Timothy D. Jones

OCT4 staining in testicular tumors: A sensitive and specific marker for seminoma and embryonal carcinoma

OCT4 (POU5F1) is a transcription factor expressed in embryonic stem and germ cells and is involved in the regulation and maintenance of pluripotency. It has been detected in primary testicular germ cell tumors with pluripotent potential, seminoma, and embryonal carcinoma. We undertook immunohistochemical staining of OCT4 in a wide variety of primary testicular neoplasms (germ cell tumors and other tumors) to assess the specificity and usefulness of this marker as a diagnostic tool. We examined histologic sections from 91 primary testicular neoplasms, including 64 cases of mixed germ cell tumors containing embryonal carcinoma (54), seminoma (51), yolk sac tumor (38), mature teratoma (31), immature teratoma (20), and choriocarcinoma (15). In addition, we examined sections from spermatocytic seminomas (5), Leydig cell tumors (8), Sertoli cell tumors (6), unclassified sex-cord stromal tumors (4), adenomatoid tumors (2), testicular tumor of adrenogenital syndrome (1), and granulosa cell tumor (1). Each tumor was examined with hematoxylin and eosin staining and with antibodies to OCT4. In all cases of mixed germ cell tumor with components of embryonal carcinoma (54) and seminoma (51), there was greater than 90% nuclear staining of the embryonal carcinoma and seminoma tumor cells with little to no background staining. In all but 1 of these cases (embryonal carcinoma), there was strong (3+) staining intensity. The other germ cell tumor components (yolk sac tumor, mature teratoma, immature teratoma, and choriocarcinoma) showed no staining. Syncytiotrophoblast cells, which were present in 15 of the cases, were also completely negative, as were all 5 of the spermatocytic seminomas. The 22 cases of non-germ cell tumors were all immunohistochemically negative for OCT4. Fifteen of the 54 germ cell tumors containing embryonal carcinoma were also examined with antibodies to CD30. These embryonal carcinoma components were all positive for CD30 with staining of greater than 90% of the tumor cells but with variable staining intensity. We conclude that immunostaining with antibodies to OCT4 is a useful diagnostic tool in the identification of primary testicular embryonal carcinomas and “usual,” but not spermatocytic, seminomas. OCT4 immunostaining has comparable sensitivity but greater consistency compared with CD30 in the diagnosis of embryonal carcinoma.

OCT4: biological functions and clinical applications as a marker of germ cell neoplasia

Germ cell tumours (GCTs) are a heterogeneous group of neoplasms, which develop in the gonads as well as in extragonadal sites, that share morphological patterns and an overall good prognosis, owing to their responsiveness to current surgical, chemotherapeutic, and radiotherapeutic measures. GCTs demonstrate extremely interesting biological features because of their close relationships with normal embryonal development as demonstrated by the pluripotentiality of some undifferentiated GCT variants. The similarities between GCTs and normal germ cell development have made it possible to identify possible pathogenetic pathways in neoplastic transformation and progression of GCTs. Genotypic and immunophenotypic profiles of these tumours are also useful in establishing and narrowing the differential diagnosis in cases of suspected GCTs. Recently, OCT4 (also known as OCT3 or POU5F1), a transcription factor that has been recognized as fundamental in the maintenance of pluripotency in embryonic stem cells and primordial germ cells, has been proposed as a useful marker for GCTs that exhibit features of pluripotentiality, specifically seminoma/dysgerminoma/germinoma and embryonal carcinoma. The development of commercially available OCT4‐specific antibodies suitable for immunohistochemistry on paraffin‐embedded specimens has generated increasing numbers of reports of OCT4 expression in a wide variety of gonadal and extragonadal GCTs. OCT4 immunostaining has been shown to be a sensitive and specific marker for seminomatous/(dys)germinomatous tumours and in embryonal carcinoma variants of non‐seminomatous GCTs, whether in primary gonadal or extragonadal sites or in metastatic lesions. Therefore, OCT4 immunohistochemistry is an additional helpful marker both in the differential diagnosis of specific histological subtypes of GCTs and in establishing a germ cell origin for some metastatic tumours of uncertain primary. OCT4 expression has also been reported in pre‐invasive conditions such as intratubular germ cell neoplasia, unclassified (IGCNU) and the germ cell component of gonadoblastoma. Additionally, OCT4 immunostaining shows promise as a useful tool in managing patients known to be at high risk for the development of invasive GCTs. Copyright

Molecular Genetic Evidence for a Common Clonal Origin of Urinary Bladder Small Cell Carcinoma and Coexisting Urothelial Carcinoma

