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. 2023 Mar 3;18(3):e0278454. doi: 10.1371/journal.pone.0278454

Liver function indicators in patients with breast cancer before and after detection of hepatic metastases-a retrospective study

Carmen Leser 1,*, Georg Dorffner 2, Maximilian Marhold 3, Anemone Rutter 1, Mert Döger 1, Christian Singer 1, Deirdre Maria König-Castillo 4, Christine Deutschmann 1, Iris Holzer 1, Daniel König-Castillo 5, Daphne Gschwantler-Kaulich 1
Editor: Surinder K Batra6
PMCID: PMC9983906  PMID: 36867604

Abstract

Background

Liver metastases are common in patients with breast cancer, and determining the factors associated with such metastases may improve both their early detection and treatment. Given that liver function protein level changes in these patients have not been determined, the aim of our study was to investigate liver function protein level changes over time, spanning 6 months before the detection of liver metastasis to 12 months after.

Methods

We retrospectively studied 104 patients with hepatic metastasis from breast cancer who were treated at the Departments of Internal Medicine I and the Department of Obstetrics and Gynecology at the Medical University of Vienna between 1980 and 2019. Data were extracted from patient records.

Results

Aspartate aminotransferase, alanine aminotransferase, gamma-glutamyltransferase, lactate dehydrogenase and alkaline phosphatase were significantly elevated when compared to normal range 6 months before the detection of liver metastases (p<0.001) Albumin was decreased (p<0.001). The values of aspartate aminotransferase, gamma-glutamyltransferase, and lactate dehydrogenase were significantly increased at the time of diagnosis compared to 6 months prior (p<0.001). Patient- and tumor-specific parameters had no influence on these liver function indicators. Elevated aspartate aminotransferase (p = 0.002) and reduced albumin (p = 0.002) levels at the time of diagnosis were associated with shorter overall survival.

Conclusion

Liver function protein levels should be considered as potential indicators when screening for liver metastasis in patients with breast cancer. With the new treatment options available, it could lead to prolonged life.

Introduction

Breast cancer accounts for 30% of all malignancies in women with a mortality rate second only to that of lung cancer in the United States [1] and causes the highest number of cancer-related deaths among women globally [2]. While the prognosis is generally favorable owing to the advances in medical treatments achieved over recent decades, 20–30% of patients with breast cancer develop secondary metastases [3–5]. The incidence of metastases has been rising, and once it occurs, the patient’s prognosis deteriorates significantly; the 5-year survival rate drops from 80% to 23% [6–8].

The impact of metastases on survival signifies the importance of their early detection, as prompt intervention can improve patient outcomes. The liver is one of the most common sites of metastasis in patients with breast cancer; furthermore, the 5-year survival rate of patients who develop hepatic metastases is only 8.5%, which is one of the poorest [9, 10]. When performed early, surgical treatment of liver metastases has been shown to be effective in prolonging life expectancy [11, 12]; as such, the early detection of liver metastases is of high importance.

Previous studies have found that liver function is poor in 92% of all patients diagnosed with breast cancer liver metastasis (BCLM), with gamma-glutamyl transferase (GGT) and alkaline phosphatase (AP) showing the strongest positive correlations [13]. Cao et al. showed that stage III cancer or c-erbB-2-positivity are positively associated with liver metastases [14]. Moreover, alanine aminotransferase (ALT), aspartate aminotransferase (AST), GGT, AP, lactate dehydrogenase (LDH), and cancer antigen 15–3 levels are significantly higher in patients with BCLM than in those without [10, 11]. Thus, tumor marker and liver function tests combined may be helpful in screening parameters for BCLM. Furthermore, serum albumin and total bilirubin levels can help predict the survival of patients with liver metastases [14, 15].

The purpose of this study was to identify serum levels of liver function-associated proteins (via common screening tests) that would predict BCLM even before symptoms occur or metastatic lesion are detected. To the best of our knowledge, our study is the first to investigate liver protein levels not only at the time of BCLM diagnosis and the following months, but also prior to diagnosis. We aimed to investigate serum levels of these proteins during the timeframe of 6 months before diagnosis until 12 months after.

Methods

Patient selection

In this retrospective analysis of prospectively collected data, patients between the ages of 18 and 100 years who were treated for breast cancer at the Department of Gynecology and the Department of Internal Medicine I (Oncology) of the Medical University of Vienna between January 1980 and May 2019 were included.

Liver function protein levels measured 6 months prior to the diagnosis of BCLM, at the time of diagnosis, and 12 months after diagnosis were acquired. These included AST, ALT, GGT, LDH, AP, albumin, and bilirubin. Moreover, data on tumors including molecular subtype (luminal A, luminal B, HER2-positive, or triple negative), tumor type (invasive ductal carcinoma or invasive lobular carcinoma), and grading (I–III); age (years); body mass index (BMI in kg/m2); history of smoking (yes/no); history of alcohol consumption (yes/no); and whether any prescribed medications were potentially toxic to the liver (yes/no) were collected. Normal ranges of values were defined as our institution’s local standard (Table 1). The study was approved by a local ethics committee (number EK 1488/2019).

