|Ahead of print
|Correlation of salivary lactate dehydrogenase with histopathological findings and tumor staging of squamous cell carcinomas in the head and neck region
Paul Simon1, Arnadi Ramachandrayya Shivashankara2, Raees Tonse3, Thomas George1, Pratima Rao4, Suresh Rao5, Manjeshwar Shrinath Baliga6
1 MBBS Student, Father Muller Medical College, Mangalore, Karnataka, India
2 Department of Biochemistry, Father Muller Medical College, Mangalore, Karnataka, India
3 Radiation Oncology, Father Muller Medical College, Mangalore, Karnataka, India
4 Department of Orodental Pathology, Mangalore Institute of Oncology, Mangalore, Karnataka, India
5 Department of Radiation Oncology, Mangalore Institute of Oncology, Mangalore, Karnataka, India
6 Research Unit, Mangalore Institute of Oncology, Mangalore, Karnataka, India
Click here for correspondence address and email
|Date of Submission||08-Feb-2020|
|Date of Acceptance||19-Apr-2020|
Background: Lactate dehydrogenase (LDH) is an important enzyme and is recently being investigated in various orodental pathologies. The present study was carried out to investigate whether there is any correlation between LDH with the various histopathological gradings. Materials and Methods: Saliva was collected between 9 and 10 AM from patients of head and neck cancers (HNCs). Concomitantly, saliva was also collected from age matched healthy volunteers for comparing the results of the study population. Saliva collected from both the cohorts were processed and evaluated for LDH levels using standard kit. The data were analyzed using analysis of variance and with Bonferroni multiple comparison. A value of P < 0.05 was considered statistically significant. Results: The results indicate that when compared to the healthy individuals, a significant increase was observed in the salivary LDH of cancer patients (280.55 ± 17.48 vs. 353.58 ± 30.19; P < 0.001). A significant difference was also observed with differentiation, size, nodal status and metastasis (P < 0.0001). Conclusions: The results indicate that salivary LDH is helpful as an adjunct to the standard histopathological grading in the diagnosis and prognosis of HNCs.
Keywords: Head and neck cancers, lactate dehydrogenase, tumor-node-metastasis stage
|How to cite this URL:|
Simon P, Shivashankara AR, Tonse R, George T, Rao P, Rao S, Baliga MS. Correlation of salivary lactate dehydrogenase with histopathological findings and tumor staging of squamous cell carcinomas in the head and neck region. Hamdan Med J [Epub ahead of print] [cited 2021 Dec 7]. Available from: http://www.hamdanjournal.org/preprintarticle.asp?id=289736
| Introduction|| |
Recent data for the year 2012 indicate that, at a global level, the cancers of the head and neck (H and N) region are the 6th most common and account for an estimated 300,000 new cases and 145,000 deaths every year. Cancers of the H and N are a major health issue in India and accounts for the highest amount of cancer related deaths., From a histological perspective, majority of the oral cancers are squamous cell carcinomas and the tumor-node-metastasis (TNM) classification that ascertains the extent of the primary tumor (T), the absence or presence and extent of regional lymph node (N) and distant metastasis (M) is clinically important. The TNM histopathological grading helps the oncologist in deciding the most suitable treatment modalities.
In the recent past, attempts made have been towards developing novel markers that can provide additional information and saliva has been found to be useful in various malignancies. Lactate dehydrogenase (LDH), involved in catalyzing the reversible conversion of pyruvate and lactate in the penultimate step of glycolysis, is an important enzyme in mammals.,, Structurally, LDH is a tetramer consisting of two major subunits A and/or B, and exists in five isozyme forms: A4 (LDH-5), A3B1 (LDH-4), A2B2 (LDH-3), A1B3 (LDH-2) and B4 (LDH-1). In healthy condition, LDH is present in the cytoplasm of the cell, but is released into the extracellular environment upon injury, necrosis, hypoxia, hemolysis, and myocardial infarction in to the tissue.
LDH has been investigated extensively in neoplastic diseases and is reported to be elevated in many cancers.,,, The principal reason for this increase is that when compared to normal cells that rely mainly on mitochondrial oxidative phosphorylation to generate energy needs, most cancer cells are dependent on aerobic glycolysis for their energy needs. This biochemical phenomenon is known as oxidative glycolysis or “the Warburg effect” and studies have shown it to influence tumor initiation, tumor maintenance and progression in the process of carcinogenesis.
