Originally published as JCO Early Release 10.1200/JCO.2006.05.9501 on August 8 2006

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Lactate Dehydrogenase 5 Expression in Operable Colorectal Cancer: Strong Association With Survival and Activated Vascular Endothelial Growth Factor Pathway—A Report of the Tumour Angiogenesis Research Group

Michael I. Koukourakis, Alexandra Giatromanolaki, Efthimios Sivridis, Kevin C. Gatter, Adrian L. Harris

From the Departments of Pathology, and Radiotherapy/Oncology, Democritus University of Thrace, Alexandroupolis, Greece; Department of Pathology, Nuffield Department of Clinical Laboratory Sciences, John Radcliffe Hospital; Cancer Research UK, Molecular Oncology Laboratories, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, United Kingdom

Address reprint requests to Michael I. Koukourakis, MD, Tumor and Angiogenesis Research Group, PO BOX 12, Alexandroupolis 68100, Greece; e-mail: targ{at}her.forthnet.gr

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... Author Contributions GLOSSARY REFERENCES

 
Purpose: Lactate dehydrogenase 5 (LDH-5) regulates, under hypoxic conditions, the anaerobic transformation of pyruvate to lactate for energy acquisition. Several studies have shown that serum LDH may be an ominous prognostic marker in malignant tumors. The clinical significance of tissue LDH-5, however, remains largely unexplored.

Patients and Methods: We investigated the immunohistochemical expression of LDH-5 in a series of 128 stage II/III colorectal adenocarcinomas treated with surgery alone. In addition, markers of tumor hypoxia (hypoxia-inducible factor 1 alpha [HIF1]), angiogenesis (vascular endothelial growth factor [VEGF] and phosporylated kinase domain receptor [pKDR]/flk-1 receptor) and the tumor vascular density (CD31 positive standard vascular density [sVD] and pKDR positive activated vascular density [aVD]) were assessed.

Results: The expression of LDH-5, together with that of HIF1 and pKDR, was both nuclear and cytoplasmic. Assessment, with minimal interobserver variability, was achieved using a previously described scoring system. LDH-5 was significantly associated with HIF1 (P = .01), aVD (P = .001) and, particularly, with pKDR expression in cancer cells (P = .0001). Tissue LDH-5 expression was linked with elevated serum LDH levels, but serum levels failed to reflect tissue expression in 71% of LDH-5 positive cases. In univariate analysis tissue LDH-5 was associated with poor survival (P = .0003, HR 15.1), whereas in multivariate analysis this isoenzyme was the strongest independent prognostic factor (P = .0009). VEGF, pKDR, aVD, sVD and vascular invasion were all significantly related to unfavorable prognosis.

Conclusion: The immunohistochemical assessment of tissue LDH-5 and pKDR provides important prognostic information in operable colorectal cancer. The strong association between LDH-5 and pKDR expression would justify their use as surrogate markers to screen patients for tyrosin kinase inhibitor therapy.

	INTRODUCTION

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Colorectal cancer is a common malignant disease.¹ Patients with operable nonmetastatic disease are effectively treated with surgery, whereas adjuvant chemotherapy and radiotherapy confer a small, still important, benefit in terms of local control and overall survival.^2,3 The presence of positive lymph nodes is certainly one of the most important features defining prognosis and is the only indisputable criterion indicating the necessity of adjuvant chemotherapy.⁴ It is, however, disappointing that, despite chemotherapy, 40% of patients with stage III and approximately 20% of those with stage II disease will eventually relapse.^5,6

It is therefore imperative to evaluate biologic targets that would identify (i) patients curable by local therapy alone, and (ii) patients who would benefit from combining cytotoxic therapy with novel targeted therapies. The achieved prolongation of survival by the addition of bevacizumab, an anti–vascular endothelial growth factor (VEGF) monoclonal antibody, to chemotherapy in patients with metastatic colorectal cancer⁷ reinforced our hopes for a similarly successful application of antiangiogenic policies to the earlier stages of disease. The important benefit conferred by bevacizumab resulted in approval of the antibody for first-line chemotherapy without demanding the signature of an active VEGF pathway in tumors.

