Ulrico Schmid

Microarrays of bladder cancer tissue are highly representative of proliferation index and histological grade

The number of genes suggested to play a role in cancer biology is rapidly increasing. To be able to test a large number of molecular parameters in sufficiently large series of primary tumours, a tissue microarray (TMA) approach has been developed where samples from up to 1000 tumours can be simultaneously analysed on one glass slide. Because of the small size of the individual arrayed tissue samples (diameter 0.6 mm), the question arises of whether these specimens are representative of their donor tumours. To investigate how representative are the results obtained on TMAs, a set of 2317 bladder tumours that had been previously analysed for histological grade and Ki67 labelling index (LI) was used to construct four replica TMAs from different areas of each tumour. Clinical follow‐up information was available from 1092 patients. The histological grade and the Ki67 LI were determined for every arrayed tumour sample (4×2317 analyses each). Despite discrepancies in individual cases, the grade and Ki67 information obtained on minute arrayed samples were highly similar to the data obtained on large sections (p<0.0001). Most importantly, every individual association between grade or Ki67 LI and tumour stage or prognosis (recurrence, progression, tumour‐specific survival) that was observed in large section analysis could be fully reproduced on all four replica TMAs. These results show that intra‐tumour heterogeneity does not significantly affect the ability to detect clinico‐pathological correlations on TMAs, probably because of the large number of tumours that can be included in TMA studies. TMAs are a powerful tool for rapid identification of the biological or clinical significance of molecular alterations in bladder cancer and other tumour types. Copyright

High-Throughput Tissue Microarray Analysis of Cyclin E Gene Amplification and Overexpression in Urinary Bladder Cancer

Studies by comparative genomic hybridization revealed that the 19q13 chromosomal region is frequently amplified in bladder cancer. The cyclin E gene (CCNE), coding for a regulatory subunit of cyclin-dependent kinase 2, has been mapped to 19q13. To investigate the role of cyclin E alterations in bladder cancer, a tissue microarray of 2,317 specimens from 1,842 bladder cancer patients was constructed and analyzed for CCNE amplification by fluorescence in situ hybridization and for cyclin-E protein overexpression by immunohistochemistry. Fluorescence in situ hybridization analysis showed amplification in only 30 of the 1,561 evaluable tumors (1.9%). Amplification was significantly associated with stage and grade (P: < 0.0005 each). Immunohistochemically detectable cyclin E expression was strong in 233 (12.4%), weak in 354 (18.9%), and negative in 1, 286 of the 1,873 interpretable tumors. The majority (62.1%) of CCNE-amplified tumors were strongly immunohistochemistry-positive (P: < 0.0001). The frequency of protein expression increased from stage pTa (22.2%) to pT1 (45.5%; P: < 0.0001) but then decreased for stage pT2-4 (29.4%; P: < 0.0001 for pT1 versus pT2-4). Low cyclin E expression was associated with poor overall survival in all patients (P: < 0.0001), but had no prognostic impact independent of stage. It is concluded that cyclin E overexpression is characteristic to a subset of bladder carcinomas, especially at the stage of early invasion. This analysis of the prognostic impact of CCNE gene amplification and protein expression in >1,500 arrayed bladder cancers was accomplished in a period of 2 weeks, illustrating how the tissue microarray technology remarkably facilitates the evaluation of the clinical relevance of molecular alterations in cancer.

Cutaneous follicular lymphoid hyperplasia with monotypic plasma cells. A clinicopathologic study of 18 patients.

Twenty cases of cutaneous follicular lymphoid hyperplasia with monotypic plasma cells are presented in a clinicopathologic study on 18 patients. The plaque-like or nodular lesions were solitary in 10 and multiple in eight patients. Immunohistochemistry showed well-defined B-and T-cell areas. Sheets of monotypic plasma cells occurred either interfollicularly or adjacent to the sclerotic stroma, with expression of lgG/κ in 14 and lgG/κ in six cases. In one patient with multiple lesions, one sample contained polyclonal plasma cells, whereas the other specimen showed light chain restriction. In another patient, disease recurred with a polytypic cutaneous plasma cell infiltrate. Polymerase chain reaction (PCR) revealed clonal immunoglobulin heavy chain gene rearrangements in eight of 13 cases, which was confirmed by Southern blot analysis in three samples. Clonal T-cell receptor chain gene rearrangements were not detected. Disease progression to overt malignant lymphoma did not occur within the follow-up period of up to 12 years, but recurrent disease was seen in three patients. Our data indicate that cutaneous lymphoid hyperplasia with monotypic plasma cells is a biologically distinct clinicopathological entity.

