0.83 before vs. -1.55 ± 0.61, -1.91 ± 0.62, and -1.97 ± 0.52; cytotoxic: 0.73 ± 0.79 before vs. -0.69 ± 0.81, -0.71 ± 0.68, and -0.75 ± 0.65 after 3, 6, and 9 months, respectively). T2 signal intensity was strongly correlated with tumor contrast enhancement (r = 0.71; p<0.001). Intra-observer agreement for T2-signal intensity was substantial (κ = 0.75). The agreement for tumoral contrast enhancement between the readers was considerably lower (κ = 0.39).

Conclusion

Liver metastases exhibit considerably hypointense in T2-weighted imaging after treatment with bevacizumab, in contrast to conventionally treated liver metastases. Therefore, T2-weighted imaging seems to reflect the effect of bevacizumab.

Introduction

Established image-based criteria for response assessment of solid tumors following systemic cytotoxic chemotherapy are mainly based on changes of tumor diameter (i.e. Response Evaluation Criteria in Solid Tumors [RECIST]) [1–4]. Modern chemotherapy regimens, however, often include antiangiogenic drugs, such as bevacizumab, a monoclonal antibody against vascular endothelial growth factor (VEGF) [5]. Bevacizumab primarily inhibits the formation of new blood vessels and leads to a regression of already existing immature tumor vascularization. A solid body of scientific literature indicates that these anti-angiogenic effects of bevacizumab do not cause a decrease in tumor size [6–8]. Consequently, commonly used size-based response assessment, especially RECIST, can fail to precisely monitor the effect of antiangiogenic therapy [9, 10]. Moreover, functional and often impractical as well as still metrics-based criteria, e.g., quantitative EASL (qEASL) [11], have several deficiencies and provide no practical assessment of the tumor response in clinical routine [12]. Therefore, a crucial clinical need on practical methods for tumor response assessment persists if therapy contains antiangiogenic drugs.

Magnetic resonance imaging (MRI) provides various methods for assessing the tissue functionality [13], including diffusion weighted imaging (DWI) [14] and dynamic contrast enhancement (DCE) [15]. In multiple studies, tumor enhancement showed to directly reflect the tumor vascularization [16, 17], whereas DWI does not exclusively reflect the vascularization but influenced by many factors [18, 19].

We observed, that liver metastases treated with bevacizumab containing chemotherapy appear considerably hypointense on T2-weighted sequences already after initial treatment. Therefore, the aim of this study was to investigate the changes in T2 signal intensity (T2-SI) after bevacizumab-containing chemotherapy (B-CT) and cytotoxical chemotherapy (C-CT). Moreover, these changes were compared to changes of tumor enhancement on DCE sequences, as an established functional imaging biomarker.

Materials and methods

This institutional review board–approved retrospective study (Independent Ethics Committee of the RWTH Aachen University; EK 105/17) was conducted at an academic comprehensive cancer center and written informed consent was waived.

Patients, target lesions, and tumor size

44 consecutive patients with a total of 67 liver metastases (26 patients with 43 metastases of colorectal cancer (CRC) and 18 patients with 24 metastases of breast cancer) were examined with standardized liver MRI from July 2010 to November 2016.

MRI was performed at baseline prior to therapy and at 3 follow-ups (FU) (average time 3, 6 and 9 months after initial treatment) under standard of care systemic therapy. Half of this cohort (n = 22) underwent B-CT, whereas the remaining patients (n = 22) underwent C-CT. Per patient, up to 3 liver metastases were investigated by one radiologist (XX blinded for review). In cases of more than 3 metastases, three target lesions (largest metastases or those metastases with the best assessability regarding motion-artifacts) in different liver segments were determined.

The sizes (mm) of the target lesions at baseline and in all FU were measured along the longest diameter by one radiologist (XX blinded for review). Only patients with newly diagnosed metastases were rated as progressive, as rating of tumor response according to size-based criteria (e.g., RECIST) might fail the outcome of patients treated with bevacizumab [9, 10].

MRI protocol

MRI was performed on a clinical 1.5 Tesla scanner (Ingenia, Philips, Best, The Netherlands) using a multi-channel surface receiver coil. As part of a standardized pulse sequence protocol, all examinations included a T2-weighted turbo spin echo sequence and a DCE series; further details are given in Table 1. For the DCE examinations 0.1 mmol/kg body weight gadobutrol (Gadovist, Bayer Schering Pharma, Leverkusen, Germany) was intravenously administered and images were acquired in pre-contrast, arterial, portal venous and late phase.

Table 1. MRI sequence parameters.

Typ of scanner 1,5-T Ingenia, Philips Healthcare
Surface coil Multielement 16-channel coil (Sense Torso XL)
T2-weighted pulse sequence Dynamic series
Pulse sequnence typ 2D turbo spin echo T1-weighted 3D gradient echo
TR/TE [ms] 2500/80 4.3/1.3
Orientation transverse transverse
Acquisition matrix 304 x 233 268 x 174
Field of view 310 mm 330 mm
Slice thickness 5 6
Breath compensation Respiratory triggering; in case of motion artefacts additionally breath-hold Breath-hold
Sense factor 1.4 2
Dynamic phases n.a. pre-contrast, arterial, portal-venous, and equilibrium phase
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T2-signal intensity assessment

T2-SI was assessed twice, in a practical reader-based (qualitative) and in a region-of-interest (ROI)-based (semi-quantitative) manner for reference purpose.

For reader-based assessment of T2-SI, three radiologists (all more than 3 years of experience in oncologic liver MRI: XX, XX, XX blinded for review) rated the signal intensity of the referring liver lesion on a 5-point scale from -2 (clearly hypointense compared to the splenic parenchyma), over 0 (isointense to the splenic parenchyma) to +2 (clearly hyperintense compared to the splenic parenchyma). In case of a disagreement in the rating of a lesion, a consensus reading carried out. The spleen was chosen as reference tissue, as the signal intensity of the liver parenchyma may change due to side effects of systemic chemotherapy.

A fourth radiologist (XX blinded for review) assessed the T2-SI of liver metastases within a representative area of the tumor excluding cystic transformed or necrotic tissue. The measured signal intensities of the metastases were intra-individually referenced to the signal intensity of the spleen by measuring round shaped ROIs on the same image.