Studies in the recent past have shown salivary LDH to be important in various oral pathogenesis.,, The profile of salivary LDH has been reported to be divergent from that of plasma and these observations indicate that the oral milieu contribute to the majority of the total salivary LDH., To substantiate this, reports also indicate that the total salivary LDH is a result of combination of secretions from both major and minor salivary glands, fluids diffused through the oral epithelium and gingiva, material originating from gastrointestinal reflux., In lieu of these observations, salivary LDH is regarded to be a useful body fluid in studying various H and N pathologies., In the present study an attempt was made to ascertain correlation between TNM tumor staging and levels of LDH.
| Materials and Methods|| |
Place and subjects
The present study was carried out at Father Muller Medical College (FMMC), Mangalore, Karnataka, India. The inclusion and exclusion criteria are enlisted in [Table 1]. In addition to this age matched volunteers (N = 40; males 22 and females 18) comprising mostly of teaching faculty of the institute without any habits, were healthy and not affected by any chronic ailments like diabetes, hypertension or were on any medication for acute ailments (like infectious diseases for the past 4 weeks) had good oral health were included as a comparator cohort for the study group. The study was undertaken only after obtaining necessary permission from the (FMMC/IEC/877/2012) and was performed as stipulated by the Helsinki declaration on research with humans and Indian Council of Medical Research guidelines for Biomedical research on human participants.
|Table 1: Details on the criteria used for the selection of patients for the study|
Click here to view
The tumor stage was performed by senior pathologists in accordance to the standard guidelines prescribed in the tenets of the 7th edition of the classification of malignant tumors of the American Joint Committee on Cancer. The tumor grade was classified following the criteria suggested by WHO.
During the course of the study the investigators approached the H and N cancer (HNC) patients admitted for the proposed treatment (before initiation of the treatment). The investigator explained the objective of the study to both the patient and the attending caregiver in their mother tongue. A written informed consent was taken from the willing patient. Information regarding the patient's age and tumor details was collected from the patient file. Unstimulated saliva was collected between 9 and 10 AM as described in detail earlier., Every subject was asked to rinse the mouth with distilled water thoroughly to remove any food debris and then after 10 min, requested to salivate into a sterile plastic. The collected saliva was centrifuged at 3000 rpm for 10 min, and the supernatants were stored in cold refrigerator (−20°C).
Estimation of lactate dehydrogenase in saliva
The stored saliva was removed from cold refrigerator thawed and analyzed using appropriate blanks, controls and standards by using the ultraviolet, visible spectrophotometer (Shimadzu, Japan). The LDH assay was performed by the kinetic spectrophotometric method described by Demetriou et al., using the reagent kit of Roche diagnostics. The assay is based on LDH-catalyzed reduction of pyruvate with Nicotinamide adenine dinucleotide (NADH) to form nicotinamide adenine dinucleotide (NAD+). The rate of oxidation of NADH to NAD+ was measured as a decrease in absorbance at 340 nm and expressed in terms of units/mg protein. The quality control programme of Biorad was used to ensure accuracy and precision of LDH values.
The data were stratified based on the tumor size (T), node (N), and metastasis (M) status and significance of the difference of the values between the groups was evaluated by analysis of variance, Bonferroni multiple comparison using the IBM SPSS 23 statistical program (SPSS Inc., Chicago, IL., USA). A value of P < 0.05 was considered statistically significant.
| Results|| |
One hundred and four clinically diagnosed patients with squamous cell carcinomas in the H and N region were included in the study. The study had 24 females and 79 males, and the mean age of the patients was 54.6 ± 10.82. The tumor site, details (TNM) and differentiation are enlisted in [Table 2]. The biochemical analysis results indicate that when compared to the controls, there was a significant difference in the levels of LDH (280.55 ± 17.48 vs. 353.58 ± 30.19) cancer patients (P < 0.0001) [Table 3]. The data was stratified based on the site of tumor (oral, pharangeal), differentiation, tumor size (T), the nodal status (N) and metastasis and analysed for the LDH levels [Table 3] and [Figure 1]. The results showed that the levels of salivary LDH was dependent on the differentiation, tumour size and was significant [P < 0.0001; [Table 3]; [Figure 1].
