Lactate Dehydrogenase (LDH) Response to First-Line Treatment Predicts Survival in Metastatic Breast Cancer: First Clues for a Cost-Effective and Dynamic Biomarker

Background: Elevated plasmatic lactate dehydrogenase (LDH) levels are associated with worse prognosis in various malignancies, including metastatic breast cancer (MBC). Nevertheless, no data are available on the prognostic role of LDH as a dynamic biomarker during first-line treatment in unselected MBC. Methods: We reviewed data of 392 women with MBC to evaluate the association between LDH variation after 12 weeks of first-line treatment and survival. The prognostic impact was tested by multivariate Cox regression analysis. Results: Plasmatic LDH was confirmed as an independent prognostic factor in MBC. Patients who maintained elevated LDH levels after 12 weeks of first-line treatment experienced worse progression-free survival (PFS, HR 2.88, 95% CI: 1.40–5.89, p = 0.0038) and overall survival (OS, HR 2.61, 95% CI 1.16–5.86, p = 0.02) compared to patients with stable normal LDH levels, even after adjustment for other prognostic factors. Notably, LDH low-to-high variation emerged as an unfavorable prognostic factor for PFS (HR 3.96, 95% CI 2.00–7.82, p = 0.0001). Conclusions: Plasmatic LDH and its variation during first-line treatment predict PFS and OS in MBC, providing independent prognostic information. It would be worthwhile to prospectively evaluate the association between LDH variation and therapeutic benefit in MBC, and explore how it may affect treatment strategies.


Introduction
Breast cancer (BC) is the most common cancer among women and the second leading cause of cancer-related death [1]. About 6% of all breast tumors present with distant metastases at diagnosis, and 30% of patients with early BC will experience local or distant recurrence [2]. BC is a heterogeneous disease, including distinct subgroups with different prognosis based on histological and molecular

Patient's Characteristics
A consecutive series of 392 women with MBC were included in the analysis, 219 with a plasmatic LDH evaluation at baseline. The median age was 62 years (range 29-88), with 42.9% of patients older than 65 years and 10.7% younger than 45 years. Invasive ductal carcinoma was the most common histology (80.4% of cases), and post-menopausal women accounted for 59.4% of patients. Approximately 60.5% of patients had HR-positive tumors (11.2% were luminal A, 38.3% luminal B, and 11.0% luminal HER2-positive; see Section 4.2. for classification details), 8.7% had HR-negative/HER2-positive disease, and 9.4% TNBC. At MBC diagnosis, nearly half of the patients presented with a single metastatic site, and about 20% had three or more localizations. Bone metastases were detected in half of the cases (20% of patients had a bone-only disease), while patients with liver, lung, or central nervous system localizations (CNS) were about 25%, 28%, and 6.4%, respectively. Overall, nearly 60% of patients received chemotherapy as first-line treatment, and the remaining 40% received hormonal therapy. Additional baseline clinical and pathologic characteristics of patients are listed in Table 1.  (Figure 1). The prognostic role of LDH plasma levels was also confirmed when evaluated as a continuous variable for both PFS (p = 0.0002) and OS (p < 0.0001). These findings were confirmed for both PFS (HR 1.51, 95% CI: 1.02-2.26, p = 0.039) and OS (HR 1.64, 95% CI: 1.05-2.55, p = 0.027) after multivariate adjustment for molecular profiles, Eastern Cooperative Oncology Group Performance Status (ECOG PS), baseline ALP level, number of metastatic sites, central nervous system (CNS), and liver and bone localizations (Tables 2 and 3).  These findings were confirmed for both PFS (HR 1.51, 95% CI: 1.02-2.26, p = 0.039) and OS (HR 1.64, 95% CI: 1.05-2.55, p = 0.027) after multivariate adjustment for molecular profiles, Eastern Cooperative Oncology Group Performance Status (ECOG PS), baseline ALP level, number of metastatic sites, central nervous system (CNS), and liver and bone localizations (Tables 2 and 3).  The role of LDH as an adverse prognostic factor was consistent in all examined subgroups: Age, profile, number of metastatic sites, type of first-line treatment (hormonal therapy or chemotherapy), baseline ALP level, and liver and bone involvement (    LDH value after 12 weeks of first-line treatment was available in 126 patients (32%). Among them, 54.7% had stable low LDH levels, 15.0% had stable high levels, and in approximately 30% of cases, LDH levels changed over time across the upper normal limit (12% had a drop under the upper normal limit, while 18.2% had a rise over the upper normal limit).
According to plasmatic LDH variation, we were able to detect significant differences of both median PFS (stable low levels: 18.71 months, high-to-low levels: 10.92 months, low-to-high levels: 5.13 months, stable high levels: 4.27 months, p < 0.0001) and median OS (stable low levels: 54.64 months, high-to-low levels: 30.87 months, low-to-high levels: 29.49 months, stable high levels: 14.83 months, p < 0.0001) (Figure 3 and Table 4).