Table 1. Normal ranges of liver function proteins.

  Normal range
AST U/L <35
ALT U/L <35
GGT U/L <40
LDH U/L <250
AP U/L <105
Albumin g/L 35–52
Bilirubin mg/dL <1.2
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Normal ranges of liver function proteins as used in the University of Vienna.

AST, aspartate aminotransferase; ALT, alanine aminotransferase; GGT, gamma-glutamyl transferase; LDH, lactate dehydrogenase, AP, alkaline phosphatase.

Statistical analysis

To test elevated levels of liver function as measured 6 months prior to the diagnosis of BCLM, a chi-square test was used to compare the percentage of cases above the respective normal value with the expected 5% for one-sided ranges (or 2.5% for two-sided ranges, respectively) assuming that upper normality levels mark the 95th (or 97.5) percentile of a distribution of values considered to be normal. Values at the time of diagnosis and 12 months thereafter were compared to values 6 months prior to diagnosis using a Mann-Whitney U-test (owing to the skewed non-normal distribution of each variable). A significance level of 0.05 was corrected by the Bonferroni method given the number of 21 tests, yielding a significance level of α = 0.0024.

On a purely exploratory basis, dependencies of liver function protein levels on other variables were tested using either a correlation analysis (Pearson correlation for metrical variables, Spearman correlation for ordinal variables) or a Mann-Whitney U-test (for nominal variables), using a significance level of α = 0.05.

Results

Patient characteristics

Four hundred patients with metastases arising from breast cancer were screened; after 296 were excluded owing to the lack of liver metastases, 104 patients with BCLM were included in this study. Among them, 69 (66.3%) had serum liver protein data both 6 months before the diagnoses of BCLM and at the time of diagnosis. Furthermore, 74 patients (71.2%) had laboratory data acquired at the time of BCLM diagnosis and 12 months after, and 40 patients (38.5%) had laboratory results available for all 3 time points. The median BMI was 24.4 kg/m2 (18.0–42.9 kg/m2). Five patients were excluded due to a lack of data.

In terms of risk factors for elevated liver function proteins (Fig 1), 98 patients (94.2%) were taking at least one medication with hepatotoxic potential (on average, each patient was taking 4.3 medications with hepatotoxic potential simultaneously). Fourteen patients (13.5%) had pre-existing liver diseases such as liver cirrhosis, hepatomegaly, hepatitis, cholangitis, steatohepatitis, cholestasis, liver tissue damage, and/or Morbus Hodgkin. Smaller percentages of patients for whom data were available engaged in smoking and drinking habits.

Fig 1. Risk factors for elevated liver proteins.

Fig 1

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Of the total, 94.2% of patients were taking at least one type of medication with hepatotoxic potential; 13.5% had pre-existing liver diseases such as liver cirrhosis, hepatomegaly, hepatitis, cholangitis, steatohepatitis, cholestasis, liver tissue damage, and/or Morbus Hodgkin; 11% are used to daily alcohol consumption and 12% were daily smokers.

Tumor characteristics

The average age of patients at time of breast cancer diagnosis was 51.4 years (range, 26–82 years). The average time between initial diagnosis of breast cancer and the detection of BCLM in the cohort was 5.2 years.

Ninety-seven patients (93.3%) had immunohistochemistry data of the metastasis available: 15 (15.7%) had luminal A, 30 (30.9%) luminal B, 32 (33.0%) HER-2 positive, and 20 (20.6%) triple-negative molecular subtypes. The immunohistochemistry results of seven patients were unknown.

Ninety-nine patients (95.2%) had tumor types that were histologically verified. Invasive ductal carcinoma was present in 80.8% of the tumors (n = 80), whereas invasive lobular carcinoma was detected in 17.2% (n = 17). Mixed carcinoma was present in two (2.0%) of the cases. Eighty-four of the patients (80.8%) had metastases in at least one other organ. The most common site of metastasis was bone (n = 65; 77.4% of the 84 with metastases in other organs), followed by lung (n = 40; 47.6%), brain (n = 15; 17.9%), skin (n = 12; 14.3%), peritoneum (n = 4; 4.8%), and ovary (n = 1; 1.2%). The remaining 20 patients (19.2%) had liver metastases only. Thirty-nine patients (37.5% of all patients) had metastases in at least two organs other than the liver. The median largest intrahepatic lesion was 20 mm (mean = 28.9 mm). Solitary lesions were found in 14 patients (14.1%): 34 (34.3%) had 2–5 metastases, 11 (11.1%) had 6–10 metastases, and 40 (40.4%) exhibited a miliary pattern.

Liver function indicator values

Liver function test results are summarized in Fig 2.

Fig 2. Mean liver function protein level changes over time.