In most cases, small-cell carcinoma of the urinary bladder is admixed with other histological types of bladder carcinoma. To understand the pathogenetic relationship between the two tumor types, we analyzed histologically distinct tumor cell populations from the same patient for loss of heterozygosity (LOH) and X chromosome inactivation (in female patients). We examined five polymorphic microsatellite markers located on chromosome 3p25-26 (D3S3050), chromosome 9p21 (IFNA and D9S171), chromosome 9q32-33 (D9S177), and chromosome 17p13 (TP53) in 20 patients with small-cell carcinoma of the urinary bladder and concurrent urothelial carcinoma. DNA samples were prepared from formalin-fixed, paraffin-embedded tissue sections using laser-assisted microdissection. A nearly identical pattern of allelic loss was observed in the two tumor types in all cases, with an overall frequency of allelic loss of 90% (18 of 20 cases). Three patients showed different allelic loss patterns in the two tumor types at a single locus; however, the LOH patterns at the remaining loci were identical. Similarly, the same pattern of nonrandom X chromosome inactivation was present in both carcinoma components in the four cases analyzed. Concordant genetic alterations and X chromosome inactivation between small-cell carcinoma and coexisting urothelial carcinoma suggest that both tumor components originate from the same cells in the urothelium.

Molecular Evidence Supporting Field Effect in Urothelial Carcinogenesis

Purpose: Human urothelial carcinoma is thought to arise from a field change that affects the entire urothelium. Multifocality of urothelial carcinoma is a common finding at endoscopy and surgery. Whether these coexisting tumors arise independently or are derived from the same tumor clone is uncertain. Molecular analysis of microsatellite alterations and X-chromosome inactivation status in the cells from each coexisting tumor may further our understanding of urothelial carcinogenesis. Experimental Design: We examined 58 tumors from 21 patients who underwent surgical excision for urothelial carcinoma. All patients had multiple separate foci of urothelial carcinoma (two to four) within the urinary tract. Genomic DNA samples were prepared from formalin-fixed, paraffin-embedded tissue sections using laser-capture microdissection. Loss of heterozygosity (LOH) assays for three microsatellite polymorphic markers on chromosome 9p21 (IFNA and D9S171), regions of putative tumor suppressor gene p16, and on chromosome 17p13 (TP53), the p53 tumor suppressor gene locus, were done. X-chromosome inactivation analysis was done on the urothelial tumors from 11 female patients. Results: Seventeen of 21 (81%) cases showed allelic loss in one or more of the urothelial tumors in at least one of the three polymorphic markers analyzed. Concordant allelic loss patterns between each coexisting urothelial tumor were seen in only 3 of 21 (14%) cases. A concordant pattern of nonrandom X-chromosome inactivation in the multiple coexisting urothelial tumors was seen in only 3 of 11 female patients; of these 3 cases, only one displayed an identical allelic loss pattern in all of the tumors on LOH analysis. Conclusion: LOH and X-chromosome inactivation assays show that the coexisting tumors in many cases of multifocal urothelial carcinoma have a unique clonal origin and arise from independently transformed progenitor urothelial cells, supporting the “field effect” theory for urothelial carcinogenesis.

Overexpression of Pim-1 during progression of prostatic adenocarcinoma

Aims: Pim-1 is a serine/threonine kinase that has been shown to play an integral role in the development of a number of human cancers, such as haematolymphoid malignancies. Recently, evidence has shown Pim-1 to be important in prostatic carcinogenesis. In order to further our understanding of its role in prostate cancer, we investigated Pim-1 expression in normal, premalignant, and malignant prostate tissue. Methods: Using immunohistochemistry, Pim-1 expression was analysed in prostate tissue from 120 radical prostatectomy specimens. In each case, Pim-1 staining was evaluated in benign prostatic epithelium, high grade prostatic intraepithelial neoplasia (PIN), and prostatic adenocarcinoma. The number of positively staining cells was estimated, and the intensity of staining was scored on a scale of 0 to 3+. Results: Pim-1 immunoreactivity was identified in 120 cases (100%) of adenocarcinoma, 120 cases (100%) of high grade PIN, and 62 cases (52%) of benign glands. The number of cells staining in benign epithelium (mean 34%) was much lower than that in high grade PIN (mean 80%; p<0.0001) or adenocarcinoma (mean, 84%; p<0.0001). There was no significant difference between high grade PIN and adenocarcinoma in the percentage of cells staining positively for Pim-1 (p = 0.34). The staining intensity for Pim-1 was significantly lower in benign prostatic epithelium than in PIN and adenocarcinoma (p<0.001). There was no statistically significant correlation between the level of Pim-1 expression and Gleason score, patient age, tumour stage, lymph node metastasis, perineural invasion, vascular invasion, surgical margin status, extraprostatic extension, or seminal vesicle invasion. Conclusions: Pim-1 expression is elevated in PIN and prostatic adenocarcinoma compared with benign prostatic epithelium. This finding suggests that upregulation of Pim-1 may play a role in prostatic neoplasia.