This phenomenon, however, should not discourage research to identify reliable predictors for the administration of targeted therapies, because such therapies should always be addressed to tumors bearing the target, just as in the case of antiher-2 monoclonal antibodies.^8,9 Nevertheless, the identification of the appropriate target becomes cumbersome especially when dealing with pharmaceutical inhibitors of growth factor receptors recognizing multiple targets on the same cell. The recent failure of two large randomized trials to show a clear overall benefit with the multi-VEGF tyrosine-kinase receptor inhibitor PTK787/ZK222584 (PTK/ZK),¹⁰ when added to fluorouracil/oxaliplatin chemotherapy, stressed the importance of such biomarkers suitable for identifying patients who would benefit from antiangiogenic therapies. In this later case, analysis of a subgroup with high levels of serum lactate dehydrogenase (LDH) showed that PTK/ZK administration led to a significant 40% reduction in the risk of disease progression (unpublished data), bringing forward an eventual role of LDH in angiogenesis-dependent tumor growth and progression.

In this study, we investigate the expression and prognostic relevance of LDH-5, a tumor-related LDH isoenzyme, in a series of patients with operable colorectal cancer, and we provide evidence of a link between tumor LDH, hypoxia and activated VEGF pathway.

	PATIENTS AND METHODS

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Formalin-fixed, paraffin-embedded tissues from 128 consecutive patients with colorectal adenocarcinoma treated with surgery alone were retrieved from the files of the Nuffield Department of Pathology (Oxford, United Kingdom). Sixty-three cases were staged as IIA (T3N0) and 65 as III (T3-N1,2) according to the TNM, American Joint Committee on Cancer/International Union Against Cancer staging system. None of the cases examined had direct extension of adjacent organs (T4 cases were excluded). Fifty of 128 cases had tumors with rectal location. Fifty-six of the cases were female and 72 male. The median age was 67 years (range, 41 to 88 years).

In addition, paraffin blocks from 20 patients with available preoperative LDH serum levels were drawn from the archives of the Department of Pathology, Democritus University of Thrace (Alexandroupolis, Greece). These were immunostained for LDH-5 to compare serum versus tissue LDH levels.

Immunohistochemistry
The expression of LDH-5, hypoxia-inducible factor 1 alpha (HIF1), vascular endothelial growth factor, and phosphorylated (activated) VEGF receptor KDR (pKDR) was assessed by immunohistochemistry on 3-µm tissue sections. The standard vascular density (sVD) was evaluated using immunohistochemistry for the CD31 panendothelial cell marker. Table 1 displays the primary antibodies used for detecting the expression of the various proteins.^11-15

Scoring the Cases
The percentage of cancer cells expressing the proteins under investigation was assessed in all optical fields at magnification x200. The percentage of positive cells per optical field was recorded, and the mean value of all fields was used to obtain the final score for each case. LDH-5, HIF1, and pKDR reactivity is both nuclear and cytoplasmic. The cytoplasmic and the nuclear expression of these proteins was first assessed separately and subsequently combined in a grading system (Table 2), as previously proposed and tested.^16,17,19 Nonetheless, a separate nuclear and cytoplasmic scoring was also used in this series for continuous variable analysis. The pattern of staining was cytoplasmic for VEGF so that the median value of the percentage of positive cancer cells present in the tissue sample was used to define groups of low versus high VEGF reactivity.

All staining scoring was perfomed separately by two independent observers who were blinded to the clinical outcome of patients. Any discrepancies were resolved on the conference microscope.

Assessment of Vascular Invasion
The presence of cancer cells within vascular channels of the primary tumor (inner tumor areas and tumor periphery) was detected in hematoxylin and eosin–stained sections.

Assessment of Necrosis
Areas of necrosis covering more than 10% of the tissue section examined was scored as "extensive," whereas all other cases were scored as being of "limited/no" necrosis.

Statistical Analysis
Statistical analysis was performed using the GraphPad Prism 4.0 and the Instat 3.1 package (GraphPad Software Inc, San Diego, CA). A Fisher's exact test was used for testing relationships between categoric variables (contingency tables) as appropriate. Linear regression analysis was used to assess correlation with continuous variables. Kappa statistics were used to assess the observer agreement on grouping cases. The Kaplan-Meier survival curves were used to assess the impact of various variables in the overall survival of patients. A Cox proportional hazards model was used to assess the effect of assessed parameters on death events. A P value of less than .05 was used for significance.

