The value of the biopsy of the contralateral testis in patients with testicular germ cell cancer: the recent German experience

PURPOSE: Testicular intraepithelial neoplasia (TIN; so-called carcinoma in situ of the testis), the precursor of testicular germ cell neoplasms can be detected by testicular biopsy many years before the clinical manifestation of the tumour. This study looked at the prevalence of contralateral TIN in patients with testicular germ cell cancer. The purpose was to evaluate this new approach of early detection of testicular cancer and to evaluate the current management strategies. Patients, methods: 1954 consecutive patients with unilateral testicular germ cell tumour underwent contralateral biopsy. All specimens were examined immunohistologically with staining for placental alkaline phosphatase. Patients with TIN were usually submitted to low-dose radiotherapy of the testis. A rebiopsy was performed after 3 months. Endocrinological evaluations were done before, during and after treatment. RESULTS: TIN was observed in 4.9% (95% confidence intervals 3.95%-5.91%). Testicular atrophy constitutes a 4.3 fold increased risk of having contralateral TIN. 64% of the cases with TIN were found in clinically normal testes. Patients with TIN were significantly younger than those without (p < 0.017). No case with TIN was found in patients older than 50 years. Three patients developed a second testicular tumour during follow-up despite a negative biopsy. After radiotherapy, all of 23 patients had complete disappearance of TIN in the rebiopsy. After chemotherapy, 3 of 10 patients had persistent TIN histologically. After radiotherapy, 12 of 41 patients required testosterone replacement. CONCLUSION: The prevalence of contralateral TIN accords well with the known prevalence of bilateral testicular tumours. Testicular atrophy is a strong indicator for the presence of TIN but about 60% of TIN-cases occur without atrophy. Local radiotherapy to the testis with 18-20 Gy is efficaceous in eradicating TIN, but it causes significant damage to almost one quarter of these patients. Chemotherapy is an unsafe treatment for TIN. This study shows the feasibility of early detection of testicular cancer in a high-risk population by means of searching for TIN. Although the management of the condition still needs refinement, the TIN-concept offers an avenue for the early detection of testicular cancer and early conservative management.     

Developmental arrest of germ cells in the pathogenesis of germ cell neoplasia

Clinical observations and epidemiological evidence suggest that important aetiopathological events that cause neoplastic transformation of the male germ cell may occur in fetal life or early infancy. The incidence of germ cell neoplasia is high in individuals with various disorders of gonadal development and sexual differentiation, such as gonadal dysgenesis or androgen insensitivity syndrome. Increased risk has also been noted in individuals with trisomy 21, idiopathic infertility and low birth weight. Infertility is sometimes associated with small aberrations of sex chromosomes (e.g. low frequency mosaicism XY/XO) which can also be found in patients with testicular cancer. The variety of conditions that predispose to testicular neoplasia and the rise in its incidence in many countries speaks for the influence of environmental factors which may affect genetically predisposed individuals. We hypothesise that if the development of the testis is disturbed or delayed, primordial germ cells or gonocytes undergo maturation delay or differentiation arrest which may render them susceptible to neoplastic transformation. Morphologically homogenous premalignant carcinoma in situ (CIS) cells have the potential to differentiate into a variety of histological forms of overt testicular tumours. Analysis of cell surface antigens expressed by CIS cells found in the vicinity of pure and mixed tumours demonstrates that CIS cells are phenotypically heterogeneous. Comparison of the phenotypes of CIS cells, primordial germ cells, human embryonal carcinoma cells and closely related primate embryonal stem cells reveals various similarities but also differences. We speculate that phenotypical heterogeneity of CIS cells may be associated with their potential to give rise to different tumour types, and may be related to the developmental stage of the early germ cell which has undergone malignant transformation.     