Prognostic Role of Plasmatic LDH Response during First-Line Treatment.
LDH value after 12 weeks of first-line treatment was available in 126 patients (32%). Among them, 54.7% had stable low LDH levels, 15.0% had stable high levels, and in approximately 30% of cases, LDH levels changed over time across the upper normal limit (12% had a drop under the upper normal limit, while 18.2% had a rise over the upper normal limit).
According to plasmatic LDH variation, we were able to detect significant differences of both median PFS (stable low levels: 18.71 months, high-to-low levels: 10.92 months, low-to-high levels: 5.13 months, stable high levels: 4.27 months, p < 0.0001) and median OS (stable low levels: 54.64 months, high-to-low levels: 30.87 months, low-to-high levels: 29.49 months, stable high levels: 14.83 months, p < 0.0001) (Figure 3 and Table 4).   The prognostic relevance of LDH response to first-line treatment was then assessed using a Cox regression multivariate model. Stable elevated LDH levels after 12 weeks of first-line treatment was confirmed as an independent negative prognostic factor for both PFS (HR 2.88, 95% CI: 1.40-5.89, p = 0.0038) and OS (HR 2.61, 95% CI 1. 16-5.86, p = 0.02) after multivariate adjustment for molecular profile, ECOG PS, number of metastatic sites, CNS, liver, bone localizations, and plasmatic ALP variation at 12 weeks. Moreover, a rise in plasmatic LDH levels after 12 weeks of first-line treatment (low-to-high variation) also emerged by multivariate analysis as an independent negative prognostic factor for PFS (HR 3.96, 95% CI 2.00-7.82, p = 0.0001) with a trend for worse OS (HR 2.02, 95% 0.89-4.56, p = 0.08). The complete Cox regression model is reported in Table 5.

Discussion
Many studies reported elevated plasmatic LDH levels to be associated with poor outcomes in various tumors [30]. A recent meta-analysis, including 76 studies conducted in patients with several cancer types, confirmed that high LDH plasmatic levels were associated with shorter PFS and OS [31]. Although the prognostic role of LDH in cancer is well-established, the underlying biological mechanisms are still unclear, and some possible explanations have been hypothesized. Firstly, high LDH plasmatic concentrations sustain anaerobic metabolism during tumor growth and metastatic spread, supporting the energetic requirements in hypoxic conditions [32]. Secondly, LDH exerts an inflammatory action on tumor microenvironment, activating interleukin (IL)-23 and IL-17 and modulating the activity of arginase I. It inhibits CD8+ T lymphocytes and natural killer (NK) activation, allowing cancer cells to evade immune response [33]. Moreover, high LDH levels promote tumor angiogenesis, cell migration, and metastatization by inhibiting the degradation of HIF-1 alpha and increasing the production of vascular endothelial growth factor (VEGF) [34]. Thirdly, preliminary evidence suggests that increased LDHA expression and lactate overproduction might also play a role in drug resistance [35].
The present study investigated the prognostic impact of plasmatic LDH levels on survival outcomes in MBC patients at first-line treatment.
Approximately 31% of evaluated patients had high baseline LDH levels and about 32% had an LDH variation during first-line treatment. In particular, 15% of patients had a stable high LDH and 18% had a low-to-high variation.
The results confirmed that elevated baseline LDH levels were independently associated with shorter PFS (6.87 vs. 13.12 months, adjusted HR 1.51, 95% CI: 1.02-2.26, p = 0.039) and OS (19.23 vs. 46.19 months, adjusted HR 1.64, 95% CI: 1.05-2.55, p = 0.027). These data were also confirmed when LDH plasma levels were evaluated as a continuous variable (PFS, OS), so our results were not dependent on the pre-specified cut-off for normal LDH plasmatic concentrations. To the best of our knowledge, this is the first study to demonstrate that LDH changes during first-line treatment significantly impact both PFS and OS in unselected MBC patients. Specifically, patients with elevated baseline plasmatic LDH who maintained high LDH levels after 12 weeks of first-line treatment experienced worse PFS and OS compared to patients with stable normal LDH levels, even after adjustment for other prognostic factors (HR 2.88, 95% CI: 1.40-5.89, p = 0.0038 and HR 2.61, 95% CI 1. 16-5.86, p = 0.02 for OS and PFS, respectively). Interestingly, since elevated plasmatic LDH levels may also reflect the presence of high tumor burden, bone localizations, liver metastases, and ALP levels variations, it is noteworthy that their prognostic value was maintained after including these covariates in the multivariate Cox regression model.
Additionally, plasmatic LDH elevation during first-line treatment emerged as an independent prognostic factor for PFS (HR 3.96, 95% CI 2.00-7.82, p = 0.0001) with a trend for OS (HR 2.02, 95% 0.89-4.56, p = 0.08). In accordance with our findings, a recent study conducted in TNBC patients confirmed that LDH changes after two cycles of first-line chemotherapy correlate with objective response rate and PFS [36].
Therefore, LDH can predict survival in patients with MBC and provides independent and dynamic prognostic information during first-line treatment. Given our results, patients with stable high LDH levels or with LDH elevation during first-line therapy may be monitored more frequently for disease progression, as they might experience shorter PFS. Conversely, patients with stable normal LDH levels will experience prolonged PFS and OS. Nevertheless, since these findings are not prospectively validated, LDH variation must not be considered an indirect proof of tumor progression or response, even if it offers additional prognostic information.
In our study, LDH-A tissue expression was not tested. However, its relationship with plasmatic LDH may be useful to define whether LDH plasmatic elevation is primarily tumor-related or not, exploring the biological significance and the prognostic value of their concordance or discordance. According to previous studies, elevated tissue LDH-A expression is associated with elevated Ki-67, high proliferation rates, and CNS metastases in TNBC [37].
The main strength of our study is the identification of a dynamic, easy-to-use, inexpensive, and reproducible prognostic biomarker in patients with unselected MBC. However, this is a retrospective and single-center study. Thus, prospective and external validation is mandatory. Moreover, the LDH cut-off value for normality implemented in this study may differ in other centers; consequently, its reproducibility has to be confirmed. Lastly, we did not consider the potential interaction of several other non-neoplastic diseases (e.g., heart failure, anemia, hypothyroidism, autoimmune, and lung disorders), which might influence plasmatic LDH levels.
On the basis of these observations, it would be of great value to prospectively evaluate the potential correlation between LDH variation and response to treatment in MBC, and explore the prognostic role of this long-standing biomarker in the modern era of immunotherapy and targeted therapy.

