**3.6 Other prognostic markers**

#### **3.6.1 Ki-67**

One of the most well-known markers of cell proliferation includes the Ki-67 protein, also known as MKI67. The respective gene (MKI67) coding for this protein is located on chromosome 10q25. The expression of the Ki-67 human protein is strictly associated with cell proliferation. During interphase Ki-67 can be easily detected within the cell nucleus, whereas in mitosis most of the protein is relocated to the surface of the chromosomes. The Ki-67 protein is present during all active phases of the cell cycle (G1, S, G2 and mitosis), but its expression is basically absent from resting cells (G0). That is the reason why Ki-67 can be identified as an excellent marker to determine the growth fraction of a given cell population. This growth fraction of Ki-67-positive tumour cells (Ki-67 index) is often correlated with clinical stage of various malignant diseases. The best-studied examples in this context are prostatic and breast carcinomas. For these types of tumours the prognostic value for survival and tumour recurrence have repeatedly been proven in uni- and multivariate analyses.

MIB-1 is a commonly used monoclonal antibody that detects the Ki-67 antigen. One of its primary advantages is that it can be used on formalin-fixed paraffin-embedded sections, which is the reason why it has essentially supplanted the original Ki-67 antibody for clinical use. Recently the use of the Ki-67 protein as proliferation markers in laboratory animals has been expended to embrace the preparation of new monoclonal antibodies prepared from rodents. Although the molecular level of the Ki-67 protein has been wellstudied and its application as a proliferation marker is widely used, its functional meaning is still not fully clear. Nevertheless, there is obvious evidence that the Ki-67 protein expression is indispensable for the cell division process to be successful (Scholzen Gerdes, 2000).

Most endometrial carcinomas demonstrate a low Ki-67 proliferation index with a favourable prognosis, while most serous and clearly cellular tumours demonstrate a high proliferation index with poor prognosis. The correlation with grading, stage of the disease and histopathological type of tumour has been confirmed by many studies (Ferrandina et al., 2005, Markova et al., 2010).

#### **3.6.2 β-catenin**

14 Cancer of the Uterine Endometrium – Advances and Controversies

Numerous losses of heterozygosity are typical for nonendometrioid carcinomas (Albertson

A certain degree of genetic instability that may, as a result of defects in mitotic segregation or recombination during cell division, lead to significant changes in the genome is typical for the genetic material in tumour cells. Normal somatic cells with 46 chromosomes (23 pairs) are called diploid cells, while extra or missing chromosomes are identified as aneuploidy. Chromosomal instability causing structural or numeric aberrations occurs in early as well as later and more invasive stage of tumour development and is typical for various types of malignant tumours. These cytogenetic changes indicate that defects in genes associated with maintaining chromosomal stability and integrity and assuring the exact mitotic segregation represent a significant element of tumour progression (Nussbaum

In endometrial carcinoma the aneuploid changes occur in 25 - 30% of cases. According to a number of studies approximately 67% of endometrioid carcinomas are diploid, whereas about 55% of nonendometrioid carcinomas demonstrate aneuploid changes (Mutter Baak, 2000). Diploid tumours are usually well differentiated tumours with only surface invasion to myometrium and are associated with longer survival than aneuploid tumours. Aneuploid tumours are in general associated with a poorer prognosis, higher number of recurrences and shorter disease free survival. The percentage of disease free survival for tumours in stage I, which is 94%, versus 64% in aneuploid tumours shows a clear difference. The important fact remains that in the vast majority of studies the ploidity is mentioned as

One of the most well-known markers of cell proliferation includes the Ki-67 protein, also known as MKI67. The respective gene (MKI67) coding for this protein is located on chromosome 10q25. The expression of the Ki-67 human protein is strictly associated with cell proliferation. During interphase Ki-67 can be easily detected within the cell nucleus, whereas in mitosis most of the protein is relocated to the surface of the chromosomes. The Ki-67 protein is present during all active phases of the cell cycle (G1, S, G2 and mitosis), but its expression is basically absent from resting cells (G0). That is the reason why Ki-67 can be identified as an excellent marker to determine the growth fraction of a given cell population. This growth fraction of Ki-67-positive tumour cells (Ki-67 index) is often correlated with clinical stage of various malignant diseases. The best-studied examples in this context are prostatic and breast carcinomas. For these types of tumours the prognostic value for survival and tumour recurrence have repeatedly been proven in uni- and multivariate

MIB-1 is a commonly used monoclonal antibody that detects the Ki-67 antigen. One of its primary advantages is that it can be used on formalin-fixed paraffin-embedded sections, which is the reason why it has essentially supplanted the original Ki-67 antibody for clinical use. Recently the use of the Ki-67 protein as proliferation markers in laboratory animals has been expended to embrace the preparation of new monoclonal antibodies

independent prognostic factor (Pradhan et al., 2006, Suehiro et al., 2008).

et al., 2003, Tashiro et al., 1997).