Immunohistochemical pattern of insulin-like growth factor (IGF) I, IGF II and IGF binding proteins 1 to 6 in carcinoma in situ of the testis

AIM: To study the immunohistochemical localisation of insulin-like growth factor (IGF) I, IGF II, and IGF binding proteins 1-6 in intratubular germ cell neoplasia in the vicinity of solid germ cell tumours of the testis. METHODS: Testes were obtained from 13 patients (20-35 years old) who had undergone orchidectomy for treatment of a solid germ cell tumour. Tumour cells were verified histologically by their distinctive morphology and by visualisation of placental alkaline phosphatase immunoreactivity. RESULTS: The majority of carcinoma in situ (CIS) cells were immunopositive for IGF I, whereas no CIS cells stained for IGF II. Of all the IGF binding proteins investigated, CIS cells showed intense immunoreactivity for IGF binding protein 5 and lower expression of all other IGF binding proteins. CONCLUSIONS: These results suggest that the action of IGF binding protein 5 in CIS cells may modulate the activity of IGF I. This may be related to a proliferative advantage that could facilitate tumour development.     

The origin and biology of CIS cells: general discussion

Participants at the 4th Copenhagen Workshop on Carcinoma in situ and Cancer of the Testis, representing cell biologists and tumour biologists, met together to discuss the similarities and differences between primordial germ cells (PGCs) of the embryo, and the carcinoma in situ (CIS) stem cell of human testicular germ cell tumours (GCTs). Much has been discovered about PGCs in the last 10 years and we still do not know the exact nature of CIS cells. Knowledge of PGCs comes mainly from mouse experiments and knowledge of CIS comes from the study of human tumours. A mouse model of human GCT would help to investigate the nature of CIS cells. Grafting mouse male genital ridges into mouse fetal testes results in the development of testicular tissue and the formation of teratomatous tumour components. Amplification of PGCs in culture is possible but this results in their transformation into embryonic germ (EG) cells. CIS cells die by apoptosis if they are isolated, and short-term culture is only possible if the CIS cells are cultured in their normal environment within seminiferous tubules. It may be possible for CIS cells to differentiate in culture although they cannot be maintained in culture as isolated cells. Human CIS cells are likely to be formed as a result of in utero factors rather than agents acting on normal adult testicular germ cells. EG cells stimulate feeder cells by paracrine factors but it is not known if these cells produce autocrine factors.     

Alteration of Sertoli cell differentiation in the presence of carcinoma in situ in human testes

PURPOSE: We investigated Sertoli cells in testicular biopsies with carcinoma in situ (CIS) in respect to cytokeratin expression to elucidate the status of Sertoli cell differentiation adjacent to CIS in human testes. Cytokeratin 18 intermediate filaments indicate a state of undifferentiation usually observed in Sertoli cells of prepubertal testes. MATERIALS AND METHODS: 29 testicular biopsies presenting CIS were investigated by means of immunohistochemistry, using a polyclonal antibody against placental-alkaline phosphatase to detect CIS cells and a monoclonal antibody against human cytokeratin 18 to show expression of cytokeratin 18 intermediate filaments in Sertoli cells. RESULTS: All tubules bearing CIS showed positive cytokeratin expression of Sertoli cells if tubules were devoid of normal germ cells. However, a total of 13 specimen revealed CIS cells together with normal germ cells. In the presence of CIS cells together with round or elongated spermatids, adjacent Sertoli cells did not express cytokeratin immunoreactivity. In the case of combined presence of CIS and spermatogonia and primary spermatocytes, Sertoli cells could be found either immunopositive or immunonegative, and were positive in tubules with CIS and spermatogonia only. CONCLUSIONS: Sertoli cells associated with CIS cells undergo a process of dedifferentiation, seen by the re-expression of cytokeratin intermediate filaments. We suggest that this dedifferentiation results in a loss of Sertoli cell function and leads to a cessation of spermatogenic activity.     

Embryonal carcinoma of the testis in elderly men

A rare case of pure embryonal carcinoma of the testis in a black man in the tenth decade of life is presented, as well as a review of the literature on germ cell testicular tumors in men 60 years old or more. It is noteworthy that nearly 9 per cent of testicular tumors occur in this age group and a third of these tumors are of germ cell origin.     