	RESULTS

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Results of Immunohistochemistry
The nuclear expression of LDH-5 ranged from 0% to 80% (median, 5%), whereas strong cytoplasmic expression was noted in 0% to 100% (median, 50%) of cells. The distribution of cases, according to the scoring system used is shown in Table 2. Ninty-nine (77.3%) of 128 of cases exhibited high LDH-5 reactivity. Typical immunohistochemical images reflecting the different patterns of LDH-5 expression are shown in Figure 1.

View larger version (83K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 1. Typical immunohistochemical images of LDH-5 expression patterns in colorectal cancer. (A, B) Weak expression (magnification a: x100, b: x200). (C, D) Strong cytoplasmic expression (magnification c: x100, d: x200). (E, F) Strong cytoplasmic and nuclear expression (magnification e: x100, f: x200). (G, H) Predominantly nuclear expression accompanied with weak cytoplasmic reactivity (magnification g: x100, h: x200).

The pKDR nuclear expression ranged from 0% to 60% (median, 0%), and the cytoplasmic from 0% to 100% (median, 50%). According to the grading system 82 (64%) of 128 of cases were grouped in the high-pKDR category.

Cytoplasmic VEGF expression ranged from 0% to 100% (median, 45%). Using this cutoff point, 64 (50%) of 128 cases showed high VEGF reactivity.

The sVD assessed with the anti-CD31 monoclonal antibody at the invading tumor edge ranged from 10 to 117 vessels per x200 optical field (median, 45 vessels). Using the median sVD, 62 (48.4%) of 128 of cases showed high sVD. The median density of vessels reactive to the anti-pKDR antibody at the invading tumor edge (aVD) ranged from three to 30 (median, 11.5). Using this cutoff point, 64 (50%) of 128 of cases had high aVD.

Interobserver variability assessed by Kappa statistics showed no important variation between observers for all the markers used (kappa coefficients between 0.79 to 0.89 and agreement range of 0.90 to 0.95).

Association of LDH-5 With Histopathologic Features
Table 3 shows the association of LDH-5 expression with histopathologic features. There was a significant association between LDH-5 overexpression and poor differentiation (P = .02).

View larger version (18K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 2. Linear regression analysis of lactate dehydrogenase 5 (LDH-5) and phosporylated kinase domain receptor (pKDR) expression status. (A) Nuclear expression; (B) cytoplasmic expression.

View larger version (15K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 3. Immunohistochemical expression of lactate dehydrogenase 5 (LDH-5) in association with LDH serum levels in 20 patients with colorectal adenocarcinoma.

View larger version (10K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 4. Kaplan-Meier overall survival curves in 128 patients with stage IIA and III colorectal adenocarcinomas. (A) All cases; (B) stage IIA patients; (C) stage III patients.