Analysis of contrast enhancement

Tumoral enhancement of target lesions was qualitatively evaluated by three radiologists using a 5-point scale ranging from 1 (no visible arterial or late contrast enhancement) to 5 (exception arterial or late contrast enhancement). In case of a disagreement in the rating of a lesion, a consensus reading carried out.

Statistical analysis

Continuous variables are expressed as mean values ± standard deviation. The Spearman correlation coefficient (ρ) assessed the degree of monotonic association between T2-SI and tumor enhancement. Interobserver agreement between the three blinded radiologists regarding T2-SI and tumor enhancement was evaluated by using Fleiss' kappa (κ). κ was categorized according to Landis and Koch [20].

Student’s t-test was used to detect statistically significant differences in the outcomes of the investigated imaging parameters (paired t-test for intergroup (1. FU, 2. FU, and 3. FU vs. baseline) and unpaired t-test for intergroup (B-CT vs. C-CT) comparison. All test results were analyzed in an explorative way, thus p values of p≤0.05 were regarded as statistically significant. The interpretation of Spearman’s ρ followed the guidelines according to Altman [21]. Statistical calculation was carried out on standard PC with IBM SPSS statistics V22 (SPSS Inc., IBM, New York, USA).

Results

Patients, target lesions, and tumor size

Forty-four patients (28 females, 16 males) were evaluated. Patient ages ranged from 36 to 84 years (63 ± 11). The patient cohort included 26 cases of CRC as primary tumor and 18 cases of breast cancer. In total 67 consecutive target lesions were assessed (average number of lesions per patient: 1.52). Table 2 gives detailed information of the metastatic location.

Table 2. Location of liver metastases.

Type of Primary Number of left hepatic metastases (n) Number of right hepatic metastases (n)
I II III IVa IVb V VI VII VIII
Colorectal Cancer 1 3 3 5 6 6 5 6 8
Breast Cancer 1 1 3 4 5 2 1 4 3
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Location of liver metastases according to hepatic segmentation by Couinaud classification.

Both groups (B-CT and C-CT) did not differ regarding the distribution of age, gender primary cancer, number of progressive diseases and average time between follow-up examinations (Table 3).

Table 3. Demographic and oncological details of the study population.

Of note, no significant differences between both groups were documented.

Study Population (n = 44)
Chemotherapy / Bevacizumab-containing chemotherapy Cytotoxic-chemotherapy p-values
Cohort Patients n 22 22 /
Number of lesions n 33 34 /
Demographic details Age y 65 ± 11 61 ± 11 0.482
Gender m 13 15 0.493
f 9 7 0.542
Type of Primary Number of patients with colorectal cancer n 13 13 1.0
Number of colorectal metastases n 22 21 0.964
Number of patients with breast cancer n 9 9 1.0
Number of breast cancer metastases n 11 13 0.753
Patients with progressive disease 1. follow up n 0 0 /
2. follow up n 7 8 0.757
3. follow up n 15 13 0.542
Time of imaging Average time in days between MRI n 102 ± 25 86 ± 24 0.132
94 ± 22 86 ± 23 0.249
88 ± 21 89 ± 20 0.867
Additional agents in chemotherapy Agents (colorectal cancer) / 5-Fluorouracil Oxaliplatin /
Irinotecan Trifluridine
Capecitabine 5-Fluorouracil
Irinotecan
Capecitabine
Agents (breast cancer) / Paclitaxel Doxorubicin /
Epirubicin
Paclitaxel
5-fluorouracil
Cyclophosphamide
Carboplatin
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Average size of liver metastases at baseline, first follow-up (1. FU), second follow-up (2. FU), and third follow-up (3. FU) in the Bevacizumab-containing chemotherapy (B-CT) group and the cytotoxic chemotherapy (C-CT) group.

The average size of the metastases increased continuously from baseline to the 3. FU in both groups. For B-CT, the metastasis size change was significant between baseline vs. 1. FU, 2. FU, and 3. FU, respectively. No significant differences in tumor size were found in the C-CT group during the course of observation. In an intergroup comparison (Table 4), no significant differences in tumor size were observed.

Table 4. Changes of liver metastases in size, T2 signal intensity, contrast enhancement.

Baseline 1. follow up 2. follow up 3. follow up p-values (1. follow up vs. baseline) p -values (2. follow up vs. baseline) p -values (3. follow up vs. baseline)
Average size (mm) of liver metastases
B-CT group 15.27 19.80 22.37 26.43 0.001 0.001 0.001
± 11.41 ± 13.48 ± 14.62 ± 14.78
C-CT group 15.88 18.34 19.62 20.63 0.331 0.101 0.056
± 9.11 ± 9.46 ± 12.86 ± 12.70
p -values (B-CT vs. C-CT) 0.811 0.613 0.423 0.163
Average reader-based metastatic T2 signal intensity
B-CT group -0.70 -1.55 -1.91 -1.97 0.001 0.001 0.001
± 0.83 ± 0.61 ± 0.62 ± 0.52
C-CT group -0.73 -0.39 -0.33 -0.39 0.072 0.058 0.063
± 0.79 ± 0.81 ± 0.68 ± 0.65
p -values (B-CT vs. C-CT) 0.136 0.001 0.001 0.001
Average ROI-based metastatic T2 signal intensity
B-CT group 0.83 0.58 0.48 0.4 0.001 0.001 0.001
± 0.2 ± 0.16 ± 0.14 ± 0.11
C-CT group 0.82 0.91 0.89 0.91 0.052 0.065 0.061
0.22 ± 0.21 ± 0.19 ± 0.16
p -values (B-CT vs. C-CT) 0.769 0.001 0.001 0.001
Average tumor contrast enhancement
B-CT group 2.42 1.58 1.48 1.42 0.001 0.001 0.001
± 0.78 ± 0.74 ± 0.66 ± 0.65
C-CT group 2.48 2.36 2.42 2.33 0.432 0.702 0.454
± 0.69 ± 0.75 ± 0.68 ± 0.65
p -values (B-CT vs. C-CT) 0.696 0.001 0.001 0.001
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bevacizumab-containing chemotherapy (B-CT); cytotoxical chemotherapy (C-CT).