Molecular pathogenesis and prognostic factors in testicular tumor

Aim of this review article was to critically analyse the recently described zytogenetic and molecular markers for testicular germ cell tumors with regard to their clinical utility. The isochromosome i(12p) represents the most common and characteristic cytogenetic finding which already appears in testicular carcinoma in situ. A number of proto-oncogenes (Cyclin D and PTHLH) as well as putative tumor suppressor genes are localized on 12p; however, their role in pathogenesis and prognosis of testicular germ cell tumors has not been defined yet. Clinical characteristics of patients with familial testicular germ cell tumors indicate a genetic background for the development of testicular tumors. Although a number of chromsomal loci encoding potential testicular tumor susceptibility genes have been identified, the genetic basis of testicular cancer pathogenesis is still unknown. With regard to molecular prognostic risk factors most of the reported data on proliferation markers, tumor suppressor genes, proteases and adhesion molecules have to be confirmed in prospective randomized trials prior to their widespread clinical use. Based on the available data on prospective studies percentage of embryonal carcinoma and vascular invasion appear to be the most significant prognosticators. Investigation and identification of those factors determining the aggressive biologic behavior of embryonal carcinoma compared to all other histological components appear to be most promising in research for prognosticators of metastatic disease. In conclusion, the increasing knowledge of molecular genetic events involved in pathogenesis and prognosis of testicular germ cell tumors will not only help to better understand development and progression of testicular cancer, but it also will define new approaches to classification and management of germ cell tumors.     

Detection of human papillomavirus DNA with in situ hybridisation in oral squamous carcinoma in a rural black population

Intra-oral carcinoma is the third most common malignancy among men in developing countries, and carries a high mortality rate, particularly in Africa, where patients often present initially with lesions at an advanced stage. The present study was undertaken to determine the prevalence of human papillomavirus (HPV) DNA in oral squamous carcinoma in the west of the Northern Transvaal, an area where a large number of new cases has been diagnosed over the past few years. Paraffin blocks from 66 cases (51 men, 15 women; mean age 58.7 years) of oral squamous carcinoma were randomly selected. Blocks contained samples of both tumour and adjacent normal epithelium. The presence of HPV antigen was established by means of immunocytochemistry and HPV DNA by in situ hybridisation with radiolabelled probes for HPV-6, 11, 16 and 18. Immunocytochemistry for viral antigen was negative in all the specimens. HPV-18 was detected in normal epithelium adjacent to the tumour in one case only. It appears from our study that HPV is of limited importance in oral squamous cell carcinogenesis in the population studied.     

Vascular endothelial growth factor expression is increased in renal cell carcinoma

PURPOSE: To compare the expression of VEGF in renal cell carcinoma (RCC) and normal kidney. MATERIALS AND METHODS: RT-PCR and Western blot analysis were performed on tumour and normal adjacent kidney collected from 31 patients (29 RCC and 2 oncocytomas) as well as proliferating vascular endothelial cells (VEC) in culture. RESULTS: Expression of 3 VEGF isoforms was detected in normal renal parenchyma and all ROC by RT-PCR, but was not apparent in proliferating VEC. In 27 RCC, Western blot analysis demonstrated 3-37 fold increases in VEGF expression when compared to normal parenchyma. Immunohistochemistry demonstrated VEGF staining of both tumour cells and adjacent vascular endothelium. Normal kidney showed no staining for VEGF. In the 2 remaining RCC and both oncocytomas VEGF was not increased. CONCLUSIONS: VEGF expression is increased in RCC and may have a paracrine effect in these tumours in stimulating angiogenesis.     

Association of Down's syndrome and testicular cancer

PURPOSE: We present additional clinical evidence for the suspected association of Down's syndrome and testicular germ cell tumors. MATERIALS AND METHODS: Four cases of Down's syndrome and testicular cancer are reported. The literature was reviewed for previous cases and analysis regarding common features. RESULTS: The 4 patients were 29 to 35 years old and had clinical stage I seminoma of the testis. Two patients received prophylactic abdominal radiotherapy, 1 is being followed and 1 received adjuvant carboplatin treatment. There was no relapse at followup of 1 to 8 years. One patient also had contralateral cryptorchidism. A total of 16 cases with the association of Down's syndrome and testicular germ cell cancer was documented previously. CONCLUSIONS: Evidence for the suspected association of Down's syndrome and testicular cancer is now accumulating. Etiologically it is suspected that, along with genetically determined malformations in many other organs in trisomy 21, the gonads also undergo maldevelopment, thus creating the conditions for step 1 of germ cell tumor oncogenesis in utero. Physicians caring for patients with Down's syndrome should be aware of the possible association with testicular neoplasms.     