**3.6 Other prognostic markers** 

**3.5 Aneuploidy** 

et al., 2004).

**3.6.1 Ki-67** 

analyses.

β-catenin is a submembranous protein that is encoded by the CTNNB1 gene located on chromosome 3p21. β-catenin is a part of a complex of proteins that constitute adherens junctions which are necessary for the creation and maintenance of epithelial cell layers by regulating the cell growth and adhesion between cells. Therefore, it takes part in maintaining tissue architecture. It is known that β-catenin is able to bind to various proteins. For example, it creates complexes with cadherines, which are transmembrane proteins functioning as transcription factors, so it plays an important role in regulating transcription. It is also known that it represents an integral component of the Wnt signal pathway, which is a network of proteins with a significant role in embryogenesis and tumorigenesis (Bullions Levine, 1998).

Under defects of the above functions β-catenin can function as an oncogene. An increased level of β-catenin and mutations of the CTNNB1 gene have been described in various tumours - basal cell carcinoma, colorectal carcinoma, medulloblastoma or ovarian carcinoma. In endometrial carcinoma the nuclear accumulation of β-catenin and, simultaneously, mutations of its CTNNB1 gene have been described in may studies. The nuclear β-catenin has been identified in 16 to 38% of endometrial carcinomas, while its expression was significantly higher in the endometrioid (type I) (31 - 47%) than in nonmetrioid (type II) (0 - 3%) carcinoma. Mutations of CNNTB1 in endometrioid carcinoma have been described in 15 to 25%, while in nonendometrioid carcinoma none has been identified. Accumulation of β-catenin in cell nucleus has been found in less aggressive tumours with low metastasizing potential and, similarly, mutations of CNNTB1 are associated with well differentiated carcinomas (Machin et al., 2002, Scholten et al., 2003).

#### **3.6.3 Steroid receptors**

Endometrium is the target tissue of steroid hormones produced by ovaries. Oestrogen (ER) and progesterone (PR) receptors are present in both epithelial and stromal endometrial cells. It is generally known that ovarian steroids, oestrogen and progesterone, have the critical importance for regulating the growth and differentiation in endometrial cells. A normal course of the menstrual cycle (proliferation, differentiation and degeneration of endometrium) reflects cyclic changes in sex steroid levels. The proliferation stage of the cycle is mostly under the influence of oestrogens that stimulate proliferation of epithelial and stromal endometrial components, whereas during the secretory stage the main function of progesterone is glandular differentiation and glycogenesis with inhibition of oestrogenmediated proliferation. Just as the ovarian steroids play an indispensable role in normal endometrium, they also significantly influence the development of endometrial carcinoma (Graham & Clarke, 1997).

ER and PR belong among a group of nuclear receptors with typically immunohistochemically detectable cyclic changes in their expression based on the cycle stage. After their activation they bind to specific target places in DNA where they modulate an expression of respective genes. In addition to this direct activation of target genes an indirect mechanism of their effect via relation to transcription factors, such as AP-1 (c-fos, c-jun) or NF-κB, has been described (Oehler et al., 2000).

Oestrogen receptors (ER) belong among the group of receptors subject to 17β-estradiol activation. ER primarily function as a transcription factor regulating the expression of other genes. Two subtypes of ER, ERα and ERβ, have recently been described; each of them is encoded by a different gene. The ESR1 gene for ERα is located on chromosome 6q24-q27, the ESR2 gene for ERβ on chromosome 14q21-q22. ER play an important role in the development of various malignancies, primarily in breast cancer (an increased expression is indicated in about 70% of cases) cancer of the ovaries, colon, prostate and, of course, endometrial carcinoma. While ERα is the dominant receptor in endometrium and participates primarily in increased proliferation, ERβ's effect is anti-proliferating and it apparently modulates the ERα function. The imbalance between the expression of ERα and ERβ is considered to be the critical moment in oestrogen-dependent carcinogenesis (type I). In endometrial carcinoma a decreasing level of the ERα mRNA expression and protein has been described, together with dedifferentiation of this tumour from grade 1 to grade 3. Under the unchanged expression of ERβ the ERα/ERβ ratio decreases (Jazaeri et al., 2001). In addition to the changed ratio of ER isoforms, incorrectly transcribed proteins derived from ERα or ERβ take part in endometrial carcinogenesis. For example, they include 5 ERα, which has been described in endometrial carcinoma but has not been detected in normal endometrium, or ER βcx with a dominant negative effect on ERα (Skrzypczak et al., 2004).