Fig 2

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Mean liver function protein level changes over time. The concentrations (Y-axis) of each enzyme, including AST (A), ALT (B), GGT (C), LDH (D), AP (E), and proteins, including Albumin (F), and Bilirubin (G), are plotted as bar graph, showing values 6-month prior, at the time of diagnosis, and 12-month post-diagnosis, represented on X-axis. The light grey belt shows normal ranges of protein level. Statistical analysis was performed using SPSS software and the p-values are represented as significant with a level of 0.05. After corrected by the Bonferroni method given the number of 21 tests, yielding a significance level of α = 0.0024.

AST. Six months before the diagnosis of BCLM, 19 out of 68 patients (27.9%) had elevated AST levels, which was significantly more than the 5% initially expected (p<0.001). Among them, the median AST level was 26.0 U/L (mean 34.2 U/L). At the time of diagnosis with BCLM, AST levels were significantly higher than 6 months before with a median of 36.0 U/L (mean 65.4 U/L; p<0.001). Twelve months after diagnosis, AST levels remained higher when compared to six months prior to diagnosis, with a median of 35 U/L (mean 77.7 U/L). This difference was not significant after correction (p = 0.004).

The number of liver metastases (encoded by the four ordinal levels reported above) was positively correlated with AST levels at the time of BCLM diagnosis (Spearman r = 0.525, p<0.001). Moreover, we found that mortality was dependent on the AST level, as patients who died during the period of observation (12 months after diagnosis of BCLM) had significantly higher AST levels at the time of diagnosis than those who survived the study period (p = 0.002) (Fig 3).

Fig 3. Correlation between aspartate aminotransferase levels at the time of diagnosis with breast cancer liver metastasis and its dependency on survival.

Fig 3

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Patients who died during the period of observation (12 months after diagnosis of breast cancer associated liver metastasis) had significantly higher aspartate aminotransferase levels at the time of diagnosis, than those who survived the study period (p = 0.002).

No significant correlations or dependencies were found between increased AST levels and different molecular subtypes, tumor types, grading, alcohol consumption, smoking, BMI, age at initial diagnosis of breast cancer, or age at the time of BCLM diagnosis.

ALT. Six months before the diagnosis of BCLM, the median ALT level was 22 U/L (mean 32.8 U/L). Nineteen out of 69 patients (27.5%) had levels above normal range, again significantly more than the expected 5% (p<0.001). At the time of diagnosis the median ALT level was 30 U/L (mean 47.4 U/L), which was found to not be significantly different from the timepoint before diagnosis of BCLM after correction (p = 0.006). Twelve months after diagnosis, ALT was found to exhibit a median of 28 U/L (mean 41.3 U/L), which was above levels from 6 months prior to diagnosis (p = 0.083).

Similar to AST, a positive correlation between the number of metastatic lesions and ALT level at the time of diagnosis was found (Spearman r = 0.418, p<0.001).

GGT. Six months before diagnosis of BCLM, GGT levels were elevated in 29 out of 69 patients (42.0%), with a median of 36.0 U/L (mean, 70.3 U/L). This was significantly higher than the expected 5% in a healthy population (p < 0.001). At the time of BCLM diagnosis, GGT levels showed a median of 53 U/L (mean 251.9 U/L) and were above the normal range in 63.1% of all patients. After 12 months, the mean GGT was found to be lower with a median of 83 U/L (mean 232.7 U/L). At both time points levels were significantly higher than 6 months prior to diagnosis (p<0.001).

GGT levels at the time of diagnosis were significantly correlated with the number of metastatic lesions (Spearman r = 0.494, p<0.001).

LDH. With a median of 198.0 U/L (mean 261.2 U/L) 6 months prior to BCLM, LDH levels were above normal range in 16 out of 58 patients (27.6%), which is significantly more than the expected 5%. At the time of diagnosis of BCLM, a median LDH level of 266 U/L (mean 398.0 U/L) was found. A median value of 237.5 (mean 529.5 U/L) for the timepoint after 12 months was determined. Levels at the time of diagnosis were significantly higher than 6 months prior to diagnosis (p<0.001), but not 12 months after diagnosis (p = 0.17).

LDH levels at the time of diagnosis were significantly correlated with the number of metastatic lesions (Spearman r = 0.268, p = 0.012).

Albumin. Six months prior to BCLM diagnosis, albumin levels were below normal range in 8 out of 48 cases (16,7%), which was significantly higher than the expected 2.5%, with a median level of 39.6 U/L (mean 39.2 U/L). The median and mean levels at the time of diagnosis and 12 months thereafter remained similar but decreased (39.6 and 38.7, as well as 36.45 and 35.8, respectively), whereas the percentage of patients below the normal range increased to 24.7% and 40.3%, respectively. The decrease after 12 months was not significant after correction (p = 0.015) when compared to 6 months prior to diagnosis.

Patients who survived less than 12 months after being diagnosed with BCLM had significantly lower levels of albumin at the time of diagnosis than patients with an overall survival of >12 months (p = 0.002).

