Towards Optimal Diagnosis of Type II Germ Cell Tumors

The aim of the work described in this thesis is to improve the understanding of the pathobiology of testicular cancer (type II Germ Cell Tumors) to create possibilities for optimalization of diagnosis for this type of malignancy in routine pathology laboratories. The different studies presented here show valuable additional information on the microscopic diagnostics in daily practice. This enables proper and complete diagnosis of this relative rare variant of cancer ensuring the best possible treatment for the patient. A number of novel sensitive and specific immunohistochemical markers are presented, of value for (early) diagnostics. In addition an enzymhistochemical method is presented informative to detect the presence of (pre-)malignant cells during a surgical intervention. Based on these data, a decision can be made regarding a testis sparing procedure. The studies included in this thesis are part of a diagnostically decision tree in which immuno- and enzymhistochemistry plays an important role. General Testicular cancer is a relative rare variant of cancer with raising incidence in the Western population, in contrast to the rest of the world. Within the Netherlands, about 750 new patients are diagnosed each year. In particular this affects young males between the age of 15 – 45 years The current treatment possibilities is successful in approximately 95% of the patients. It consists of removing of the affected testicle (radical orchiectomy) and possibly additional treatment using irradiation and/or chemotherapy. In spite of the excellent prognosis, patients often will suffer from long-term side effects of the treatment like fatigue, disorders of the metabolism, heart diseases, neurotoxicity and infertility. A small group of patients (5%) will shows an insensitivity of the cancer treatment given, and will possibly die of the disease. An early and accurate diagnosis can prevent under- or overtreatment, ensure an optimal balance between the pro and conts of the treatment. This will have significant impact on their quality of life, for both short- and long term. Specific The cell of origin from which testicular cancer arises is already present during the first months of pregnancy (embryogenesis), as demonstrated in Chapter 3. It is a primitive germ cell formed after the first month after fertilization, which provide spermatozoa after puberty by spermatogenesis. In case this primitive germ cell does not maturate completely and keeps the properties of the embryonal cell. This cell can become carcinoma in situ (CIS), being the progenitor of testicular cancer. These CIS cell is located in the testis on the same place as the spermatogonium, below the Sertoli cells on the basal lamina. These cells start to CHAPTER 14 multiply in an uncontrolled manner after puberty and finally they can develop cancer. In chapter 4 the totipotent character of this cancer is described for the first time in literature, shown by the presence of new formed germ cells. In other words testicular cancer is the only real totipotent cancer that can give rise to different germ lines the same as in embryogenesis. The application of a unique panel of immunological markers permitted this finding. In chapter 5 the value of the presence of OCT3/4 in CIS, seminoma and embryonal carcinoma is for the first time described. This study was initiated by the presence of this marker in embryonal cells and germ cells of the mouse. The diagnostic significance of this extremely sensitive and specific marker is huge as described in chapter 6. Diagnostics of testicular germ cell tumors requires in most cases surgical removed tissue. In chapter 7 a new method is described that offers the possibilities to detect OCT3/4 positive cells in semen. This method introduces the possibility to screen men with a higher risk for developing testicular germ cell tumor for CIS cells without surgical intervention. The robustness of this detection method for OCT3/4, even in sub optimal material, is illustrated in chapter 8. The malignant germ cells in the here described patient where only detectable by the presence of OCT3/4. The two groups of testicular cancer: seminomatous and nonseminomatous tumors, differs in sensitivity for irradiation and chemotherapy. Therefore differentiation of these two groups is of great importance for an optimal treatment. The nonseminomas can be subdivided in different groups of which embryonal carcinoma is the stem cell population. In chapter 9 detection of two SOX proteins (SOX2 and SOX17) is shown to be informative. Seminoma cells show expression of OCT3/4 and SOX17, embryonal carcinoma cells of OCT3/4 and SOX2. The other subgroups of the nonseminomas (yolk sac tumor, choriocarcinoma and teratoma) show a more heterogeneous expression. A chance of overdiagnosis of CIS in the testicle based on the presence of OCT3/4 is risk in young children, especially in the first year after birth, a result of a possible delay in maturation of the normal developing germ cells. This is Important because maturation delay is common also in cases of an increased risk for the development of testicular cancer, for instance an undescended testicle. In chapter 10 it is demonstrated how to distinguish a germ cell with maturation delay from a (pre-) malignant germ cell (CIS). Because SCF is only present in the direct neighbourhood of (pre-)malignant germ cells. This finding has an additional value in case of possible maturation delay when OCT3/4 positive cells are present. There is also a significant value for this finding concerning that specific DNA variants related to this gene, associated with an increased risk for development of testicular cancer in the entire population. SUMMARY/SAMENVATTING Organ sparing surgery may be preferred for different reasons, even so in cases of testicular cancer. Prevention of a second surgical intervention requires proper histopathological examination of frozen sections. In testis cancer this is mainly based on the detection of the presence of CIS. Unfortunately morphologic analysis of frozen material is not that easy, therefore formalin fixed tissue is needed which excludes often a single operation procedure. In chapter 11 a direct enzymhistochemical method is described which makes it possible to detect the presence of these CIS cells in a few minutes on frozen tissue. By using this direct alkaline phosphatase (dAP) technique it is possible to have certainty about the presence of CIS cells in the available tissue. Based on these results a decision is possible about performing a testis sparing operation. Chapter 12 describes the low DNA methylation status of both CIS cells and embryonal germ cells in their natural environment. This is performed by the use of immunohistochemistry. Interesting is the possible connection between cancer cells which are sensitive to cisplatin and the status of methylation. DNA of the resistant tumors seems to be hypermethylated compared to sensitive tumors. Analysis of cultured cell lines indicates c-FLIP gene as a potential candidate. This gene regulates the sensitivity of cells for a programmed cell death (apoptosis). Conclusions The studies presented give possibilities to perform optimal diagnostics of testicular cancer, eventually in certain circumstances, even in a non-invasive set up. These findings demonstrate that understanding of the normal development of germ cells, especially during embryogenesis, is of importance to carry out these kind of studies. The results give a solid base for implementation in a diagnostic set up and for further translational research.

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