Gonadal function in men with testicular cancer: biological and clinical aspects

This paper reviews current knowledge about the effect of testicular germ cell cancer (TGCC) on gonadal function and of cancer treatment on spermatogenesis and Leydig cell function. It is well documented that testicular cancer is associated with impaired spermatogenic function and some patients already have impairment of Leydig cell function before orchidectomy. The degree of spermatogenic dysfunction is higher than what can be explained by local tumour effect and by a general cancer effect, since patients with other malignant diseases have normal, or only slightly decreased, semen quality. Furthermore, sperm counts after orchidectomy are further reduced to less than half of the values in healthy men, even in patients cured from the cancer disease after orchidectomy alone. These observations are supported by histological investigations which have shown a high prevalence of abnormalities of spermatogenesis in the contralateral testis in patients with unilateral TGCC. The association between testicular cancer and poor gonadal function is very interesting both from a biological and from a therapeutic point of view. Firstly, the increase in incidence of testicular cancer has been suggested to be associated with a general decline in male reproductive health and it seems likely that the development of TGCC shares common aetiologic factors with development of other types of testicular dysfunction. This suggestion is supported by the observation that men with various types of gonadal dysfunction such as testicular dysgenesis, androgen insensitivity syndrome, and cryptorchidism have increased risk of testicular cancer. Secondly, the general cure rate in patients with testicular cancer exceeds 90% and the quality of life, including fertility aspects, is therefore important in the management of these patients. Spermatogenesis is already so severely impaired before treatment that fertility is lower than in healthy men. Moreover, radiotherapy and chemotherapy both induce dose-dependent impairment of spermatogenesis and recovery of spermatogenesis after treatment may be long lasting even more than five years in some patients. Sufficient androgen production is seen in the majority of the patients, but some patients suffer from testosterone deficiency. The effect of chemotherapy on Leydig cell function also seems to be dose-dependent. In conclusion there is no doubt that testicular cancer is associated with poor gonadal function even before treatment. Furthermore, the treatment of testicular cancer may have a serious impact on the gonadal function in these patients, most of whom are in the reproductive age. Moreover, the epidemiological and clinical data indicate a common aetiology between testicular germ cell cancer and other abnormalities in male reproductive health (such as infertility and cryptorchidism). These observations are in agreement with the suggestions of hormonal involvement in the aetiology of testicular cancer. Generally, men with TGCC need counselling about their reproductive function with respect to semen cryopreservation, chance of recovery of spermatogenesis, fertility, and the possible need for androgen replacement.     

Interaction of C-reactive protein with alpha-chain of interleukin-2 receptor

BACKGROUND: Approximately 5% of patients with testicular cancer harbour carcinoma in situ (CIS) in the contralateral testis. CIS will progress into invasive tumour in about 50% of cases within five years. The present study evaluated the effect of platinum containing chemotherapy on CIS. PATIENTS AND METHODS: Thirty-three patients with disseminated germ-cell cancer and biopsy proven CIS of the testis were evaluated. RESULTS: CIS had disappeared in the first follow-up biopsy in 30 patients. Six patients had a relapse of CIS with or without invasive cancer after 30, 31, 47, 51, 76 and 95 months from start of chemotherapy. Two relapses were among six patients who initially received cisplatin, vinblastine and bleomycine and four among 27 patients who initially received cisplatin, etoposide and bleomycine. The estimated cumulative risk of CIS five and 10 years after chemotherapy was 21% and 42%, respectively. The estimated cumulative incidence of spermatogenesis was 64% and 81% at five and 10 years of follow-up, respectively. CONCLUSION: Platinum containing chemotherapy may eradicate CIS. However, patients with CIS may develop invasive cancer in spite of chemotherapy. In the light of the present data, we recommend radiotherapy to the affected testicle in patients with CIS in the contralateral testis and in patients with bilateral testicular CIS. In patients with extragonadal disease and CIS in one testicle, orchiectomy of the affected testicle is recommended. In patients for whom future fertility is an important issue, follow-up including repeated biopsies can be offered for a period of at least 10 years.     