AP. AP levels were above normal range in 27 out of 68 patients (39.7%) 6 months prior to BCLM diagnosis, which was significantly more than the expected 5% in a normal population (p<0.001). It showed a median of 93 U/L (mean 111.9 U/L). The levels exhibited had a median of 113 U/L (mean 193,7 U/L) and 116 U/L (mean 217.4 U/L) at the time of diagnosis and 12 months thereafter, respectively. Differences between timepoints were not significant after correction (p = 0.17 and p = 0.008, respectively).

AP levels at the time of diagnosis were significantly correlated with the number of metastatic lesions (Spearman r = 0.405, p<0.001).

Bilirubin. Five out of 68 patients (7.4%) had bilirubin levels above the normal range, which was found to not be significantly different from the expected 5% threshold (p = 0.373). The percentage of patients exhibiting elevated bilirubin levels was shown to be significantly higher (12.9%; 22 out of 89, p<0.001) at the time of diagnosis, and 12 months after diagnosis (19.2%; 14 out of 73, p<0.001), while medians (0.47 U/L 6 months prior, 0.47 U/L at time of diagnosis, 0.49 U/L 12 months thereafter) and means (0.6, 0.9, 1.6 U/L, respectively) did not increase significantly (p = 0.582 and p = 0.012, respectively).

Bilirubin levels at the time of diagnosis were significantly correlated with the number of metastatic lesions (Spearman r = 0.255, p = 0.012).

Discussion

Our analysis revealed that 40.2% of patients exhibited a significant elevation in GGT levels 6 months prior to diagnosis. The level increased at the time of BCLM and correlated with the number of metastases present. BCLM and increased GGT and AP shows increased biliary injury.

GGT is the most sensitive liver enzyme, even though its fluctuations lack specificity [16]. Additionally, nonalcoholic fatty liver disease (NAFLD) and cholestasis, both of which are possible causes of increased GGT values, share some of the same risk factors as breast cancer [17]. Interestingly, a case-control study of 540 patients by Kwak et al. found that NAFLD was significantly associated with breast cancer in non-obese women; this might be one explanation for the elevated GGT levels observed in our study and is therefore a confounder.

Even though it is limited to a single organ, liver metastasis indicates poor prognosis [18].

Cao et al. reported the median survival time with liver metastasis to be 11.2 months [14]. Furthermore, O’Reilly et al. showed a correlation between elevated AST levels and mortality [13], while Cheung et al. described the correlation of albumin levels with shorter survival time [19]. In addition to elevated AST, low albumin levels were significantly associated with earlier death in our study. Patients who survived for less than 12 months after being diagnosed with BCLM had significantly lower levels of albumin (p = 0.002).

The implication of the normal bilirubin at baseline, i.e. 6 months before BCLM diagnosis, despite all other LFTs reported here being elevated should be discussed. This observation is potentially due to hepatocellular damage rather than cholestasis.

Liver enzyme levels can be a good indicator of BCLM. Therefore, to improve medical treatment, with new therapy options like selective internal radiation therapy [20], and long-term prognosis, we recommend screening for liver metastases in women with breast cancer who have elevated liver enzyme levels.

The increasing elevation of AST, LDH, AP, and bilirubin, as well as the decreasing albumin levels 12 months after BCLM diagnosis could be because of disease recurrence or the hepatotoxic drugs given.

Limitations

Our study was limited owing to its small sample size. Hepatotoxic medications and pre-existing liver disease, such as hepatitis B infection [21], and NAFLD in patients is a potential confounder. The same is true for the patients with bone lesions which could elevate AP. We are planning a large-cohort prospective study in breast cancer patients with liver metastases, including AP isoenzyme levels to see how much AP is from bone tissue and how much from the liver. Nevertheless, this is the first study to investigate liver protein level changes before versus after detection of BCLM.

Conclusion

We recommend performing liver function tests in women with breast cancer as a screen for potential BCLM. Due to the new treatment options available, the survival time could increase. A prospective multicenter study should be conducted to verify our findings and to determine suitable intervals of checking liver function-associated protein levels during follow-up care.

Supporting information

S1 File. Additional information about mean, standard deviation, median and patients included for each time point and liver function protein.

(DOCX)

Click here for additional data file. (12.4KB, docx)

Data Availability

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

Funding Statement

The author(s) received no specific funding for this work.

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Surinder K Batra

20 Jul 2022

PONE-D-22-13541Liver function indicators in patients with breast cancer before and after detection of hepatic metastasesPLOS ONE

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The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In Liver function indicators in patients with breast cancer before and after detection of hepatic metastases, the authors set out to retrospectively assess LFTs, in particular proteins, as well as demographic and tumour characteristics over 18 months in breast cancer patients w/metastatic liver disease (BCLM). They documented values at 3 time points: 6 months before BCLM diagnosis, at BCLM diagnosis, and 12 months after BCLM diagnosis in 104 patients gathered over 39 years.