|Table 3: Salivary lactate dehydrogenase levels in the squamous cell carcinomas diagnosed in the head and neck region|
Click here to view
|Figure 1: Correlation of salivary LDH with Tumor size (a), Nodal status (b), metastasis status (c) and Differentiation (d)|
Click here to view
| Discussion|| |
In the recent past, research on ascertaining the changes in the salivary constituents has been on a rise and this is principally because it is a useful body fluid and its collection is non-invasive., Salivary LDH has been investigated in oral pathogenesis and found to be of possible prognostic value in ailments pertaining to inflammatory reactions that lead to cell death and tissue breakdown. Cancer is an inflammatory disease and it was observed that when compared to the healthy controls, the levels of LDH was high in oral cancer patients and in agreement to earlier observations.,,, An increased level of LDH indicates increased glycolysis and possible high rate of cell division.,,,
The prominent result of this study was that when analysis of the LDH was based on the tumor size (T) a strong association was observed [Table 3] and [Figure 1]. When compared to the smaller tumors, the levels of LDH were higher in patients with large advanced tumors (T3 to T4) [Table 3] and [Figure 1]. As far as the authors are aware, this is the first time a study to analyze the role of tumor size on the salivary LDH has been performed. However there are reports that the LDH in serum was increased in people with various advanced cancers.,, Squamous cell carcinomas of H and N region are known to be highly hypoxic and are dependent on aerobic glycolysis to provide Adenosine triphosphate (ATP). The increased expression of LDH observed in people with big tumors indicates their glycolytic dependency, and suggest that patients with this type of tumor may be at high risk for metastasis and regrowth.,
In this study it was also observed that when compared to both well (P < 0.0001) and moderately (P < 0.0002) differentiated tumors, the levels of salivary LDH was high in the poorly/undifferentiated cells and are in agreement to the reports of D'Cruz and Pathiyil. Additionally, previous studies have also shown that poorly differentiated HNCs have high levels of LDH in blood, and together all these observations affirm that aerobic glyolysis may be the underlying cause for this increase.,, Tumor differentiation is an important prognostic marker in the histopathological grading and earlier studies have shown that poorly differentiated tumors generate more and larger nodal metastases., In our study a similar trend was observed and together all these observations conclusively suggest that poorly/undifferentiated tumors had more salivary LDH.,,
| Conclusions|| |
Our data strongly adds an important layer of information in concordance with the literature that salivary LDH is an important endpoint to ascertain orodental health. To the best of our knowledge, no previous studies have been reported observing the association between tumor size and salivary LDH with the squamous cell carcinomas grade and stage. However, larger and longer studies are needed to validate the data to be of clinical relevance as a possible prognostic marker.
This study which was conducted at Father Muller Medical College (FMMC), Mangalore, Karnataka, India and was approved by the institutional ethics committee (FMMC/IEC/877/2012).
Informed consent was taken from all the volunteers, after having been informed of the study details and provided with clarifications. All patients and participants have agreed to use their information anonymously for the purpose of this study in a written consent as per the regulation of FMMC ethics committee.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Gupta B, Johnson NW, Kumar N. Global epidemiology of head and neck cancers: A continuing challenge. Oncology 2016;91:13-23.
Mishra A, Meherotra R. Head and neck cancer: Global burden and regional trends in India. Asian Pac J Cancer Prev 2014;15:537-50.
Nagler RM, Lischinsky S, Diamond E, Klein I, Reznick AZ. New insights into salivary lactate dehydrogenase of human subjects. J Lab Clin Med 2001;137:363-9.
Gatenby RA, Gillies RJ. Why do cancers have high aerobic glycolysis? Nat Rev Cancer 2004;4:891-9.
Sun W, Zhang X, Ding X, Li H, Geng M, Xie Z, et al
. Lactate dehydrogenase B is associated with the response to neoadjuvant chemotherapy in oral squamous cell carcinoma. PLoS One 2015;10:e0125976.
Lokesh K, Jayanthi K. Lactate dehydrogenase: An enzymatic biomarker in oral health and disease. Int J Contem Dent Med Rev 2015;1:1-3.
Kayser G, Kassem A, Sienel W, Schulte-Uentrop L, Mattern D, Aumann K, et al
. Lactate-dehydrogenase 5 is overexpressed in non-small cell lung cancer and correlates with the expression of the transketolase-like protein 1. Diagn Pathol 2010;5:22.
Kolev Y, Uetake H, Takagi Y, Sugihara K. Lactate dehydrogenase-5 (LDH-5) expression in human gastric cancer: Association with hypoxia-inducible factor (HIF-1alpha) pathway, angiogenic factors production and poor prognosis. Ann Surg Oncol 2008;15:2336-44.
Koukourakis MI, Giatromanolaki A, Sivridis E, Bougioukas G, Didilis V, Gatter KC, et al
. Lactate dehydrogenase-5 (LDH-5) overexpression in non-small-cell lung cancer tissues is linked to tumour hypoxia, angiogenic factor production and poor prognosis. Br J Cancer 2003;89:877-85.
Koukourakis MI, Giatromanolaki A, Sivridis E, Gatter KC, Harris AL; Tumour Angiogenesis Research Group. 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. J Clin Oncol 2006;24:4301-8.
Vander Heiden MG, Cantley LC, Thompson CB. Understanding the Warburg effect: The metabolic requirements of cell proliferation. Science 2009;324:1029-33.
Fantin VR, St-Pierre J, Leder P. Attenuation of LDH-A expression uncovers a link between glycolysis, mitochondrial physiology, and tumor maintenance. Cancer Cell 2006;9:425-34.