Germ cells and germ cell transplantation

The germ cell lineage in mice is established about a week after fertilization, in a group of cells that have left the epiblast and moved to an extraembryonic site. They migrate back into the embryo, along the hind gut and into the gonads. Germ cells in male and female embryos then pursue different pathways: in the testis the germ cells cease proliferating and enter mitotic arrest, while germ cells in the ovary, like those in male embryos that remain outside the gonads, enter meiotic prophase. Studies on explanted germ cells suggest that all germ cells may enter meiosis at a certain stage of their development, unless prevented from doing so by some inhibitory influence of the testis. Germ cells during the migratory stage can be cultured, but do not enter meiosis unless embedded in somatic tissue. Addition of certain growth factors and cytokines to the culture medium allows germ cells to proliferate indefinitely in vitro: Like embryonic stem cells, these immortalized EG (embryonic germ) cells will colonize all cell lineages if introduced into a blastocyst. After birth, germ cells undergo gametogenesis; oogenesis in the female, spermatogenesis in the male. Brinster and his colleagues have shown that spermatogonial stem cells injected into a germ-cell depleted testis will repopulate the seminiferous tubules and undergo spermatogenesis, giving rise to functional spermatozoa. Stem cells from frozen testicular tissue are still capable of giving rise to spermatogenesis in a host testis. Rat testicular tissue can undergo spermatogenesis in a mouse testis, to form morphologically normal rat spermatozoa, even though the Sertoli cells that support them are of endogenous mouse origin. These findings are of fundamental importance for our understanding of spermatogenesis and the interactions between germ cells and Sertoli cells; but they also have significant practical implications, in relation to both agricultural practice and clinical treatment of infertility.     

Early stage of development in testicular choriocarcinomas

Choriocarcinoma is the most malignant among germ cell tumors in the testis. However little is known about the early stage of its development. To understand the development of testicular choriocarcinomas, twenty cases of testicular choriocarcinoma were studied histologically and immunohistochemically. It was found that in the early stage of development, choriocarcinomas imitate the morphologic or functional differentiation of normal trophoblasts. It was also found that some choriocarcinomas regress spontaneously in the early stage. The majority of choriocarcinomas seemed to develop by first going through the embryonal carcinoma phase. However, there were some choriocarcinomas that showed no relationship with embryonal carcinoma.     

Bezafibrate down-regulates fibrinogen biosynthesis in human hepatoma HepG2 cells

Cellular distribution of HIV-1 proviral DNA has been studied, by in situ PCR hybridization, in the testes of infected men who died at various stages of the disease. In seropositive asymptomatic subjects, HIV-1 proviral DNA was present in the nuclei of germ cells at all stages of their differentiation. The presence of provirus did not induce germ cell damage, was associated with normal spermatogenesis, and was not accompanied by morphologic signs of immune response. The observed HIV hybridization pattern of germ cells suggests clonal infection. Mechanisms responsible for HIV penetration in testicular germ cells remain to be clarified; however, the possibility of a direct infection of the germ cells by cell-free virus is suggested. In the testes of AIDS-deceased men, histologic features of hypoplasia with arrested spermatogenesis were evident, and few infected spermatogonia and spermatocytes were observed. The whole of these data demonstrates that the testis is a site of early viral localization that fails to elicit an immunological response, and that HIV-seropositive men produce infected spermatozoa that are released in the genital tract.     

Germ cell cancer and disorders of spermatogenesis: an environmental connection?

Why is there a small peak of germ cell tumours in the postnatal period and a major peak in young age, starting at puberty? And, paradoxically, small risk in old age, although spermatogenesis is a lifelong process? Why is this type of cancer more common in individuals with maldeveloped gonads, including undescended testis, gonadal dysgenesis and androgen insensitivity syndrome? Why has there, during the past 50 years, been a quite dramatic increase in testicular cancer in many developed countries? These are just a few of many questions concerning testicular cancer. However, the recent progress in research in the early stages of testicular cancer (carcinoma in situ testis (CIS)) allows us to begin to answer some of these questions. There is more and more evidence that the CIS cell is a gonocyte with stem cell potential, which explains why an adult man can develop a non-seminoma, which is a neoplastic caricature of embryonic growth. We consider the possibility that CIS cells may loose their stem cell potential with ageing. Along these lines, a seminoma is regarded a gonocytoma where the single gonocytes have little or no stem cell potential. The Sertoli and Leydig cells, which are activated postnatally and during and after puberty, may play a crucial role for both the development of the CIS gonocyte and progression of the neoplasm to invasiveness. The reported increase in testicular cancer is not the only sign that male reproductive health is at risk. There are reports that undescended testis and hypospadias have become more common. Also semen quality has deteriorated, at least in some countries. The epidemiological evidence suggests that environmental factors may play a role. Are the environmental hormone disrupters (e.g. DDT, PCB, nonylphenol, bisphenol A) to be blamed for the apparently synchronised deterioration in these aspects of male reproductive health?     