They found very significant (p<.001) elevations in AST, ALT, GGT, LDH and AP, and similarly significant (p<.001) decrease in albumin. These LFT abnormalities were independent of patient- or tumour-specific factors. Furthermore, AST and albumin levels were significantly (p <.002) associated with decreased OS

General Comments Given the prevalence of breast cancer and the poor clinical course once liver metastases have developed, this manuscript covers a highly relevant topic.

Specific Comments

1. Only 69 + 40= 109 patients had labs before and at time of dx. Yet your cohort is 104 rather than 109. Why were 5 of these patients excluded?

2. Hepatotoxic meds in the 98 patients is a potential confounder, i.e. if LFTs elevated, it could be either due to BCLM or DILI. The same is true for the 14 patients w/pre-existing liver disease.

3. In those w/BCLM and bone metastases, the bone lesions are also a confounder because they could elevate AP. Was the AP isoenzyme test done to see how much of the AP came from bone and how much from liver? This would be helpful if a prospective study is done to correlate elevated LFTs with BCLM.

4. Although you found a correlation between the number of liver metastases and the degree of elevation of the labs, unless the vast majority of your already small cohort were on the same liver-toxic drugs, the differences in lab values could be due to differences in drug toxicities rather than more advanced BCLM, i.e. greater number of liver metastases. A larger cohort is probably needed to untangle this data.

5. On page 7, under GGT, you refer to your cohort as a “healthy population.” That contradicts the data you reported above (bottom p 5 /top p 6) regarding the number of patients on hepatotoxic drugs (ca 94%), and the number of patients w/metastases involving at least one other organ (ca 80%). That could mean the cohort already had DILI and/or micrometastases to the liver in the months preceding BCLM diagnosis.

6. Again, on p 8, it should not be so surprising that the AP was elevated before BCLM diagnosis since you mentioned above that a majority of the cohort had bone metastases.

7. Please modify Table 2, subdividing each of the 3 columns into 4, then put the appropriate heading above the new sub columns, i.e. number of BCLM patients; mean lab value; what is the 3rd sub column, i.e. (22.3) under AST?; median lab value

8. From Table 2, how do you explain the increasing elevation of AST, LDH, AP, and bilirubin, as well as the decreasing albumin 12 months after BCLM diagnosis? Is this because the patients were not treated once BCLM diagnosed or were they treated and the rise represents disease recurrence or post-treatment effect from hepatotoxic drugs? At least speculate on the cause.

9. Please explain, in the Discussion, the implication of the normal bilirubin at baseline, i.e. 6 months before BCLM diagnosis, although all of the other LFTs reported here are elevated. In other words, what does this mean about the type of liver damage that the drugs may be causing? I think the damage is probably hepatocellular rather than cholestatic.

10. In the Discussion, please clarify why NAFLD was associated w/breast cancer? Is this due to underlying NAFLD or the effect of chemotherapy? This is important as NAFLD is then another confounder in LFT evaluation in these patients.

11. In the Abstract, “may improve both their early prediction and treatment,” should be “may improve both their early detection and treatment.” OR “may improve both their prediction and treatment.”

12. In Intro, first paragraph, “the incidence rate of metastasis” should be simply “the incidence of metastases”.

Reviewer #2: In the manuscript titled “Liver function indicators in patients with breast cancer before and after detection of hepatic metastases”, Leser et al. have reported a clinically relevant prognostic significance of liver functions in breast cancer liver metastasis. A retrospective temporal analysis of different liver enzymes Aspartate aminotransferase, alanine aminotransferase, gamma-glutamyltransferase, lactate dehydrogenase and alkaline phosphatase, and albumin protein are important parameters to predict liver metastasis in breast cancer patients. However, it is expected that the authors generate more figures from the data compiled in the tabular form for a better understanding of the results. In addition, the correlation plot must be shown if both aminotransferase and albumin correlate with the patient’s survival. Irrespective of significance, both univariate and multivariate analysis must be presented to find the significance and correlation of proposed prognostic liver function proteins/enzymes. Unfortunately, the current form of the manuscript is not acceptable. However, the following are comments that must be addressed so that the manuscript can be reconsidered for publication in PLOS One. Please see the following comments:

1. Add continuous line numbers in the manuscript so that comments can be addressed to the page and line number.

2. References cited do not cover the recent publications in the field. For example, references for statistical values presented in the introduction are old, including refs.1-4. Please include the most up-to-date information with recent citations.

3. It is not clear what the treatment was for the patients diagnosed with liver metastasis for 12 months post-diagnosis. Is there information available on whether these patients were considered for surgery or any other treatment modality?

4. Description of each enzyme and protein has been given in the results. However, authors are suggested to plot the values to make them clearer and more understandable.

5. Baseline value for each parameter in normal individuals can be merged in table 2, and data can be plotted in the form of bar graphs.