Anatomic distribution and pathologic characterization of small-volume prostate cancer (<0.5 ml) in whole-mount prostatectomy specimens.

Some investigators consider small-volume prostate cancer (0.5 ml or less) without Gleason pattern 4/5 elements as clinically insignificant. The objective of this study was to characterize the anatomic distribution and pathologic features of small tumors (aggregate volume of 0.5 ml or less) in whole-mount prostatectomy specimens. Between 1999 and 2003, 371 consecutive patients underwent radical prostatectomy at the Indiana University Hospitals for localized prostate cancer. Patients who received hormonal or radiation therapy prior to the surgery were excluded from the study. A total of 62 specimens with total tumor volume of 0.5 ml or less were identified and included in this study. All specimens were embedded and whole-mounted. Tumor volume was measured using the grid method. The mean age at the time of surgery was 59 years (median, 61 years; range, 37–72 years). The mean preoperative prostate-specific antigen (PSA) was 6.5 ng/ml (range: 0.3–18 ng/ml). The mean prostate weight was 53 g (range: 16–132 g). The mean tumor volume was 0.29 ml (median, 0.35 ml; range, 0.02–0.48 ml). Tumor multifocality and bilaterality were present in 69 and 37% of cases, respectively. Three (5%) had positive surgical margins. The largest tumor was located in the peripheral zone, transitional zone, and central zone in 79, 16, and 5% of cases, respectively. The largest tumor was located in the anterior prostate in 10 cases (16%) and in the posterior prostate in 52 cases (84%). The distribution of Gleason scores was 5 (12 cases, 19 %), 6 (40 cases, 65 %), and 7 (10 cases, 16 %). One case had a primary Gleason pattern 4. None had extraprostatic extension, seminal vesicle invasion, or lymph node metastasis. Small-volume prostate cancers are often multifocal and bilateral, with predilection for the peripheral zone. Of these small-volume cases, 16% had Gleason pattern 4 and might, therefore, be clinically significant.

Molecular Genetic Evidence for the Independent Origin of Multifocal Papillary Tumors in Patients with Papillary Renal Cell Carcinomas

Purpose: In patients with papillary renal cell carcinoma, it is not uncommon to find two or more anatomically distinct and histologically similar tumors at radical nephrectomy. Whether these multiple papillary lesions result from intrarenal metastasis or arise independently is unknown. Previous studies have shown that multifocal clear cell renal cell carcinomas express identical allelic loss and shift patterns in the different tumors within the same kidney, consistent with a clonal origin. However, similar clonality assays for multifocal papillary renal cell neoplasia have not been done. Molecular analysis of microsatellite and chromosome alterations and X-chromosome inactivation status in separate tumors in the same patient can be used to study the genetic relationships among the coexisting multiple tumors. Experimental Design: We examined specimens from 21 patients who underwent radical nephrectomy for renal cell carcinoma. All patients had multiple separate papillary lesions (ranging from 2 to 5). Eighteen patients had multiple papillary renal cell carcinomas. Seven had one or more papillary renal cell carcinomas with coexisting papillary adenomas. Genomic DNA samples were prepared from formalin-fixed, paraffin-embedded tissue sections using laser-capture microdissection. Loss of heterozygosity assays were done for six microsatellite polymorphic markers for putative tumor suppressor genes on chromosomes 3p14 (D3S1285), 7q31 (D7S522), 9p21 (D9S171), 16q23 (D16S507), 17q21 (D17S1795), and 17p13 (TP53). X-chromosome inactivation analyses were done on the papillary kidney tumors from three female patients. Fluorescence in situ hybridization analysis was done on the tumors of selected patients showing allelic loss at loci on chromosome 7 and/or chromosome 17. Results: Twenty of 21 (95%) cases showed allelic loss in one or more of the papillary lesions in at least one of the six polymorphic markers analyzed. A concordant allelic loss pattern between each coexisting kidney tumor was seen in only 1 of 21 (5%) cases. A concordant pattern of nonrandom X-chromosome inactivation in the coexisting multiple papillary lesions was seen in two of three female patients. A discordant pattern of X-chromosome inactivation was seen in the tumors of the other female patient. Fluorescence in situ hybridization showed that the majority of tumors analyzed had gains of chromosomes 7 and 17. Two patients had one tumor with chromosomal gain and another separate tumor that did not. Conclusion: Our data suggest that, unlike multifocal clear cell renal cell carcinomas, the multiple tumors in patients with papillary renal cell carcinoma arise independently. Thus, intrarenal metastasis does not seem to play an important role in the spread of papillary renal cell carcinoma, a finding that has surgical, therapeutic, and prognostic implications.