Changes in T2-SI

After treatment with B-CT, the average reader-based T2-SI was significant lower in all three FU’s compared to the baseline (0.70 ± 0.83 before vs. -1.55 ± 0.61, -1.91 ± 0.62, and -1.97 ± 0.52 after treatment, respectively). Comparing the T2-SI of metastases treated with C-CT, no statistical were found between baseline and the follow-up examinations (0.73 ± 0.79 before vs. -0.69 ± 0.81, -0.71 ± 0.68, and -0.75 ± 0.65 after treatment, respectively) (Table 4).

Comparing the two groups (B-CT vs. C-CT), the T2-SI did not differ at baseline (p>0.136). At all three FU, the T2-SI of the metastases from the B-CT was significantly lower than in the C-CT group (Fig 1). Regarding the inter-reader reliability of agreement for T2-SI, κ was substantial with 0.75.

Fig 1. Signal changes of liver metastases after bevacizumab-containing chemotherapy and cytotoxic chemotherapy.

Fig 1

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a-d) T2-weighted MRI of a 56-year old man with hepatic metastases of rectal cancer. The treatment contained bevacizumab (B-CT). Patient was progressive with new liver metastases at the 3. follow up. 3 metastases are marked each with a white arrow. Please note the remarkable decrease in T2-signal intensity after bevacizumab therapy although slightly progress of steatosis hepatis. e-h) T2-weighted MRI of a 68-year old man with hepatic metastases of rectal cancer. Treatment did not contain bevacizumab (C-CT). Patient was progressive with new liver metastases at the 3. follow up.

ROI-based assessment of T2-SI revealed corresponding and significant (p<0.001) results for every FU compared to the baseline: B-CT (0.83 ± 0.20 before vs. 0.58 ± 0.16, 0.48 ± 0.14, and 0.40 ± 0.11 after treatment, respectively), and C-CT (0.82 ± 0.22 before vs. 0.91 ± 0.21, 0.89 ± 0.19, and 0.91 ± 0.16 after treatment, respectively) (Fig 2). In an intergroup comparison (B-CT vs. C-CT), the T2-SI did not differ at baseline (p>0.769). At all three FU, the T2-SI of the metastases from the B-CT group was significantly lower than in the C-CT group (Table 4).

Fig 2. Longitudinal signal changes on T2-weighted imaging after bevacizumab-containing chemotherapy and cytotoxic chemotherapy.

Fig 2

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ROI-based T2-signal intensity of liver metastases, referenced to the signal intensity of the spleen, with (B-CT; blue balls) and without (C-CT; green squares) bevacizumab containing chemotherapy. Please note the significant (both, intra- and inter-group comparison p<0.001, respectively) decrease in T2- signal intensity of liver metastases after initial bevacizumab containing chemotherapy.

Changes in tumor contrast enhancement

On average, metastases treated with B-CT exhibited less tumor enhancement at all FUs; at the 2. FU and 3. FU the differences were significant compared to baseline (2.42 ± 0.78 before vs. 1.58 ± 0.74, 1.48 ± 0.66, and 1.42 ± 0.65 after treatment, respectively). In the C-CT group, no relevant change of contrast enhancement was observed in the DCE before versus after treatment (2.48 ± 0.69 before vs. 2.36 ± 0.75, 2.42 ± 0.68, and 2.33 ± 0.65 after treatment, respectively).

The intra-group comparison revealed no statistical differences before treatment (p>0.696). At all follow-ups, the B-CT showed a significantly (p<0.001) decreased tumor enhancement (Table 4). With κ = 0.39, the inter-reader agreement of tumor enhancement on DCE sequences was rated as fair.

Correlation of imaging biomarkers

The reader-based T2-SI assessment by three radiologists and the ROI-based measurements showed an excellent correlation of r = 0.91.

Post-therapeutic changes of reader-based and ROI-based assessment of T2-SI and tumor enhancement showed a strong correlation with r = 0.71 and r = 0.65, respectively.

Tumor size did not significantly correlate with T2-SI, neither ROI- nor reader-based assessment (r = -0.05, p = 0.448; r = -0.10, p = 0.111), or tumor enhancement (r = -0.03, p = 0.628).

Discussion

Our most important finding is that liver metastases treated with bevacizumab containing systemic therapies present hypointense on T2-weighted MRI, whereas conventionally, cytotoxically treated metastases did not show relevant changes in their T2-SI.

In solid tumors, neoangiogenesis leads to an increased intra-tumoral microcirculation, tortuous micro-vessels and irregular blood flow with unstable rheology [22, 23]. Pathological examinations showed that angiogenesis inhibitors normalize the tumor vasculature, reducing the oncotic pressure, and hence normalize the interstitial water content [24]. In line to this study, it is likely that the longitudinal decrease of the T2-SI in bevacizumab treated metastases (B-CT) found in our study, may be caused by the antiangiogenic effect. In metastases that were treated cytotoxically, we did not observe such a decrease in T2-SI. This can be seen in accordance with a pathologic study where Ribero et al. [25] found significantly more vascular and sinusoidal dilation in cytotoxically treated tumors. In line with this, Shindoh et al. [4] reported a different morphological response in computed tomography of liver metastases after treatment with versus without bevacizumab containing therapies. They found that B-CT treated metastases more frequently turned into homogenous, well margined tumors (47% vs. 12%). This effect may correspond to the changes that we observed on T2-weighted MRI. However, MRI is suitable to detect distinct biopathological of the tissue and by that deriving a broader spectrum of information. Both effect–decreasing T2-SI and morphological pronounced delineation against the liver tissue on CT–might reflect the same pathological changes in the long term. Regarding the quantification of these effects, however, MRI is considered to be superior to other imaging techniques in the detection of biological changes. If a decreasing T2-SI might depict the biological changes more accurate should be further investigated.