6. Figure legends are missing for figures given in the manuscript. Please provide the figure legends, including the statistical significance.

7. Font size and type are not consistent in eh manuscript and figures. Authors are suggested to revise the manuscript with the same font type and size as per the journal’s guidelines.

8. It has not been mentioned what the label on the Y-axis of figure 2 is. Please add the Y-axis details.

9. Discussion needs to be elaborated with overall more citations of recent work in the field.

10. Authors are suggested to explain why enzyme and protein levels went down after 12 months of diagnosis of BCLM, trending towards the values observed 6 months prior to the diagnosis of BCLM. Is this an effect of treatment in these patients? Were there patients where progressive liver metastases showed a positive correlation with elevated enzyme and protein levels?

11. Page 8, Line 8, the word prediction should be replaced by diagnosis.

12. Page 8, Is the statement “Departments of Internal Medicine I and Gynecology at..” correctly mentioned in the method section of the abstract.

13. Page 8: The sentence needs to be ended after the "…detection of liver metastases (p<0.001)”. Please add a dot in the result section of the abstract or connect the following sentence properly.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Shailendra K Gautam

**********

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PLoS One. 2023 Mar 3;18(3):e0278454. doi: 10.1371/journal.pone.0278454.r002

Author response to Decision Letter 0

26 Aug 2022

Dear Editor:

We/I wish to re-submit the manuscript titled “Liver function indicators in patients with breast cancer before and after detection of hepatic metastases.” The manuscript ID is PONE-D-22-13541.

We thank you and the reviewers for your thoughtful suggestions and insights. The manuscript has benefited from these insightful suggestions. I look forward to working with you and the reviewers to move this manuscript closer to publication in the PLoS ONE.

The manuscript has been rechecked and the necessary changes have been made in accordance with the reviewers’ suggestions. The responses to all comments have been prepared and attached herewith.

Thank you for your consideration. I look forward to hearing from you.

Sincerely,

Leser Carmen, MD, PhD

Address: Waehringer Guertel 18-20, 1090 Vienna, Austria

Phone: 0043/660/2040087

Fax: 0043/1/40400 23230

Email: carmen.leser@meduniwien.ac.at

Reviewer #1: In Liver function indicators in patients with breast cancer before and after detection of hepatic metastases, the authors set out to retrospectively assess LFTs, in particular proteins, as well as demographic and tumour characteristics over 18 months in breast cancer patients w/metastatic liver disease (BCLM). They documented values at 3 time points: 6 months before BCLM diagnosis, at BCLM diagnosis, and 12 months after BCLM diagnosis in 104 patients gathered over 39 years.

They found very significant (p<.001) elevations in AST, ALT, GGT, LDH and AP, and similarly significant (p<.001) decrease in albumin. These LFT abnormalities were independent of patient- or tumour-specific factors. Furthermore, AST and albumin levels were significantly (p <.002) associated with decreased OS

General Comments Given the prevalence of breast cancer and the poor clinical course once liver metastases have developed, this manuscript covers a highly relevant topic.

Specific Comments

1. Only 69 + 40= 109 patients had labs before and at time of dx. Yet your cohort is 104 rather than 109. Why were 5 of these patients excluded?

Response: Five patients were excluded due to a lack of data.

2. Hepatotoxic meds in the 98 patients is a potential confounder, i.e. if LFTs elevated, it could be either due to BCLM or DILI. The same is true for the 14 patients w/pre-existing liver disease. Response: This has been mentioned in the limitations.

3. In those w/BCLM and bone metastases, the bone lesions are also a confounder because they could elevate AP. Was the AP isoenzyme test done to see how much of the AP came from bone and how much from liver? This would be helpful if a prospective study is done to correlate elevated LFTs with BCLM.

Response: This is planned, and we have mentioned bone lesions as a confounder.

4. Although you found a correlation between the number of liver metastases and the degree of elevation of the labs, unless the vast majority of your already small cohort were on the same liver-toxic drugs, the differences in lab values could be due to differences in drug toxicities rather than more advanced BCLM, i.e. greater number of liver metastases. A larger cohort is probably needed to untangle this data.

Response: A prospective study has been planned.

5. On page 7, under GGT, you refer to your cohort as a “healthy population.” That contradicts the data you reported above (bottom p 5 /top p 6) regarding the number of patients on hepatotoxic drugs (ca 94%), and the number of patients w/metastases involving at least one other organ (ca 80%). That could mean the cohort already had DILI and/or micrometastases to the liver in the months preceding BCLM diagnosis.

Response: Thank you for your comment. I meant that it was higher than in a healthy population, and of course this could be because of your annotations. I have rewritten it to improve clarity.

6. Again, on p 8, it should not be so surprising that the AP was elevated before BCLM diagnosis since you mentioned above that a majority of the cohort had bone metastases.

Response: I have rewritten it to improve clarity.

