A biomarker based detection and characterization of carcinomas exploiting two fundamental biophysical mechanisms in mammalian cells

The immunohistochemical detection of biomarkers in tumor tissue-sections is an essential and powerful technique to determine the malignancy of the tumor and to stratify cancer patient treatment [1]. The success of such stratification strongly depends on the use and quality of biomarkers and their capacity to characterize tumors with regard to malignancy and therapy response. Some biomarkers have already been used for immunohistochemical characterization of tumors. For example, increased proliferation detected by Ki-67 in tumor cells allows a better characterization in terms of malignancy of tumors [2].

In order to establish biomarkers applicable to all tumor entities, biomarkers for two fundamental biophysical mechanisms in mammalian cells have been selected. Despite the extreme complexity of signaling processes within and between cells, only a few principle biophysical mechanisms are known to determine the existence and death of mammalian cells.

One important biophysical mechanism which determines the fate and death of a cell is the cleavage of nuclear DNA by endonucleases [3]. Inhibition of alkaline and acid endonucleases has been identified in tumor cells leading to the suppression of apoptosis [4]. The block of endonuclease activity was due to a factor present in tumor cells [4]. Caspase-activated endonucleases are inhibited by nuclear Akt counteracting apoptosis [5]. Therefore, inhibition of endonuclease (DNase) enzyme activity represents an important biophysical mechanism leading to transformation of healthy cells to tumor cells.

Another important, if not the most important biophysical mechanism of life is the way of energy release within cells. Multicellular organisms depend on energy release either by fermentation or by oxidative phosphorylation (OxPhos). Therefore, only two ways of energy release are possible [6]. While fermentation in eukaryotes is biochemically restricted to sugar metabolites, energy release by oxidation is possible with glucose as well as with amino acids and/or fatty acids [7]. Furthermore, the end product of fermentation (lactic acid) still contains most of the energy. Thus, with regard to energy release OxPhos is superior compared to fermentation. However, despite this, fermentation is the way of choice in cells harboring extremely important DNA like (cancer) stem and germ cells due to safety issues [8]. These cells use this way of energy release to inhibit radical induced DNA damages [8‐10], which would lead to DNA mutations in all cells produced by proliferation of stem and germ cells. Cells using OxPhos, which generates fast electrons leading to radical production and DNA damages, do have to pay the price for this efficient, but dangerous way of energy release–they get DNA damages due to radical production [8]. Since radical production is completely prevented by fermentation (substrate chain phosphorylation), stem and germ cells use this way of energy release. Moreover, since fermentation leads to the production of metabolites being able to neutralize (quenching) radicals (e.g. pyruvate, lactic acid), fermentation is also used in cells exposed to a high level of radical production by sun light (retinal cells) or high oxygen concentration (endothelial cells). During evolution of higher vertebrates genome duplication led to duplication of the transketolase (TKT) gene giving rise to the transketolase-like 1 (TKTL1) precursor gene [11, 12]. This duplication was followed by an integration of the TKTLI precursor mRNA into the genome creating the intronless and active transketolase-like-2 gene (TKTL2). After this, the TKTL1 precursor gene mutated creating the recent TKTL1 gene. In comparison to the known transketolase proteins, the TKTL1 gene encodes for a TKTL1 protein isoform harboring a 38-amino acid deletion [11, 12]. It has been postulated that the altered biochemical properties of the TKTL1 protein(s) represent the basis for a sugar fermentation metabolism linking glucose and fat metabolism independent of pyruvate dehydrogenase [12]. Using metabolic flux analysis and radioactive labeling of sugar metabolites it could be demonstrated that Acetyl-CoA is generated in a TKTL1 dependent way and is incorporated into lipids (Diaz et al., submitted) thus demonstrating a new connection between glucose and lipid metabolism. TKTL1 overexpression has been found in many different cancer types like breast, lung, renal, thyroid, ovarian, colorectal cancer, in tumors of the ocular adnexa and correlates with the increase of metastasis, poor prognosis, tumor recurrence, and resistance to chemo- and radiation therapy [13‐28].

The Apo10 protein epitope is detected by the monoclonal antibody Apo10, which has been raised against a DNaseX peptide sequence. DNaseX is a member of the DNaseI-protein family consisting of DNaseI, DNaseX (DNaseI-like 1), DNaseI-like 2 and DNaseI-like 3 (DNase gamma). The Apo10 epitope is present in tumor cells and in very few non-malignant cell types [13, 29].

Our study describes the immunohistochemical evaluation of the two biomarkers Apo10 and TKTL1 for characterization of OSCC tissue sections. Furthermore, both biomarkers have been detected intracellularly in monocytes using the epitope detection in monocytes (EDIM) technique, allowing a sensitive and specific noninvasive detection of OSCC, breast and prostate cancer patients by blood samples. This new blood test is based on the EDIM technology [13, 29‐31], which utilizes the fact that activated monocytes phagocytize and present tumor-related material even in the presence of low tumor mass [32]. Those activated monocytes, which contain intracellular tumor epitopes, can be detected by CD14 and CD16 specific antibodies using flow cytometry [13, 29‐31].