In this intraindividual study, we investigated the behavior of two tumor entities with different vascularization characters, that are portal-venously supplied colon and arterially supplied breast metastases. Surprisingly, both entities are treated with bevacizumab containing chemotherapies following the guidelines. As the presence or absence of bevacizumab is the major aspect that dichotomizes the study cohort, and as these two groups significantly differ in their presentation on T2w MRI after therapy, we deduce that the described observations can be attributed to the effects of bevacizumab. However, it remains unclear whether the degree of T2-SI decrease is able to predict a positive outcome on the survival of the patient. Consequently, further studies should be conducted to identify the predictive value of this possible biomarker. Generally, changes in tumor vascularity are radiologically assessed using dynamic contrast enhanced imaging [4]. Thus, functional MRI techniques were examined to assess the effects of bevacizumab [26, 27]. In liver metastases, Detsky et al. [28] found a significant decrease in permeability and blood volume on perfusion MRI. In this study, we also found significantly reduced enhancement on DCE in lesions treated with bevacizumab compared to exclusive cytotoxic systemic therapies. However, recent literature claims a lack of standardization regarding the assessment of functional parameters on complex and ambiguous images as well as the extensive mandatory post-processing [29, 30]. In our study, the reader-based contrast enhancement agreed only fairly between the three observers (κ = 0.39). Although tumor enhancement is a morphologically used response criterion, the degree of enhancement is difficult to measure and therefore less reliable [19]. Moreover, colorectal liver metastases are considered hypo-vascular tumors [31, 32] and changes of tumor perfusion are even more difficult to assess. In contrast to this, the practical reader-based T2-SI assessment on T2 turbo-spin echo sequences revealed a substantial interobserver agreement (κ = 0.75). Underlining the validity of this reader-based assessment, the reader- and ROI-based measurement of T2-SI almost perfectly correlated (r = 0.91). In contrast to this, the quality of DCE measurements highly depends on individual patient related factors such as circulation, patient breathing, or motion. Thus, it seems to be easier and more practical to assess the signal change on T2-weighted TSE sequences rather than comparing the changes in DCE. Despite, T2-weighted imaging is not directly related to antiangiogenic effects, the changes in T2-SI most likely correlate with a therapeutic effect of bevacizumab. Additionally, this might even be of advantage, as not only the antiangiogenic effect, as in DCE, but its overall changes in the tumor metabolism and extracellular matrix are monitored.

As another functional MRI parameter, DWI has been extensively proven to serve as a technique to assess tumor response [18]. In this study, we did not compare the T2-SI changes with the DWI or the apparent diffusion coefficient (ADC), respectively. Although, similar to T2-SI, the DWI contrast/ADC are not directly related to the tumor micro-vascularity and the antiangiogenic drug effects are only indirectly reflected. However, other than T2-weighted TSE-imaging, DWI is a prone MR technique and therefore its interpretation often remains ambiguous.

The limitations of this study are its retrospective nature and the use of a DCE (4 contrast phases) instead of an over-time highly resolved perfusion technique. Moreover, respiratory artefacts may have interfered with the image evaluation, especially on the DCE images; the T2-weighted images were at least performed twice in case insufficient image quality. However, no patient had to be excluded because of minor image quality.

Although, we could show that there are distinct changes in liver metastases after treatment with bevacizumab, these results are limited in the implications for the clinical outcome. Due to the though longitudinal but retrospective setting of this study, we are not able to investigate these changes against the background of a possible change in survival. However, the benefits of a bevacizumab containing therapy are well known [6–8], and out of this our results should elucidate the effects of bevacizumab with possible practical implications on the reporting for radiologist as these changes might have an influence on the diagnostic workflow after bevacizumab treatment.

Another limitation addresses the imbalance of population size against the background of a long enrollment period (~7 years). Over this period, possible changes in patient treatment might have influenced the treatment of liver metastases. The study was designed as a longitudinal intraindividual observation within a period of up to 9 moth per patient. Moreover, no patient with any interventional tumor treatment was analyzed and a possible infestation of the metastatic rheology (e.g. portal venous embolization) was prevented within this highly controlled setting. But differences between the individual chemotherapeutical regimens during may be considered as a possible limitation.

This study shows that liver metastases treated with bevacizumab containing therapies appear hypointense on T2-weighted images. These T2 signal changes highly correlate with a reduced tumor enhancement that has been shown to reflect bevacizumab therapy effects. Towards practical efforts, reader- and ROI-based measurement of T2-SI showed a perfect correlation. Thus, T2-SI changes may serve as a functional imaging biomarker if liver metastases were treated with bevacizumab. However, future studies should evaluate the predictive value of these T2-SI changes on the clinical outcome.

Supporting information

S1 Data. Raw data of the patients with age, tumor type, and ROI-based measurements.

(XLSX)

Click here for additional data file. (53.9KB, xlsx)

Acknowledgments

The project was supported by the „START-Program” of the Faculty of Medicine of the RWTH Aachen University. The authors gratefully thank Daniel Truhn and Alexander Ciritsis for careful assistance during this investigation.

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

The authors received no specific funding for this work.

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Decision Letter 0

Michael C Burger

2 Jan 2020

PONE-D-19-27984

Signal changes in T2-weighted MRI of liver metastases under Bevacizumab‒ A practical imaging biomarker ? ‒

PLOS ONE

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Reviewer #2: Yes

Reviewer #3: Partly

**********

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Reviewer #3: Yes

**********

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Reviewer #3: No

**********

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Reviewer #1: In this study, the authors analyzed the impact of T2-weighted MRI on liver metastases from colorectal and breast primary treated with or without anti-VEGF antibody therapy. The conclusion is that T2 weighted imaging can reflect the effect of bavacizumab.

I have some comments.

1. As mentined in the discussion, the tumor regression after anti-VEGF therapy is unique that is called "morphological change" on CT reported from MDACC group. Basically, you only validated the morphological change on MRI, didn't you? You should more emphasize the superiority of this study over the previous CT-based studies because CT is more universally diffused.

2.You did not show the clincal outcome of the patients. I did not know the correlation of the change on the MRI with the clincal outcome. You need to analyze that if you want to show the benefit of T2-weighted MRI as a new biomarker.