Human skin irritation studies of a lecithin microemulsion gel and of lecithin liposomes

OBJECTIVES: (1) To examine the prevalence and extent of intramural metastasis in squamous cell carcinomas of the oesophagus so as to delineate the resection margins for these tumours; (2) to devise an appropriate method for measurement of these lesions which takes into account of the contraction of the specimens after resection. METHODS: Oesophagectomy specimens were prospectively collected from 96 patients (87 males, nine females) with primary oesophageal squamous cell carcinoma over a two year period. The sizes of the tumours were measured in situ, after resection and after application of muscle relaxant (to regain their in situ length). The specimens were then serially sectioned for histological examination. RESULTS: The sizes of the tumours measured after application of muscle relaxant roughly corresponded to those measured in situ. Intramural metastasis was observed in 26% of the cases. Sixty four per cent (16 cases) of these were on the oral side, 72% (18 cases) on the gastric side, and 25% (nine cases) on both sides of the tumours. The most distant extent of intramural metastasis from the primary tumour was from 0.5 cm to 7.7 cm (mean = 3.4 cm) on the oral side, and 0.5 to 9.5 cm (mean 4 cm) on the gastric aspect of the tumour. Intramural metastasis was seen only in patients in whom the primary cancer had deep muscle infiltration. Multiple neoplastic lesions could be detected in 33% of the patients. Both intramural metastasis and multiple neoplastic lesions were associated with extensive lymph node infiltration. However, they had different histological features and extent of infiltration. CONCLUSIONS: Intramural metastasis was frequently observed in oesophageal squamous cell carcinoma. This implies that excision with wide margins should be considered for local control of the disease.     

Different roles of prepubertal and postpubertal germ cells and Sertoli cells in the regulation of serum inhibin B levels

To elucidate the role of germ cells in the regulation of inhibin B secretion, serum inhibin B levels in prepubertal boys and adult men whom had a concurrent testicular biopsy showing either normal or impaired testicular function were compared. In addition, by immunohistochemistry the cellular localization of the two subunits of inhibin B (alpha and betaB) were examined in adult testicular tissue with normal spermatogenesis, spermatogenic arrest, or Sertoli cell only tubules (SCO) as well as in normal testicular tissue from an infant and a prepubertal boy. Adult men with testicular biopsy showing normal spermatogenesis (n=8) or spermatogenic arrest (n=5) had median inhibin B levels of 148 pg/mL (range, 37-463 pg/mL) and 68 pg/mL (range, 29-186 pg/mL), respectively, corresponding to normal or near-normal levels of our reference population (165 and 31-443 pg/mL; n=358). Men with SCO (n=9) had undetectable or barely detectable (n=1) serum levels of inhibin B. In contrast to adults, prepubertal boys with SCO (n=12) all had measurable serum inhibin B levels that corresponded to our previously determined normal range in healthy prepubertal boys (n=114). However, in postpubertal samples from the same SCO boys, inhibin B levels were undetectable as in the adult SCO men. Intense inhibin alpha-subunit immunostaining was evident in Sertoli cells in both prepubertal and adult testes. In the prepubertal testis, positive immunostaining for the betaB-subunit was observed in Sertoli cells. In the adult testis, intense immunostaining for the betaB-subunit was evident in germ cells from the pachytene spermatocyte to early spermatid stages and to a lesser degree in Leydig cells, but not in Sertoli cells or other stages of germ cells. Thus, surprisingly, in adult men the two subunits constituting inhibin B were expressed by different cell types. We speculate that during puberty Sertoli cell maturation induces a change in inhibin subunit expression. Thus, immature Sertoli cells express both alpha and betaB inhibin subunits, whereas fully differentiated Sertoli cells only express the alpha-subunit. The correlation in adult men between serum inhibin B levels and spermatogenesis may be due to the fact that inhibin B in adult men is possibly a joint product of Sertoli cells and germ cells, including the stages from pachytene spermatocytes to early spermatids.