Le A, Cooper CR, Gouw AM, Dinavahi R, Maitra A, Deck LM, et al
. Inhibition of lactate dehydrogenase A induces oxidative stress and inhibits tumor progression. Proc Natl Acad Sci U S A 2010;107:2037-42.
Todorovic T, Dozic I, Vicente-Barrero M, Ljuskovic B, Pejovic J, Marjanovic M, et al
. Salivary enzymes and periodontal disease. Med Oral Patol Oral Cir Bucal 2006;11:E115-9.
Edge SB, Compton CC. The American Joint Committee on Cancer: The 7th
edition of the AJCC cancer staging manual and the future of TNM. Ann Surg Oncol 2010;17:1471-4.
Demetriou JA, Drewes PA, Gin JB. Enzymes. In: Henry RJ, Cannon DC, Winkelman JW, editors. Clinical Chemistry: Principles and Techniques. 2nd
ed. Hagerstown, Maryland, USA: Haper and Row; 1974. p. 929-37.
Sanjay PR, Hallikeri K, Shivashankara AR. Evaluation of salivary sialic acid, total protein, and total sugar in oral cancer: A preliminary report. Indian J Dent Res 2008;19:288-91.
] [Full text]
Shpitzer T, Bahar G, Feinmesser R, Nagler RM. A comprehensive salivary analysis for oral cancer diagnosis. J Cancer Res Clin Oncol 2007;133:613-7.
Shetty SR, Chadha R, Babu S, Kumari S, Bhat S, Achalli S. Salivary lactate dehydrogenase levels in oral leukoplakia and oral squamous cell carcinoma: A biochemical and clinicopathological study. J Cancer Res Ther 2012;8 Suppl 1:S123-5.
Pereira T, Shetty S, Pereira S. Estimation of serum lactate dehydrogenase level in patients with oral premalignant lesions/conditions and oral squamous cell carcinoma: A clinicopathological study. J Cancer Res Ther 2015;11:78-82.
Bigler LR, Streckfus CF, Dubinsky WP. Salivary biomarkers for the detection of malignant tumors that are remote from the oral cavity. Clin Lab Med 2009;29:71-85.
Petrelli F, Cabiddu M, Coinu A, Borgonovo K, Ghilardi M, Lonati V, et al
. Prognostic role of lactate dehydrogenase in solid tumors: A systematic review and meta-analysis of 76 studies. Acta Oncol 2015;54:961-70.
Giatromanolaki A, Koukourakis MI, Sivridis E, O'Byrne K, Cox G, Thorpe PE, et al
. Coexpression of MUC1 glycoprotein with multiple angiogenic factors in non-small cell lung cancer suggests coactivation of angiogenic and migration pathways. Clin Cancer Res 2000;6:1917-21.
Mall JW, Schwenk W, Philipp AW, Meyer-Kipker C, Mall W, Muller J, et al
. Serum vascular endothelial growth factor levels correlate better with tumour stage in small cell lung cancer than albumin, neuron-specific enolase or lactate dehydrogenase. Respirology 2002;7:99-102.
Yüce K, Baykal C, Genç C, Al A, Ayhan A. Diagnostic and prognostic value of serum and peritoneal fluid lactate dehydrogenase in epithelial ovarian cancer. Eur J Gynaecol Oncol 2001;22:228-32.
Janot F, Klijanienko J, Russo A, Mamet JP, de Braud F, El-Naggar AK, et al
. Prognostic value of clinicopathological parameters in head and neck squamous cell carcinoma: A prospective analysis. Br J Cancer 1996;73:531-8.
D'Cruz AM, Pathiyil V. Histopathological differentiation of oral squamous cell carcinoma and salivary lactate dehydrogenase: A biochemical study. South Asian J Cancer 2015;4:58-60.
Roland NJ, Caslin AW, Nash J, Stell PM. Value of grading squamous cell carcinoma of the head and neck. Head Neck 1992;14:224-9.
Ross CD, Gomaa MA, Gillies E, Juengel R, Medina JE. Tumor grade, microvessel density, and activities of malate dehydrogenase, lactate dehydrogenase, and hexokinase in squamous cell carcinoma. Otolaryngol Head Neck Surg 2000;122:195-200.
Arnadi Ramachandrayya Shivashankara,
Department of Biochemistry, Father Muller Medical College, Kankanady, Mangalore, Karnataka
Manjeshwar Shrinath Baliga,
Research Unit, Mangalore Institute of Oncology, Pumpwell, Mangalore, Karnataka
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2], [Table 3]
| Article Access Statistics|
| Viewed||808 |
| PDF Downloaded||43 |