	DISCUSSION

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In an immunohistochemical study, we showed that LDH-1 is expressed consistently in normal and carcinomatous tissues.²¹ In contrast, LDH-5 is absent from normal epithelia but is extensively expressed in cancer cells.^22,23 LDH-5 is, therefore, the enzyme linked to tumor phenotype and the increased LDH serum levels noted in our cancer patients should probably reflect LDH-5 isoenzyme release, although direct clinical confirmation is lacking. In a study of non–small-cell lung cancer, the immunohistochemical expression of LDH-5 was directly linked with high total LDH serum levels,²⁴ although 50% of tumors with high tissue LDH-5 expression had normal serum levels. In fact, the exact mechanism of LDH release from cells is obscure, because lysis (necrosis) or stress-induced increased membrane permeability are the only known pathways.^25,26 In any case, removal of the primary tumor results in a rapid drop of the LDH serum levels within 1 week from surgery, suggesting that any excess in serum LDH is a tumor product in cancer patients.²⁴ High serum LDH has been linked with poor postoperative outcome and failure of radiotherapy and chemotherapy in sarcomas, lymphomas and carcinomas,^27-40 including colorectal cancer.^41-43 Indeed, in the large PTK/ZK randomized trials, serum LDH together with performance status was the most important prognostic feature in a control group of patients with metastatic colorectal cancer receiving chemotherapy (G. Meinhardt, personal communication, October 2005). Unlike serum LDH, the analysis of isoenzyme LDH expression by specific monoclonal antibodies in tumor tissues has been studied only recently.^21,24 It was thus shown that LDH-5 expression by cancer cells is associated with poor survival in operable non–small-cell lung carcinomas and is directly linked to the expression of HIF1.²⁴ In colorectal cancer, the expression of LDH-5 was linked with distant metastases and, again, with the expression of HIF1 and 2.^16,17 HIFs are oxygen-regulated transcription factors degraded by proteolysis via von Hippel-Lindau, and have been used as endogenous markers of hypoxia, because their concentration is increased under hypoxic conditions. Translocation into the nuclei leads to the transcription of a variety of genes related to erythropoiesis, angiogenesis, and glycolysis.⁴⁴ Indeed LDH-5, just like VEGF, is directly regulated by HIFs^45,46 which explains the immunohistochemical findings of the direct association between LDH-5 and HIFs in lung²⁴ and colorectal cancer.^16,17 This finding was reproduced in the current study. The impressive association of LDH-5 expression with poor prognosis in operable colorectal cancer, as confirmed herein, could be explained, at least in part, by an important biologic pathway regulating LDH gene expression, namely hypoxia. On the other hand, lactate production by LDH-5 overexpressing cancer cells leads to acidification of the extracellular matrix triggering additional biologic events that facilitate tumor growth, invasion and metastasis.^47,48 An additional pathway has recently been described whereby lactate inhibits the prolyl hydroxylases responsible for targeting HIF for destruction, amplifying thus the expression of HIF.⁴⁹ The link between hypoxia and VEGF angiogenic pathway is well established. In fact, HIFs are directly regulating the expression of VEGF under hypoxic stimulation, in much the same way that HIFs regulate the expression of LDH-5 and glucose transporters.^45,46 LDH-5, being regulated by the same pathway as VEGF, is expected to reflect a subset of tumors with a high likelihood to bear an activated VEGF pathway. Indeed, in non–small-cell lung cancer, a direct, still weak, correlation of LDH-5 with VEGF was noted.²⁴ Certainly, VEGF expression is more frequent in LDH-5 expressing colorectal carcinomas.¹⁶ It should be stressed, however, that these proteins are also regulated through HIF-independent additional mechanisms, such as oncogenes.^50-52 The best way to assess the activated VEGF pathway in cancer and endothelial cells is to assess the complex VEGF/receptor on the cell membrane⁵³ or the phosphorylated form of KDR/flk-1 VEGF receptor.¹⁴ In this study, using an anti-pKDR monoclonal antibody,¹⁴ we were able to assess the pKDR expression in both colon cancer cells and tumor-associated vessels along the tumor periphery. pKDR was strongly expressed in the cytoplasm of cancer cells and nuclei in which it has been translocated.^14,54 An autocrine VEGF growth and antiapoptosis pathways are therefore postulated to function in colorectal cancer, which is in accordance with several experimental studies.^55-57 On the other hand, the activated pKDR vascular density could be assessed in the invading tumor front, in a similar way as we previously assessed the activated tumor vasculature using a hybrid monoclonal antibody to VEGF/KDR complex.^58,59 Both pKDR expression and pKDR-activated vascular density were independent prognostic factors in colorectal cancer, although less strong than LDH-5. An association between LDH-5 and pKDR expression in cancer cells and in tumor-associated vasculature was recorded in 128 colorectal carcinomas examined. In fact, 75% and 60% of tumors with high LDH-5 expression had high pKDR expression in cancer cells and in vessels, respectively. These percentages were clearly lower (30% and 23%, respectively), in tumors with low LDH-5 expression. Linear regression analysis confirmed that these two variables tended to increase in concert in cases analyzed. The herein-reported analysis of serum LDH versus tissue LDH-5 indicates a trend for the serum LDH levels to be elevated in patients with strong LDH-5 expression in tumors. However, 71% of patients with high LDH-5 tumor expression demonstrates serum levels within the normal limits. This is probably because the serum LDH levels for each individual patient before the development of the tumor is unknown, so that an important increase may still fall within the normal range of the general population. Assessment of LDH-5 serum levels would be an appropriate tumor marker for discriminating between highly and poorly expressing LDH-5 patients, but the above hypothesis should be tested in a large number of patients. In any case, assessing tissue LDH-5 and pKDR by immunohistochemistry is an easy and inexpensive procedure. Larger studies are required to confirm the important prognostic role of LDH-5 in colorectal cancer, as described herein. Whether these tissue markers can be used as surrogate markers for screening patients for tyrosine kinase inhibitor therapy remains to be proved. Nonetheless, experimental studies are necessary to unveil the biologic pathway linking LDH-5 with the activated pKDR status in cancer cells and tumor-associated vessels of colorectal adenocarcinomas to support the clinical links between tumor metabolism, angiogenesis and disease progression.