Papillary Urothelial Neoplasm of Low Malignant Potential: Evolving Terminology and Concepts

PURPOSE The most controversial aspect of the new WHO 2004/ISUP classification system is the creation of the PUNLMP diagnostic category. We discuss PUNLMP tumors and the WHO 2004/ISUP classification system with an emphasis on tumor morphology and heterogeneity, recurrence and progression rates, tumor genetics, interobserver variability and the usefulness of biomarkers and molecular diagnostic techniques for grading bladder tumors. MATERIALS AND METHODS A literature search using PubMed was performed. All relevant literature concerning PUNLMP and the WHO 2004/ISUP grading system for urothelial neoplasms was reviewed. RESULTS The new WHO 2004/ISUP classification reflects work in progress. Low malignant potential terminology may not reflect the true biological behavior of these tumors. Additionally, interobserver variability in making a diagnosis of PUNLMP is high despite detailed histological criteria. Urine cytopathology in the context of the WHO 2004/ISUP classification does not appear to effectively discriminate PUNLMP from low grade carcinoma. CONCLUSIONS For practical purposes patients with PUNLMP should be treated similarly to patients with low grade, noninvasive urothelial carcinoma. It is our hope that recent advances in the molecular grading of these tumors may eventually supplant traditional morphological classification, allowing a more precise and objective assessment of the biological potential of these tumors.

Molecular Evidence for the Same Clonal Origin of Multifocal Papillary Thyroid Carcinomas

Purpose: Patients with papillary thyroid carcinoma often have two or more distinct papillary tumors at thyroidectomy. Whether these multifocal papillary lesions are clonally related or whether they arise independently is unknown as previous studies have shown conflicting results. Molecular analysis of microsatellite alterations and X-chromosome inactivation status in separate tumors from the same patient can be used to define the genetic relationships among the multiple coexisting tumors. Experimental Design: We examined 64 separate tumors from 22 female patients who underwent thyroidectomy for thyroid carcinoma. All patients had multiple and separate papillary carcinomas (range, two to six). Genomic DNA samples were prepared from formalin-fixed, paraffin-embedded tissue sections using laser-capture microdissection. Loss of heterozygosity assays for three microsatellite polymorphic markers for putative tumor suppressor genes on chromosomes 3p25 (D3S1597), 9p21 (D9S161), and 18p11.22-p11 (D18S53) were done. In addition, X-chromosome inactivation analysis was done on the tumors from all patients. Results: Twenty of 22 (91%) cases showed allelic loss in one or more of the papillary lesions in at least one of the three polymorphic markers analyzed. Concordant allelic loss patterns between coexisting papillary tumors were seen in 20 of 23 (87%) cases. A concordant pattern of nonrandom X-chromosome inactivation in the multiple coexisting papillary lesions was seen in all informative cases. Conclusion: Our data suggest that the multifocal tumors in patients with papillary thyroid carcinoma often arise from the same clone. Thus, intrathryoid metastasis may play an important role in the spread of papillary thyroid carcinoma, a finding that has important therapeutic, diagnostic, and prognostic implications.

Genetically heterogeneous and clonally unrelated metastases may arise in patients with cutaneous melanoma.

Melanoma of the skin frequently metastasizes to multiple regional lymph nodes and to distant sites. It is uncertain whether all metastases originate from the same tumor clone or whether the genetic heterogeneity of the primary tumor is reflected in the multiple metastases. A total of 73 archival, formalin-fixed, paraffin-embedded, melanoma lesions, including 13 primary tumors and 60 metastases, were studied from 13 patients each having 2 or more metastatic tumors. Genomic DNA samples were prepared from tissue sections using laser-assisted microdissection. We find that the majority of melanoma metastases share a common clonal origin with the matched primary tumor. However, significant genetic divergence occurs frequently during the clonal evolution of metastatic melanoma. In addition, using X-chromosome inactivation analysis, we find that, in some cases, multiple coexisting metastases seem to be derived from different, genetically unrelated tumor clones, implying that some primary tumors may arise from more than a single transformed melanocyte.