Reviewer #2: The manuscript is well written, easy to understand and the results are clearly explained. The discussion clearly correlate the results with current litterature. For this reason I suggest that it can be published in the current form

Reviewer #3: The finality of this study was to investigate signal changes in T2-weighted magnetic resonance

imaging of liver metastases under treatment with and without bevacizumab-containing

chemotherapy and to compare these signal changes to tumor contrast enhancement.

It is true that this is a crucial clinical need on usual methods for tumor response

assessment persists if therapy contains antiangiogenic drugs like bevacizumab, TKI inhibitors, and others drugs in many type of tumors where now these modern drugs are used.

It is well known that many modern drugs and Bevacizumab, the first one adopted worldwidely in chemotherapy, primarily inhibits the formation of new blood vessels and leads to a regression of already existing tumor vascularization. Obviously a great debate is still open about

commonly used size-based response assessment, especially RECIST, can fail to precisely

monitor the effect of antiangiogenic therapy.

The basic idea of the Authors is that liver metastases exhibit considerably hypointense in T2-weighted imaging after treatment with bevacizumab, in contrast to conventionally treated liver metastases. Therefore, T2-weighted imaging seems to reflect the effect of bevacizumab

After sharing these good intentions of the Authors, it is necessary to point out some conceptual problems that seriously undermine many of the reported observations and the good work done.

First of all the Authors present a limited number of Patients that received bevacizumab-containing (n=22) or conventional cytotoxic chemotherapy (n=22). Again the recruitment is too long and in these 7 years many aspects are changed. A serious conceptual error is to consider colon cancer, known to be hypovascular, and breast cancer which are very well vascularized.

Another methodological error is to consider few metastases per patient, it is known that after chemotherapy with or without angiogenic agents the response varies in the various metastases and segments. Because 44 consecutive patients with a total of 67 liver metastases (26 patients with 43 metastases of colorectal cancer (CRC) and 18 patients with 24 metastases of breast cancer) were studied with standardized liver MRI from July 2010 to November 2016.

I agree with the finding is that liver metastases treated with bevacizumab containing

systemic therapies present hypointense on T2-weighted MRI, whereas conventionally,

cytotoxically treated metastases did not show relevant changes in their T2-SI.

As the Authors report colorectal liver metastases are considered hypo-vascular tumors and changes of tumor perfusion are even more difficult to assess.

This study present other bias: its retrospective nature and the use of a DCE (4 contrast

phases) instead of an over-time highly resolved perfusion technique.

Even if the Authors report that respiratory artefacts may have interfered with the image evaluation, especially on the DCE images, the claim that T2-weighted images were at least performed twice in case insufficient image quality is not sufficent to accept considering

minor image quality.

The conclusion that.. “T2-SI changes may serve as a functional imaging biomarker if liver

metastases were treated with bevacizumab” is not fully acceptable considering this paper.

In any case, it must be recognized that the authors' intentions are very useful in clinical practice and this work is part of a very important discussion in the evaluation of responses to chemotherapy.

The work is well written, well developed and must be reconsidered after choosing only one pathology (colon or breast) which has a homogeneous pattern of vascularization.

The lesions must be mapped for segments and at least 3 metastases per patient must be considered to have usable data and conclude as the authors say.

In conclusion, the work is well written and significant for the research it involves on a very important topic but greater conceptual changes are needed. It may be published after a major revision.

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Giammaria Fiorentini

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PLoS One. 2020 Mar 31;15(3):e0230553. doi: 10.1371/journal.pone.0230553.r002

Author response to Decision Letter 0

14 Feb 2020

Point-by-Point Reply to the Editor’s and Reviewers’ Comments

Title:

Signal changes in T2-weighted MRI of liver metastases under Bevacizumab

‒ A practical imaging biomarker ? ‒

Reference number:

PONE-D-19-27984

Journal:

PLOS ONE

General Reply:

Thank you very much for the thorough review of our manuscript. We very much appreciate the opportunity and hope to have satisfactorily answered all of your and the reviewers’ comments. We would be happy if our manuscript was chosen to be published in PLOS ONE.

In the following we would like to address the comments of the reviewers and the editor point by point. Please note, that changes to the original document are tracked with “track changes” in word. As required, we have also attached a “clean” version in which all of the changes have been accepted.

 

Reply to Reviewer #1

PONE-D-19-27984

Title:

Signal changes in T2-weighted MRI of liver metastases under Bevacizumab

‒ A practical imaging biomarker ? ‒

Comment 1.1: “In this study, the authors analyzed the impact of T2-weighted MRI on liver metastases from colorectal and breast primary treated with or without anti-VEGF antibody therapy. The conclusion is that T2 weighted imaging can reflect the effect of bavacizumab. I have some comments.”

Authors’ Response: We would like to thank the Reviewer very much for taking the time to review our manuscript and for her/his overall appreciation of our work.

Comment 1.2: ” 1. As mentioned in the discussion, the tumor regression after anti-VEGF therapy is unique that is called "morphological change" on CT reported from MDACC group. Basically, you only validated the morphological change on MRI, didn't you? You should more emphasize the superiority of this study over the previous CT-based studies because CT is more universally diffused.”

Authors’ Response: Indeed, these imprecisions in our discussion warrant further explanation: In our study liver metastases showed characteristically changes in their signal behaviour on T2-weighted MRI. These changes might be in line with the changes described by the MDACC group. However, MRI is suitable to detect a broader spectrum of information compared to CT. The changes described by MDACC group are related to the morphological appearance of the metastases (e.g. round-shaped). In contrast to this, we could show that the texture of the metastases exhibits changes that might reflect biopathological changes in the metastases itself. Moreover, this has important implications for the radiologist in the reporting workflow of patients treated with antiangiogenic therapy. In general, metastases exhibit an intermediate-to-hyperintense signal on T2-weighted imaging. Nevertheless, with this publication we want to make radiologist aware of these changes after treatment with bevacizumab to maintain the diagnostic accuracy in these cases.