In the present study, we analysed retrospectively the potential prognostic and predictive influence of Apo10 and TKTL1 expression on clinicopathological parameters and on disease-free survival rates in a large patient cohort with OSCC. In addition to the retrospectively assessed Apo10 and TKTL1 data, prospectively Apo10 and TKTL1 have been determined using the EDIM technique. EDIM-Apo10 and EDIM-TKTL1 blood test was performed in patients with primary and/or recurrent OSCC, breast cancer patients, prostate cancer patients and healthy individuals (blood donors).

In 1991 Nobel laureate Professor Harald zur Hausen initiated a genome analysis program in the German Cancer Center focusing on the genomic region Xq28. At that time there was no evidence for the presence of cancer related genes in this region except the already known MAGE genes. This genomic region was systematically cloned in cosmids, ordered in contigs and further analysed for the presence of genes, conserved between human and pig, encoding tissue-specific expressed transcripts [11]. As a result of this approach the DNaseX and TKTL1 gene have been identified [11, 12]. Both genes represent the result of a genome duplication event leading to a copy of a DNaseI and transketolase (TKT) precursor gene, each. Gene duplications played an important role in the evolution of higher vertebrates, since the gene copies allowed an evolution of the function and regulation of genes without destroying the primary function of the gene which was copied [11]. DeoxyribonucleaseI as well as transketolase represent highly conserved enzymes which were the target of gene duplications leading to new DNase and transketolase genes/proteins, with sophisticated changes of expression and function of the copied genes. Those altered functions had implications for the evolution of higher vertebrates due to better adaptions of cells and multicellular organisms, but may also have implications for arising and development of nonmalignant and malignant tumor cells.

To analyse the expression of DNaseX protein in OSCC, a DNaseX peptid has been used to generate monoclonal antibody Apo10. Although another commercially available anti-DNaseX monoclonal antibody revealed an overexpression of the detected protein in OSCC, its staining pattern is distinct from Apo10 antibody and less tumor specific. This difference in staining patterns of both antibodies could be due to differences in crossreactivity to other DNaseI protein family members or even unrelated proteins, but could also be the consequence of epitope masking by protein binding. Such selective epitope detection by monoclonal antibodies has been revealed for DNase-gamma (DNaseI-like 3) [45]. Two monoclonal antibodies have been raised against a DNase-gamma specific peptide. Whereas one antibody detected a constitutively expressed DNase-gamma protein variant, the other monoclonal antibody specifically detected a nuclear DNase-gamma protein variant as a consequence of apoptosis induction by X-ray radiation, suggesting that some molecular change(s), which triggers the activation of DNase-gamma, occurs in response to apoptotic stimuli in the detected protein domain [45].

Similar to the DNase-gamma peptide, the DNaseX peptide may represent an epitope (Apo10 epitope) which is the target of molecular and/or biochemical change(s) leading to differential accession by monoclonal antibodies. Monoclonal antibody Apo10 detects a protein epitope present in the nucleus of apoptotic benign cells, but also in the nucleus of tumor cells. Whereas benign apoptotic cells (e.g. heart muscle cells of myocarditis patients) execute apoptosis, tumor cells apparently block the endonuclease driven execution of apoptosis by expression of proteins inhibiting endonucleases [4, 5] or other apoptosis executing proteins. Although the mechanism of activation and blocking of apoptosis is not fully understood, the presence of endonuclease epitopes in the nucleus can be exploited for tumor cell detection.

The Apo10 epitope is present in neoplastic cells including carcinomas, sarcomas, glioblastomas, lymphomas, and leukemias [29], whereas only few benign cells with and without induction of apoptosis show this epitope. The presence of the Apo10 epitope in OSCC and other neoplasias is a widespread event. Apo10 protein was present in 82% of primary OSCC tumors and correlated with poor patient prognosis.

Although the overexpression of TKTL1 takes also place in carcinomas [16], sarcomas [13], glioblastomas [26], lymphomas [27], and leukemias, the percentage of tumors overexpressing TKTL1 is lower compared to Apo10. 42% of the 161 tested primary OSCC showed an overexpression of TKTL1. Almost all TKTL1 positive tumors turned out to be Apo10 positive, whereas only 4% of the 161 tested primary OSCC were Apo10-/TKTL1+. This indicates that Apo10 precedes TKTL1 activation. This is in line with the currently accepted cancer theory that arising of tumor cells is based on the acquisition of mutations in different genes controlling cellular processes like apoptosis and metabolism. By using the biomarkers Apo10 and TKTL1 it is now possible to detect neoplasia associated changes in the two fundamental biophysical processes of endonuclease/apoptosis activation and glucose/energy metabolism.