7. Please modify Table 2, subdividing each of the 3 columns into 4, then put the appropriate heading above the new sub columns, i.e. number of BCLM patients; mean lab value; what is the 3rd sub column, i.e. (22.3) under AST?; median lab value

Response: I have included these details in a figure.

8. From Table 2, how do you explain the increasing elevation of AST, LDH, AP, and bilirubin, as well as the decreasing albumin 12 months after BCLM diagnosis? Is this because the patients were not treated once BCLM diagnosed or were they treated and the rise represents disease recurrence or post-treatment effect from hepatotoxic drugs? At least speculate on the cause.

Response: Thank you for your comment. I have included this in the manuscript.

9. Please explain, in the Discussion, the implication of the normal bilirubin at baseline, i.e. 6 months before BCLM diagnosis, although all of the other LFTs reported here are elevated. In other words, what does this mean about the type of liver damage that the drugs may be causing? I think the damage is probably hepatocellular rather than cholestatic.

Response: Thank you for your comment. I have included this in the manuscript.

10. In the Discussion, please clarify why NAFLD was associated w/breast cancer? Is this due to underlying NAFLD or the effect of chemotherapy? This is important as NAFLD is then another confounder in LFT evaluation in these patients.

Response: We have yet to clarify the cause, and so we have included it as a confounder.

11. In the Abstract, “may improve both their early prediction and treatment,” should be “may improve both their early detection and treatment.” OR “may improve both their prediction and treatment.” –

Response: Thank you for your comment. I have changed this accordingly.

12. In Intro, first paragraph, “the incidence rate of metastasis” should be simply “the incidence of metastases”. –

Response: Thank you for your comment. I have changed this accordingly.

 

Reviewer #2: In the manuscript titled “Liver function indicators in patients with breast cancer before and after detection of hepatic metastases”, Leser et al. have reported a clinically relevant prognostic significance of liver functions in breast cancer liver metastasis. A retrospective temporal analysis of different liver enzymes Aspartate aminotransferase, alanine aminotransferase, gamma-glutamyltransferase, lactate dehydrogenase and alkaline phosphatase, and albumin protein are important parameters to predict liver metastasis in breast cancer patients. However, it is expected that the authors generate more figures from the data compiled in the tabular form for a better understanding of the results. In addition, the correlation plot must be shown if both aminotransferase and albumin correlate with the patient’s survival. Irrespective of significance, both univariate and multivariate analysis must be presented to find the significance and correlation of proposed prognostic liver function proteins/enzymes. Unfortunately, the current form of the manuscript is not acceptable. However, the following are comments that must be addressed so that the manuscript can be reconsidered for publication in PLOS One. Please see the following comments:

1. Add continuous line numbers in the manuscript so that comments can be addressed to the page and line number.

Response: Line numbers have been added to the manuscript.

2. References cited do not cover the recent publications in the field. For example, references for statistical values presented in the introduction are old, including refs.1-4. Please include the most up-to-date information with recent citations.

Response: Thank you for your comment. I have updated the references accordingly.

3. It is not clear what the treatment was for the patients diagnosed with liver metastasis for 12 months post-diagnosis. Is there information available on whether these patients were considered for surgery or any other treatment modality?

Response: None of them received surgery. They all remained on the medication-based therapy, but the medication which was given varied.

4. Description of each enzyme and protein has been given in the results. However, authors are suggested to plot the values to make them clearer and more understandable.

Response: We request to not make this change because we believe that these revisions will not help to better understand the data. However, we have included the results in Table 2.

5. Baseline value for each parameter in normal individuals can be merged in table 2, and data can be plotted in the form of bar graphs.

Response: We have added a figure to make the results more understandable.

6. Figure legends are missing for figures given in the manuscript. Please provide the figure legends, including the statistical significance.

Response: Thank you for your comment. We have added the figure legends and included the statistical significance.

7. Font size and type are not consistent in eh manuscript and figures. Authors are suggested to revise the manuscript with the same font type and size as per the journal’s guidelines.

Response: The font has been changed to Times New Roman throughout the manuscript.

8. It has not been mentioned what the label on the Y-axis of figure 2 is. Please add the Y-axis details. Response: The Y-axis details have been included.

9. Discussion needs to be elaborated with overall more citations of recent work in the field. Response: I have tried to include more recent work, but there have been no recent publications in that field worth mentioning.

10. Authors are suggested to explain why enzyme and protein levels went down after 12 months of diagnosis of BCLM, trending towards the values observed 6 months prior to the diagnosis of BCLM. Is this an effect of treatment in these patients? Were there patients where progressive liver metastases showed a positive correlation with elevated enzyme and protein levels?

Response: These details have been included.

11. Page 8, Line 8, the word prediction should be replaced by diagnosis.

Response: Thank you for your comment. I have made this change.

12. Page 8, Is the statement “Departments of Internal Medicine I and Gynecology at..” correctly mentioned in the method section of the abstract.

Response: Thank you for your comment. I have made this change.