So, in line with the reviewer’s suggestion, we added the following to the discussion on page 16 (ll. 252-258) in order to guide the astute reader in understanding our line of thought:

“However, MRI is suitable to detect distinct biopathological of the tissue and by that deriving a broader spectrum of information. Both effect – decreasing T2-SI and morphological pronounced delineation against the liver tissue on CT – might reflect the same pathological changes in the long term. Regarding the quantification of these effects, however, MRI is considered to be superior to other imaging techniques in the detection of biological changes. If a decreasing T2-SI might depict the biological changes more accurate should be further investigated.”

Comment 1.3: “2. You did not show the clincal outcome of the patients. I did not know the correlation of the change on the MRI with the clincal outcome. You need to analyze that if you want to show the benefit of T2-weighted MRI as a new biomarker.”

Authors’ Response: We totally agree with the reviewer’s criticism. However, this study was initiated to describe MR imaging effects that seem to be typical for bevacizumab containing chemotherapies and might further serve as an additional parameter in follow-up exams. The effects of bevacizumab regarding the patient outcome after tumor treatment are well studied and were not part of this investigation. This study was accomplished with a longitudinal and retrospective approach; therefore, we are not able to investigate whether our findings have an implication on the clinical outcome, although we would like to. As this a critical limitation, we added this to the limitations on page 17 (ll. 305-312):

“Although, we could show that there are distinct changes in liver metastases after treatment with bevacizumab, these results are limited in the implications for the clinical outcome. Due to the though longitudinal but retrospective setting of this study, we are not able to investigate these changes against the background of a possible change in survival. However, the benefits of a bevacizumab containing therapy are well known (1-3), and out of this our results should elucidate the effects of bevacizumab with possible practical implications on the reporting for radiologist as these changes might have an influence on the diagnostic workflow after bevacizumab treatment.“

 

Reply to Reviewer #2

PONE-D-19-27984

Title:

Signal changes in T2-weighted MRI of liver metastases under Bevacizumab

‒ A practical imaging biomarker ? ‒

Comment 2.1: “The manuscript is well written, easy to understand and the results are clearly explained. The discussion clearly correlate the results with current litterature. For this reason I suggest that it can be published in the current form.”

Authors’ Response: Thank you very much for your overall appreciation of our manuscript. Moreover, we agree with you and hope to find the right and interested readership in the journal PLOS ONE and hope to publish this manuscript in the journal.

 

Reply to Reviewer #3

PONE-D-19-27984

Title:

Signal changes in T2-weighted MRI of liver metastases under Bevacizumab

‒ A practical imaging biomarker ? ‒

Comment 3.1: ” The finality of this study was to investigate signal changes in T2-weighted magnetic resonance imaging of liver metastases under treatment with and without bevacizumab-containing chemotherapy and to compare these signal changes to tumor contrast enhancement. It is true that this is a crucial clinical need on usual methods for tumor response assessment persists if therapy contains antiangiogenic drugs like bevacizumab, TKI inhibitors, and others drugs in many type of tumors where now these modern drugs are used.

It is well known that many modern drugs and Bevacizumab, the first one adopted worldwidely in chemotherapy, primarily inhibits the formation of new blood vessels and leads to a regression of already existing tumor vascularization. Obviously, a great debate is still open about commonly used size-based response assessment, especially RECIST, can fail to precisely monitor the effect of antiangiogenic therapy. The basic idea of the Authors is that liver metastases exhibit considerably hypointense in T2-weighted imaging after treatment with bevacizumab, in contrast to conventionally treated liver metastases. Therefore, T2-weighted imaging seems to reflect the effect of bevacizumab”

Authors’ Response: We would like to thank the Reviewer for his/her thorough revision of our manuscript, its overall appreciation and for the numerous comments and detailed feedback that we hope to have addressed sufficiently. We are confident that the revision of our manuscript along these lines has been of great benefit to its quality. Please refer to our detailed responses to the comments below.

Comment 3.2: ”After sharing these good intentions of the Authors, it is necessary to point out some conceptual problems that seriously undermine many of the reported observations and the good work done. First of all the Authors present a limited number of Patients that received bevacizumab-containing (n=22) or conventional cytotoxic chemotherapy (n=22). Again the recruitment is too long and in these 7 years many aspects are changed.”

Authors’ Response: We totally understand the reviewers’ concerns and with her/his criticisms of the long recruitment phase. We have to point out, that there may have been differences within the chemotherapy regimens of the patients related to ongoing improvements in oncological patient care. However, the main difference between the observed groups was the presence or absence of bevacizumab. Consequently, this study was designed as an intraindividual comparison to observe the bevacizumab effects in a longitudinal manner. As a second aspect of this reviewer’s concerns, we performed a highly standardized pulse sequence protocol, that did not change within observation period and was carried out on the same 1.5 T Philips MR scanner, so we can guarantee that MRI technique did not influence the study results. Regarding the small sample size (2 x n=22) against the background of an enrollment phase of 7 years, we have to point out, that we need to have highly comparable patients in this longitudinal intraindividual study design. No patient received any interventional tumor treatment, hemihepatectomy, portal vein embolization or other tumor related interventions. As a mater of fact, this is one of the major strengths of our study, and which is in contrast to other studies on bevacizumab (4, 5).

In line with the reviewer’s concerns, we added the following to the limitations sections (page 18; ll. 313-320) to improve the interpretation of our results by the readers:

“Another limitation addresses the imbalance of population size against the background of a long enrollment period (~7 years). Over this period, possible changes in patient treatment might have influenced the treatment of liver metastases. The study was designed as a longitudinal intraindividual observation within a period of up to 9 moth per patient. Moreover, no patient with any interventional tumor treatment was analyzed and a possible infestation of the metastatic rheology (e.g. portal venous embolization) was prevented within this highly controlled setting. But differences between the individual chemotherapeutical regimens during may be considered as a possible limitation.”

Comment 3.3: ”A serious conceptual error is to consider colon cancer, known to be hypovascular, and breast cancer which are very well vascularized.”