The clinical relevance of these two fundamental biophysical processes is underlined by the fact, that Apo10 and TKTL1 presence in tumors represent two independent processes which are associated with advanced tumor stages and reduced tumor-specific survival in OSCC. This allows for the first time a two-step model of detection and characterization of tumors based on two biomarkers. OSCC tumors negative for Apo10 and TKTL1 represented tumors with favourably prognostic impact on survival, Apo10 presence indicated malignant OSCC tumors, and Apo10+/TKTL1+ OSCC tumors turned out to be more malignant tumors with an invasive/metastasing phenotype and worst prognostic impact on survival.

A large number of studies underlined the clinical relevance of increased TKTL1 expression on gene, transcript and protein level, since TKTL1 expression correlates with poor patient outcome and metastasis in many solid tumors [14‐27]. Inhibition of TKTL1 mRNA translation has been shown to inhibit cancer cell proliferation and to decrease lactate production [14, 15]. Specifically in head and neck squamous cell carcinoma TKTL1 is activated by promoter hypomethylation and drives carcinogenesis by increased aerobic glycolysis and hypoxia inducible factor 1 alpha (HIF-1α) stabilization [19]. A more recently published study describes TKTL1 to be indispensable for the function of the p53-dependent effector Tp53-induced glycolysis and apoptosis regulator (TIGAR) on hypoxia-induced cell death. Besides their diagnostic opportunity for cancer detection, these data confirm TKTL1 as an important new target for cancer treatment allowing inhibition of tumor cell viability and the increase of sensitivity towards hypoxia-, apoptosis and reactive oxygen species inducing therapies [15, 19, 24, 46].

A multistep process comprising initially six biological capabilities has been proposed as the basis for development of human tumors. The so called hallmarks of cancer include sustaining proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, and activating invasion and metastasis [47]. Our Apo10 and TKTL1 results contribute to the hallmarks of abnormal apoptosis/proliferation and increased invasion and metastasis, respectively. In addition to these intrinsic characteristics of tumor cells leading to tumor growth, the immune system based elimination of tumor cells determines whether a net gain of tumors happens or not. Therefore, a prerequisite of tumor growth is the outbalance of growth compared to the elimination of tumor cells. The hallmarks of cancer not only influence the growth rate of tumors but also influence the elimination rate by the immune system. The presence of tumor specific antigens facilitates the detection and elimination of tumor cells either by cytotoxic killing of tumor cells or by phagocytosis of tumor cells executed by macrophages. The elimination of tumor cells by cytotoxic killing of tumor cells is strongly dependent on tumor metabolism, since fermentation of glucose to lactic acid even in the presence of oxygen (aerobic glycolysis/Warburg effect [48]) prevents killing of tumor cells by natural killer cells [49]. Furthermore lactic acid excretion by tumor cells allows an acid based degradation of surrounding matrix of healthy tissue leading to invasive growth. Matrix degradation in distant organs allows disseminated tumor cells to build distant colonies thereby leading to metastases [6, 7]. Therefore, the metabolic switch from a mitochondria-based energy metabolism (OxPhos) to glucose fermentation e.g. mediated by TKTL1 is the basis of an invasive and metastasis inducing malignant phenotype of tumors as well as the basis of an immune protective strategy avoiding elimination of tumor cells by natural killer cells or cytotoxic T cells. Furthermore, since the metabolic switch from a mitochondria-based energy release to fermentation inhibits apoptosis induction (via reducing cytochrome c) and radical induction, elimination of tumor cells by apoptosis and radical inducing therapies (e.g. chemotherapy, radiation, respectively) is suppressed by TKTL1 metabolism [7, 15, 19, 24, 46].

In a proof of concept study the biomarkers Apo10 and TKTL1 have been used exploiting the epitope detection in monocytes (EDIM) technology. The EDIM technology allows a noninvasive detection of tumor proteins in blood. EDIM-Apo10 and EDIM-TKTL1 blood tests have been prospectively conducted in patients with primary or recurrent OSCC as well as in patients with primary breast and prostate cancer. In patients with histologically confirmed OSCC, breast and prostate cancers, blood samples before surgery revealed significant elevated levels of Apo10 and TKTL1 in CD14/CD16 positive monocytes compared to blood donors. By using a single cut-off for all three tumor entities it was possible to identify cancer patients by either EDIM-Apo10 or EDIM-TKTL1 blood test in a specific and sensitive manner. The combination of EDIM-TKTL1 and EDIM-Apo10 scores increased the sensitivity to 95.8% and the specificity to 97.3% in all cancer samples/entities.

The combined detection of two independent fundamental biophysical processes by the two biomarkers Apo10 and TKTL1 may allow a sensitive and specific detection of neoplasia in a noninvasive and cost-effective way. Further prospective trials are warranted to validate this new concept for the diagnosis of neoplasia and tumor recurrence.