The progesterone receptor (PR), also known as NR3C3 (nuclear receptor subfamily 3, group C, member 3), is an intracellular receptor able to specifically bind progesterone.

PR is encoded by one PGR gene located on chromosome 11q22. It also exists in two isoforms differing by their molecular weight: PR-A and PR-B. One of the main functions of PR-A in endometrium is down-regulation of oestrogen activity via ERα inhibition. On the other hand, PR-B works as an oestrogen agonist in endometrial cells. Imbalances in PR-A/PR-B ratio are similarly considered to be a critical moment in the development of endometrial carcinoma (type I) (Arnett-Mansfield et al. 2001).

A number of studies have demonstrated that the presence and quantity of these steroid receptors correlate with the stage of tumour, grading and survival. The absence of steroid receptors is seen as a negative prognostic factor of aggressive growth and poor prognosis (Ferrandina et al., 2005, Jazaeri et al., 2001, Pilka et al., 2008). Nevertheless, the mechanisms of the loss of their expression in endometrial tumours is not fully known.

#### **3.6.4 Growth factors**

Steroid hormones regulate a number of growth factors that apparently participate in the paracrine and autocrine regulation of endometrial proliferation. They primarily include

of progesterone is glandular differentiation and glycogenesis with inhibition of oestrogenmediated proliferation. Just as the ovarian steroids play an indispensable role in normal endometrium, they also significantly influence the development of endometrial carcinoma

ER and PR belong among a group of nuclear receptors with typically immunohistochemically detectable cyclic changes in their expression based on the cycle stage. After their activation they bind to specific target places in DNA where they modulate an expression of respective genes. In addition to this direct activation of target genes an indirect mechanism of their effect via relation to transcription factors, such as AP-1 (c-fos, c-jun) or NF-κB, has been described

Oestrogen receptors (ER) belong among the group of receptors subject to 17β-estradiol activation. ER primarily function as a transcription factor regulating the expression of other genes. Two subtypes of ER, ERα and ERβ, have recently been described; each of them is encoded by a different gene. The ESR1 gene for ERα is located on chromosome 6q24-q27, the ESR2 gene for ERβ on chromosome 14q21-q22. ER play an important role in the development of various malignancies, primarily in breast cancer (an increased expression is indicated in about 70% of cases) cancer of the ovaries, colon, prostate and, of course, endometrial carcinoma. While ERα is the dominant receptor in endometrium and participates primarily in increased proliferation, ERβ's effect is anti-proliferating and it apparently modulates the ERα function. The imbalance between the expression of ERα and ERβ is considered to be the critical moment in oestrogen-dependent carcinogenesis (type I). In endometrial carcinoma a decreasing level of the ERα mRNA expression and protein has been described, together with dedifferentiation of this tumour from grade 1 to grade 3. Under the unchanged expression of ERβ the ERα/ERβ ratio decreases (Jazaeri et al., 2001). In addition to the changed ratio of ER isoforms, incorrectly transcribed proteins derived from ERα or ERβ take part in endometrial carcinogenesis. For example, they include 5 ERα, which has been described in endometrial carcinoma but has not been detected in normal endometrium, or ER βcx with a dominant negative effect on ERα

The progesterone receptor (PR), also known as NR3C3 (nuclear receptor subfamily 3, group

PR is encoded by one PGR gene located on chromosome 11q22. It also exists in two isoforms differing by their molecular weight: PR-A and PR-B. One of the main functions of PR-A in endometrium is down-regulation of oestrogen activity via ERα inhibition. On the other hand, PR-B works as an oestrogen agonist in endometrial cells. Imbalances in PR-A/PR-B ratio are similarly considered to be a critical moment in the development of endometrial

A number of studies have demonstrated that the presence and quantity of these steroid receptors correlate with the stage of tumour, grading and survival. The absence of steroid receptors is seen as a negative prognostic factor of aggressive growth and poor prognosis (Ferrandina et al., 2005, Jazaeri et al., 2001, Pilka et al., 2008). Nevertheless, the mechanisms

Steroid hormones regulate a number of growth factors that apparently participate in the paracrine and autocrine regulation of endometrial proliferation. They primarily include

C, member 3), is an intracellular receptor able to specifically bind progesterone.

of the loss of their expression in endometrial tumours is not fully known.