13. Page 8: The sentence needs to be ended after the "…detection of liver metastases (p<0.001)”. Please add a dot in the result section of the abstract or connect the following sentence properly. Response: I have included this accordingly.

Attachment

Submitted filename: Review_leber.docx

Click here for additional data file. (19.3KB, docx)

Decision Letter 1

Surinder K Batra

25 Oct 2022

PONE-D-22-13541R1Liver function indicators in patients with breast cancer before and after detection of hepatic metastasesPLOS ONE

Dear Dr. Leser,

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 minor points raised by the reviewers during the review process. Please submit your revised manuscript by Dec 09 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

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If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

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We look forward to receiving your revised manuscript.

Kind regards,

Surinder K. Batra

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

Reviewer #3: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: Overall, the manuscript looks good. Authors have revised the manuscript and addressed the comments and concerns satisfactorily. The revised manuscript can be considered for publication in the journal PLOS One. However, few more corrections need to be made to further improve the manuscript:

1. Each enzyme or protein plot can be relabeled in alfa-numeric format in the Figure 2 and accordingly it can be mentioned in the figure legend and result section. Authors are suggested to label each graph from 2A-2G.

2. Legend and result have been combined for figure 2, which must be separated (Line 162-179). The description given in the revised manuscript is fitting well as result. Authors are advised to write figure legend after labeling each graph, as suggested in comment 1. Moreover, Authors must consider revising the legends of each figure, as the purpose of legend is to describe the figure, its axes, dimensions, and statistics, not to explain the results. Please consider revising the legends following legend template given below for figure 2:

Figure 2: Mean liver function protein level changes over time. The concentrations (Y-axis) of each enzyme, including AST (A), ALT (B), GGT (C), LDH (D), AP (E), and proteins, including Albumin (F), and Bilirubin (G), are plotted as bar graph, showing values 6-month prior, at the time of diagnosis, and 12-month post-diagnosis, represented on X-axis. Statistical analysis was performed using………..software and the p-values are represented as…………….

Reviewer #3: This is a revision manuscript entitled “Liver function indicators in patients with breast cancer before and after detection of hepatic metastases” led by Carmen et al., and have significantly revised based on previous concerns raised by the reviewers. The manuscript is highly relevant topic given the poor clinical course of liver metastasis with breast cancer patients.

Minor comments to improve the manuscript.

a. Authors can consider revising their title to reflect that this is a retrospective

b. Also, BCLM and increased GGT and AP show that there increased biliary injury can be added in the manuscript. Further biliary injury has correlated with metastatic lesions has been observed in the manuscript

c. Table 2 is confusing; number of samples can be in a separate column. Also, a dotted plot would with individual data would be a great addition to enhance the clarity of the data presented

d. Authors can add the details of obesity or NAFLD as a confounding variable in their analysis, if they have the data otherwise mentioning them in the limitations is fine

e. Consider adding a subtitle of limitations before conclusion.

f. Figure 2 legends needs the details of sample number and statistical analysis

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: Yes: Shailendra K Gautam

Reviewer #3: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

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PLoS One. 2023 Mar 3;18(3):e0278454. doi: 10.1371/journal.pone.0278454.r004

Author response to Decision Letter 1

4 Nov 2022

We thank you and the reviewers for your thoughtful suggestions and insights. The manuscript has benefited from these insightful suggestions. I look forward to working with you and the reviewers to move this manuscript closer to publication in the PLoS ONE.

The manuscript has been rechecked and the necessary changes have been made in accordance with the reviewers’ suggestions. The responses to all comments have been prepared and attached herewith.

Attachment

Submitted filename: Review Leber 2.docx

Click here for additional data file. (13.7KB, docx)

Decision Letter 2

Surinder K Batra

17 Nov 2022

Liver function indicators in patients with breast cancer before and after detection of hepatic metastases - a retrospective study

PONE-D-22-13541R2

Dear Dr. Leser,

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

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Surinder K. Batra

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: All the comments have been addressed satisfactorily and the manuscript can be considered for publication.

Reviewer #3: Authors have satisfactorily improved their manuscript based on all three reviewers comments.

Manuscript has improved significantly

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: Yes: Shailendra K Gautam

Reviewer #3: No

**********

Acceptance letter

Surinder K Batra

15 Dec 2022

PONE-D-22-13541R2

Liver function indicators in patients with breast cancer before and after detection of hepatic metastases-a retrospective study

Dear Dr. Leser:

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Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Prof. Surinder K. Batra

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 File. Additional information about mean, standard deviation, median and patients included for each time point and liver function protein.

    (DOCX)

    Click here for additional data file. (12.4KB, docx)
    Attachment

    Submitted filename: Leser et al 2022.docx

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    Attachment

    Submitted filename: Review_leber.docx

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    Attachment

    Submitted filename: Revision comments PLOS One Leser et al.docx

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    Attachment

    Submitted filename: Review Leber 2.docx

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    Data Availability Statement

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

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