Study Design
This observational, retrospective, no-profit, monocentric cohort study examined data of 392 consecutive MBC patients treated between 2007 and 2017 at the Department of Oncology of the University Hospital of Udine (Italy). The study was conducted under the Declaration of Helsinki, and the Regional Ethics Committee approved the protocol (N • Protocol 14571 ratified in May 2018). Informed consent was obtained for the use of clinical data, rendered anonymous, for purposes of clinical research, epidemiology, training, and study of diseases.

Blood Sample Analysis
Serum LDH and ALP data were retrospectively evaluated. Blood samples data were eligible for review if performed within one month before first-line treatment administration (baseline pre-treatment sample) and after 12 weeks ± 1 week after first treatment dose (post-treatment sample). The quantitative determination of LDH and ALP was performed using the Roche Cobas 8000 c702 system (Roche Diagnostics, Indianapolis, IN, USA). The LDH and ALP cut-off value for normality was the normal upper limit (NUL) defined by the analytical system used (480 IU/L and 104 IU/L, respectively).

Statistical Analysis
The study was designed in order to explore the prognostic role of LDH response after 12 weeks of first-line treatment in unselected MBC, with a hierarchical design: The independent prognostic impact of plasmatic LDH was first evaluated at baseline and then for its variation at 12 weeks, using a multivariate Cox regression model for both PFS and OS with 95% confidence interval (95% CI). A two-sided p < 0.05 was considered statistically significant. The multivariate model included the following covariates: The molecular profile, ECOG PS, number of metastatic sites, CNS, liver and bone localizations, and plasmatic ALP levels (at baseline and its variation at 12 weeks). Baseline clinicopathological characteristics were summarized through descriptive analysis. OS was defined as the time elapsed between the start of first-line treatment and death or last follow-up. PFS was defined as the interval between the start of first-line treatment and disease progression or death for any cause. Differences in survival were tested by a log-rank test and represented by Kaplan-Meier survival curves. Statistical analysis was performed with STATA (StataCorp, www.stata.com (2015) Stata Statistical Software: Release 14.2. College Station, TX: StataCorp LP).

Conclusions
LDH is a routinely used biomarker with a well-established prognostic role in several solid tumors and hematological malignancies. Our study confirmed that LDH is an independent prognostic factor also in MBC and explored its value as a dynamic biomarker. To the best of our knowledge, this is the first study to demonstrate that LDH response to first-line treatment significantly impacts both PFS and OS in unselected MBC patients. If validated in prospective studies, LDH could represent a cost-effective biomarker to stratify patient's prognosis, monitor treatment efficacy, and to implement treatment strategies in MBC.
Author Contributions: G.P. contributed to the concept and design of the study, acquisition of data, statistical analysis, and interpretation of data. Furthermore, he contributed to the drafting and revision of the article and the final approval of the version to be published. L.G. contributed to the design of the study, to statistical analysis, and to interpretation of data. S.Z. and F.C. contributed to LDH and ALP analytical evaluation and revised the article. C.L., Michele Bartoletti, L.B., and S.B. contributed to the drafting and revision of the article. D.B., M.G.V., V.F., and M.C. contributed to acquisition of data and revised the article. M.B., M.M., and G.F. contributed to the revision of the article. A.M.M. and F.P. contributed to the concept and design of the study, interpretation of data, revision of the article, and the final approval of the version to be published.