Authors’ Response: The Reviewer is right to remark this and in line with the Reviewer we have to admit that this seems intuitively sound. However, anti-angionetic drugs are applied in both highly vascularized breast cancer and hypovascular colon cancer. As this longitudinal study observes the intraindividual effects, these physiological cancer properties are eliminated. Moreover, we did not focus on dynamic contrast enhanced MRI to observe perfusion effects that are highly variable even at baseline and that can only insufficiently be monitored using often quite variable DCE sequences. Instead, we focused on the reproducible T2-weighted TSE sequence to observe possible chemotherapeutical induced effects. In line with our conception we have to admit, that first admission for bevacizumab was granted for hypovascularized colon cancer (2005) and hereafter an admission for the hypervascularized breast cancer was granted (2007). We have considered this in our initial design for this study and wanted to point out that the similar behavior in T2-weighted TSE could overcome possible masking effects in to date shortcomings of dynamic enhancement imaging. T2-weighted MRI might be suitable to depict changes of antiangiogenic therapy regardless of the initial metastatic vascularization which is significantly demonstrated in this study. To discuss this crucial issue, we added the following to the discussion section on page 17 (ll. 259-268):

“In this intraindividual study, we investigated the behavior of two tumor entities with different vascularization characters, that are portal-venously supplied colon and arterially supplied breast metastases. Surprisingly, both entities are treated with bevacizumab containing chemotherapies following the guidelines. As the presence or absence of bevacizumab is the major aspect that dichotomizes the study cohort, and as these two groups significantly differ in their presentation on T2w MRI after therapy, we deduce that the described observations can be attributed to the effects of bevacizumab. However, it remains unclear whether the degree of T2-SI decrease is able to predict a positive outcome on the survival of the patient. Consequently, further studies should be conducted to identify the predictive value of this possible biomarker.”

Comment 3.4: ”Another methodological error is to consider few metastases per patient, it is known that after chemotherapy with or without angiogenic agents the response varies in the various metastases and segments. Because 44 consecutive patients with a total of 67 liver metastases (26 patients with 43 metastases of colorectal cancer (CRC) and 18 patients with 24 metastases of breast cancer) were studied with standardized liver MRI from July 2010 to November 2016.”

Authors’ Response: We do agree with the reviewer’s statement. Thus, we assessed all metastases, up to 3, in all cases. If the number of metastases was >3 and the lesions were spread over the liver, we selected metastases all from different segments. To address this valuable point, we clarified this in the method and results section (page 5 ll. 94-95 and 10 ll. 152-156) and added another table (table 2) which summarizes the exact location of all metastases:

“In cases of more than 3 metastases, three target lesions (largest metastases or those metastases with the best assessability regarding motion-artifacts) in different liver segments were determined.”

“Table 2 gives detailed information of the metastatic location.”

Table 2: Location of liver metastases

Type of Primary Number of left hepatic metastases (n) Number of right hepatic

metastases (n)

I II III IVa IVb V VI VII VIII

Colorectal Cancer 1 3 3 5 6 6 5 6 8

Breast Cancer 1 1 3 4 5 2 1 4 3

Table 2: Location of liver metastases according to hepatic segmentation by Couinaud classification.

Comment 3.5: ”I agree with the finding is that liver metastases treated with bevacizumab containing systemic therapies present hypointense on T2-weighted MRI, whereas conventionally, cytotoxically treated metastases did not show relevant changes in their T2-SI.

As the Authors report colorectal liver metastases are considered hypo-vascular tumors and changes of tumor perfusion are even more difficult to assess.”

Authors’ Response: We would like to thank the reviewer for sharing his/her considerations on the role of perfusion-related imaging in general and by this support the clinical need of a practical imaging biomarker in the daily clinical routine.

Comment 3.6: ” This study present other bias: its retrospective nature…”

Authors’ Response: We agree, that a retrospective design is a general limitation. In line with the qualified criticism of Reviewer 1 we have addressed this issue in our limitations and by this hope to drive future prospective studies on that persisting clinical need.

Comment 3.7: ”…the use of a DCE (4 contrast phases) instead of an over-time highly resolved perfusion technique.”

Authors’ Response: Thank you for this thoughtful remark. We agree that perfusion imaging is the state of art technique in an experiential setting. Although, perfusion is technically highly challenging and thus prone to errors. Moreover, it is challenging for the patient compliance (breath hold etc.). As a matter of fact, it is rarely part of a standard clinical examination. We strive to aim with this study for a practical imaging biomarker in everyday patient care. However, we absolutely agree with these concerns, and hope to yield the scientific basis for a possible prospective study, in which perfusion MRI should be part of the study MRI protocol. We hope that we have addressed these concerns sufficiently with our limitations section on page 17.

Comment 3.8: “Even if the Authors report that respiratory artefacts may have interfered with the image evaluation, especially on the DCE images, the claim that T2-weighted images were at least performed twice in case insufficient image quality is not sufficent to accept considering

minor image quality.”

Authors’ Response: Generally, follow-up MRI exams of the abdomen are challenging for the majority of patients. In particular, as in our population we examined patients with a crucial illness and thus optimal compliance was hard to achieve. Being part of a cancer comprehensive center with standardize examination routines, we consequently perform T2-weighted-TSE sequences with respiratory gating, and in case of minor image quality, in breath hold. Using DCE, all these challenges (shortness of breath etc.) become even more critical. However, as we performed measurements in a ROI-based manner, we were able to avoid measurements in artifacts. Against this background, we have to state - according to our discussion section (page 17) - that no patient was excluded due to insufficient image quality, neither in T2-weighted imaging nor on DCE imaging.

Comment 3.9: “The conclusion that.. “T2-SI changes may serve as a functional imaging biomarker if liver metastases were treated with bevacizumab” is not fully acceptable considering this paper. In any case, it must be recognized that the authors' intentions are very useful in clinical practice and this work is part of a very important discussion in the evaluation of responses to chemotherapy.”

Authors’ Response: We share the intention of the reviewer and in line with his astute criticism and the considerations of Reviewer #1 we hope to sufficiently address this issue in the discussion section. On and above, we would like to point out that our results underpin the highly published results of Shindoh and Chun et al. (4, 5). The results we would like to publicize with this manuscript are the first descriptions of changes in MRI after bevacizumab therapy. These changes in T2-weighted MRI are correlated to some extend to the perfusion and consecutively to the rheology of the metastases, which is in line with the above-mentioned publications. By increasing use of antiangiogenic drugs, radiologist should be aware of these changes in everyday patient care. We do not strive to overvalue the implications of T2-weighted imaging in a context of a deeper pathological tumorbiological response. We hope to donate certain guidance to the readers through this meaningful scientific discussion on antiangiogenetic therapy by our discussion.