(Graham & Clarke, 1997).

(Oehler et al., 2000).

(Skrzypczak et al., 2004).

**3.6.4 Growth factors** 

carcinoma (type I) (Arnett-Mansfield et al. 2001).

epidermal growth factor (EGF) and transforming growth factor alpha (TGF- α), which influence the endometrial cells through an EGF receptor. Both growth factors and their receptor stimulate cell growth in endometrial carcinoma in vitro. Other growth factors involved in endometrial carcinogenesis (type I) include transforming growth factor beta (TGF- β), basic fibroblast growth factor (bFGF) and insulin-like growth factor I(IGF-I) (Myeroff et al., 1995).

#### **3.6.5 Matrix metalloproteinase**

Matrix metalloproteinase belongs among the family of enzymes of zinc-dependent endopeptidases that are capable of degrading extracellular matrix. So far, more than 25 subtypes of these enzymes have been identified; based on their structure they are further classified into 8 different classes and their production is induced by an inflammatory or tumorous process (Nagase et al., 1999). One of the important members of the metalloproteinase family with an epithelial expression concerns MMP-7 (matrilysin-1), expression of which has been detected in both normal and malign epithelial cells. Only a limited number of studies focused on the MMP-7 expression in endometrial carcinoma has been published. In his study Ueno et al. demonstrated an increased expression of MMP-7 correlating with the worse clinical stage of the disease and presence of lymphatic metastases (Ueno et al., 1999). A similar trend is also described in the study carried out by (Graesslin et al. 2006, Wang et al., 2005). Markova et al. describes a significant relation between age and MMP-7 as in patients older than 65 the expression of MMP-7 was significantly lower (Markova et al., 2010).

Another member of the matrilysin enzymes subfamily is identified as MMP-26 (matrilysin-2). Likewise, MMP-26 is also generated in various tissues, both normal and malignant, including endometrial carcinoma. The outcomes of studies carried out by various authors indicate that despite MMP-26 belonging among the same subfamily of metalloproteinases as MMP-7, its function may apparently be different. It is known that the expression of MMP-26 specifically fluctuates during the menstrual cycle. The detection of high levels in the middle of the cycle and in hyperplastic endometrium, and, on the other hand, low levels in the late stage of the cycle and endometrial carcinoma indicate the correlation with oestrogen receptors. Isaka et al. and Pilka et al. demonstrated a significantly reduced expression of MMP-26 in endometrial carcinoma, which goes against the results of study carried out by Tunuguntla et al., who describes an increased immunohistochemical expression of MMP-26 in low-differentiated endometrial carcinoma (Isaka et al., 2003, Pilka et al., 2004, Tunuguntla et al., 2003).

#### **4. Conclusion**

The efficient treatment of malignancies requires an early and accurate diagnosis enabling to optimize therapy and minimize adverse effects. Early diagnosis of cancer, together with individual "custom-made" therapy, may reduce mortality and improve the prospects and quality of the patient's life. Gynaecological malignant tumours represent a group of diseases where the prognosis depends on subtle genomic, epigenetic and proteomic changes. The application of molecular biology techniques, including analysis of methylation and acetylation, and preoteomic techniques have become an important tool not only in basic research but also when determining the appropriate therapy.

The significance of various immunohistochemical parameters for the prognosis in patients suffering from endometrial carcinoma has not been unambiguously determined yet. The aim is, by applying the information acquired based on the expression of tumour biomarkers, to limit the radicalism in surgical and radiation therapy. The future objective is to further classify the subtypes of endometrial carcinoma based on their genetic alterations, in particular those that are significant in terms of prognosis. It is probable that future histological classifications will be based more on a molecular basis. In addition to clinical pathological factors, the molecular biological prognostic factor may improve the characteristics of tumours and provide a more accurate definition of their clinical behaviour. Although these factors will apparently be more important in managing the endometrial carcinoma treatment in the near future, any practical diagnostic and therapeutic application of biological factors will require more detailed studies.

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