	Authors' Disclosures of Potential Conflicts of Interest

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Although all authors completed the disclosure declaration, the following authors or their immediate family members indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of the investigation. For a detailed description of the disclosure categories, or for more information about ASCO’s conflict of interest policy, please refer to the Author Disclosure Declaration and the Disclosures of Potential Conflicts of Interest section in Information for Contributors.

Authors Employment Leadership Consultant Stock Honoraria Research Funds Testimony Other Michael I. Koukourakis SCHERING DE (A)

Dollar Amount Codes (A) < $10,000 (B) $10,000-$99,999 (C) $100,000 (N/R) Not Required

	Author Contributions

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Conception and design: Michael I. Koukourakis, Adrian L. Harris Administrative support: Michael I. Koukourakis, Efthimios Sivridis Provision of study materials or patients: Alexandra Giatromanolaki, Efthimios Sivridis, Kevin C. Gatter Collection and assembly of data: Alexandra Giatromanolaki, Efthimios Sivridis Data analysis and interpretation: Michael I. Koukourakis, Alexandra Giatromanolaki, Adrian L. Harris Manuscript writing: Michael I. Koukourakis, Alexandra Giatromanolaki, Efthimios Sivridis, Kevin C. Gatter, Adrian L. Harris Final approval of manuscript: Michael I. Koukourakis, Alexandra Giatromanolaki, Efthimios Sivridis, Kevin C. Gatter, Adrian L. Harris

 

	GLOSSARY

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Angiogenesis:

The process involved in the generation of new blood vessels. While this is a normal process that naturally occurs and is controlled by "on" and "of" switches, blocking tumor angiogenesis (antiangiogenesis) disrupts the blood supply to tumors, thereby preventing tumor growth.

HIF (hypoxia-inducible factor):

HIF is a transcriptional factor that regulates the adaptive responses of mammalian cells to low oxygen (hypoxia). It is composed of HIF-1, which is upregulated in conditions of hypoxia, and HIF-1β (or, aryl hydrocarbon receptor nuclear translocators), which is expressed constitutively. Dimerization of HIF-1 with HIF-1β leads to transcription of genes such as VEGF and PDGF.

KDR/Flk-1:

Also known as VEGFR. VEGFRs are transmembrane tyrosine kinase receptors to which the VEGF ligand binds. VEGFR-1 (also called Flt-1) and VEGFR-2 (also called KDR/ Flk-1[murine homologue]) are expressed on endothelial cells, while VEGFR-3 (also called Flt-4) is expressed on cells of the lymphatic and vascular endothelium. VEGFR-2 is thought to be principally responsible for angiogenesis and for the proliferation of endothelial cells. Typically, most VEGFRs have seven extracellular immunoglobulin-like domains, responsible for VEGF binding, and an intracellular tyrosine kinase domain.

LDH (lactate dehydrogenase):

An enzyme found in the blood and other body tissues, and involved in energy production in cells. High levels of LDH in the blood may indicate tissue damage, cancer, or another disease.

PTK787/ZK222584:

A small molecule that inhibits the tyrosine kinase activity of VEFGR-1, VEGFR-2, VEGFR-3, and PDGFRs.

Pyruvate:

Pyruvate can be either converted to lactate under anaerobic conditions, or broken down to water and carbon dioxide in the presence of oxygen, generating large amounts of ATP.

VEGF (vascular endothelial growth factor):

VEGF is a cytokine that mediates numerous functions of endothelial cells including proliferation, migration, invasion, survival, and permeability. VEGF is also known as vascular permeability factor. VEGF naturally occurs as a glycoprotein and is critical for angiogenesis. Many tumors overexpress VEGF, which correlates to poor prognosis. VEGF-A, -B, -C, -D, and -E are members of the larger family of VEGF-related proteins.

	NOTES

 
published online ahead of print at www.jco.org on August 7, 2006.

Supported by the Tumour and Angiogenesis Research Group and Cancer Research UK.

Terms in blue are defined in the glossary, found at the end of this article and online at www.jco.org.

Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.

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Submitted January 30, 2006; accepted May 30, 2006.

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