Comment 3.10: “The work is well written, well developed and must be reconsidered after choosing only one pathology (colon or breast) which has a homogeneous pattern of vascularization.”

Authors’ Response: We would like to thank the reviewer for her/his commendation. Regarding focusing on only one entity, we would like to refer to our reply of comment 3.3 and its consequences. We still agree, that only one entity seems more intuitive in the context of an interindividual setting, however we could show that the observed effect can be attributed to bevacizumab and are independent form the tumor entity and its initial stet of perfusion in our intraindividual longitudinal observation.

Comment 3.11: “The lesions must be mapped for segments and at least 3 metastases per patient must be considered to have usable data and conclude as the authors say.”

Authors’ Response: In line with the reviewer’s suggestion we provided a detailed mapping (table 2; pls. be referred to authors’ response to comment 3.4) of the metastases with the location of all metastases and an exact distribution of all in the segments.

Comment 3.12: “In conclusion, the work is well written and significant for the research it involves on a very important topic but greater conceptual changes are needed. It may be published after a major revision.”

Authors’ Response: Thank you for this kind comment. We hope to have replied to your entire satisfaction, and we further hope, that you esteem this publication worthy to be published.  

Reply to the Academic Editor

PONE-D-19-27984

Title:

Signal changes in T2-weighted MRI of liver metastases under Bevacizumab

‒ A practical imaging biomarker ? ‒

Comment AE 1: “Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process."

Authors’ Response: Thank you very much for your overall appreciation of our manuscript. We hope to have addressed all issues raised by you and the reviewers. Moreover, we agree with you and hope to find the right and interested readership in the journal PLOS ONE and keen on publishing this manuscript in the journal.

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Authors’ Response: We apologize for not fulfilling the requirements of PLOS ONE style. We thoroughly revised the entire manuscript and hope to sufficiently address all formal requirements.

Comment AE 3: “2. We note you have included a table to which you do not refer in the text of your manuscript. Please ensure that you refer to Table 2 and in your text; if accepted, production will need this reference to link the reader to the Table.”

Authors’ Response: We apologize for this lack of clear presentation. The editor is right in that the table 2 was missed to mentioned in the text. Due to the response to the reviewers another table was inserted and table 3 is now mentioned in the text, as requested.

Comment AE 4: “3. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.”

Authors’ Response: Captions for the Supporting Information files have been added.

 

References:

1. Brufau BP, Cerqueda CS, Villalba LB, Izquierdo RS, González BM, Molina CN. Metastatic renal cell carcinoma: radiologic findings and assessment of response to targeted antiangiogenic therapy by using multidetector CT. Radiographics. 2013;33(6):1691-716.

2. Krajewski KM, Nishino M, Franchetti Y, Ramaiya NH, Abbeele AD, Choueiri TK. Intraobserver and interobserver variability in computed tomography size and attenuation measurements in patients with renal cell carcinoma receiving antiangiogenic therapy: implications for alternative response criteria. Cancer. 2014;120(5):711-21.

3. Smith AD, Zhang X, Bryan J, Souza F, Roda M, Sirous R, et al. Vascular Tumor Burden as a New Quantitative CT Biomarker for Predicting Metastatic RCC Response to Antiangiogenic Therapy. Radiology. 2016;281(2):484-98.

4. Shindoh J, Loyer EM, Kopetz S, Boonsirikamchai P, Maru DM, Chun YS, et al. Optimal morphologic response to preoperative chemotherapy: an alternate outcome end point before resection of hepatic colorectal metastases. Journal of Clinical Oncology. 2012;30(36):4566.

5. Chun YS, Vauthey J-N, Boonsirikamchai P, Maru DM, Kopetz S, Palavecino M, et al. Association of computed tomography morphologic criteria with pathologic response and survival in patients treated with bevacizumab for colorectal liver metastases. Jama. 2009;302(21):2338-44.

Attachment

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Decision Letter 1

Michael C Burger

4 Mar 2020

Signal changes in T2-weighted MRI of liver metastases under Bevacizumab

‒ A practical imaging biomarker ? ‒

PONE-D-19-27984R1

Dear Dr. Thüring,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

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PLOS ONE

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Reviewer #1: All comments have been addressed

Reviewer #3: All comments have been addressed

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Reviewer #3: Partly

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Reviewer #3: Yes

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Reviewer #1: The authors have successfully responded the reviewer's comments. I think this manuscript to be worth publishing in the journal of Plos One.

Reviewer #3: It is a good job, well articulated and well written. The article has been much improved and I am delighted with the authors.

Unfortunately, however, I believe that the mistake of considering two such different pathologies as colon cancer and breast cancer is unacceptable.

If it is correct to say that bevacizumab is used in both diseases, but it is associated with very different drugs in colon cancer compared to breast cancer.

This is an insuperable conceptual mistake that would generate confusion in our readers.

My very friendly and respectful advice of the excellent work done is to re-submit the same work dedicated only to one tumor and perhaps increasing the number of cases that remains poor.

I am really very sorry to reject this very appreciable work but the conceptual and methodological mistake in evaluating in the same way such different metastases under the vascular profile cannot be published

**********

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Reviewer #1: No

Reviewer #3: Yes: Giammaria Fiorentini

Acceptance letter

Michael C Burger

16 Mar 2020

PONE-D-19-27984R1

Signal changes in T2-weighted MRI of liver metastases under Bevacizumab ‒ A practical imaging biomarker ? ‒

Dear Dr. Thüring:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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With kind regards,

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on behalf of

Dr. Michael C Burger

Academic Editor

PLOS ONE

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    Supplementary Materials

    S1 Data. Raw data of the patients with age, tumor type, and ROI-based measurements.

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    Click here for additional data file. (53.9KB, xlsx)
    Attachment

    Submitted filename: Response to Reviewers.docx

    Click here for additional data file. (46.5KB, docx)

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