Cyclin D1 overexpression lacks prognostic significance in superficial urinary bladder cancer

The biological behaviour of urinary bladder neoplasms cannot be adequately predicted by histological criteria alone. Cyclin D1 is a cell‐cycle regulating protein known to be overexpressed in a proportion of bladder carcinomas. To evaluate the prognostic significance of cyclin D1 expression and its relationship with tumour phenotype, 392 bladder carcinomas were analysed by immunohistochemistry. Clinical follow‐up information was available in 337 patients with superficial bladder tumours (stages pTa/pT1). Cyclin D1 positivity was seen in 176 of 392 carcinomas. Cyclin D1 overexpression was strongly linked to papillary tumour growth, low stage, and low histological grade (p<0·005 each). Multivariate analysis showed that papillary tumour growth was the only parameter which was independently linked to cyclin D1 positivity. There was no significant difference in proliferative activity (Ki67 labelling index) between cyclin D1‐negative and ‐positive tumours. Cyclin D1 positivity was not linked to the risk of recurrence or tumour progression, either in pTa or in pT1 carcinomas. It is concluded that cyclin D1 positivity distinguishes a large subgroup of papillary bladder tumours, but there is no evidence of prognostic significance for increased cyclin D1 expression. Copyright

Metastatic signet-ring cell melanoma in a patient with an unknown primary tumor: Histologic, immunohistochemical, and ultrastructural findings***

Signet-ring cell melanoma is a rare morphologic variant of malignant melanoma. We describe a 27-year-old man with widespread cutaneous and internal metastases of an unknown primary tumor. One skin biopsy specimen and a fine-needle aspirate showed polygonal tumor cells consistent with the diagnosis of metastatic melanoma, but a second skin biopsy specimen revealed neoplastic cells with a signet-ring cell appearance. Both specimens, however, yielded identical immunohistochemical findings. Tumor cells were positive for vimentin and S-100 protein and reacted with the melanoma markers HMB-45 and NKI-C3. Ultrastructural studies revealed abundant intermediate filaments in the cytoplasm of the signet-ring cells. Based on these findings, a diagnosis of metastatic melanoma was made. In this report we describe the fourth case of metastatic signet-ring cell melanoma, and discuss how malignant melanoma may lead to metastases that have divergent morphologic appearances in the same patient.

Focal loss of CD44 variant protein expression is related to recurrence in superficial bladder carcinoma.

The majority of papillary transitional cell carcinomas of the bladder are localized tumors at initial diagnosis; identification of those developing recurrence and an aggressive behavior is important. CD44 variant proteins have been implicated in tumor progression and metastasis, and a correlation with adverse prognosis has been demonstrated in a variety of human tumors. Here, the usefulness of conventional CD44 protein immunohistochemistry as a prognostic parameter for recurrence of superficial transitional cell carcinomas was assessed in paraffin sections of 241 tumors with long-term follow-up. A highly significant association was found between focal loss of CD44v3 and -v6 immunostaining and short recurrence-free interval in noninvasive (pTa) transitional cell carcinomas (P = 0.005), but not in minimally invasive (pT1) carcinomas (P = 0.78). Our results indicate the value of conventional CD44 immunohistochemistry as an additional tool for identifying patients at high risk for recurrence of pTa transitional cell carcinomas. They also point to biological differences between noninvasive and minimally invasive transitional cell carcinomas of the bladder.

IgVH mutations in blastoid mantle cell lymphoma characterize a subgroup with a tendency to more favourable clinical outcome

Mantle cell lymphoma (MCL) is associated with a very unfavourable clinical course. This is particularly true for mantle cell lymphoma of the blastoid subtype (MCL‐b). In order to define prognostic factors, we analysed the impact of immunoglobulin heavy chain variable (IgVH) gene somatic hypermutations on clinical outcome in a series of 21 cases of morphologically, phenotypically, and genotypically well‐characterized MCL‐b. Testing and estimation were performed using log‐rank statistics and displayed on Kaplan–Meier graphs. Thirteen of 21 cases of MCL‐b revealed a homology rate of ≥99% compared to IgVH germ‐line sequences in the databases and were scored as non‐mutated. Eight of 21 cases (38%) of MCL‐b were mutated. In MCL‐b the mutation frequency was usually low and the mutation pattern was only rarely antigen‐selected, in contrast to a control group of 11 cases with morphologically almost identical, but phenotypically and genotypically clearly distinguishable, diffuse large B cell lymphoma, derived, most likely, from germinal centre B cells. In our series of 21 MCL‐b, positive IgVH mutational status, irrespective of varying homology thresholds, had no statistically significant prognostic impact on event‐free or overall survival. However, mutated MCL‐b tended to present more frequently at an earlier stage and without bone marrow involvement and to show lower rates of relapse and death, resulting in a more favourable clinical outcome. Copyright

Distribution of cytokeratin polypeptides in syringomas : an immunohistochemical study on paraffin-embedded material

The distribution of cytokeratin (CK) polypeptides expressed in syringomas (12 cases) was compared with that in normal eccrine sweat ducts using immunohistochemical techniques on paraffin-embedded tissue. Intradermal and intraepidermal segments of the eccrine duct showed reactivity with an antibody to CK 1/5/10/11 in all cell layers, whereas CK19 expression was restricted to the luminal cell layer. CK14 was expressed in all cells of the eccrine duct except for the peripheral cells of the intraepidermal duct. Expression of CK5/6 was seen in the basal cells of the dermal duct and of the lower intraepidermal duct (sweat duct ridge) exclusively. Reactivity with an antibody to CK1 was found in the intermediate cells of the uppermost part of the eccrine dermal duct. In addition, this antibody gave a strong staining of the peripheral cells of the intraepidermal duct, leaving basal cells of the sweat duct ridge and luminal cells unstained. In syringoma, CK distribution was essentially comparable with that found in the uppermost part of the dermal duct and in the sweat duct ridge. Namely, ductal luminal cells expressed CK1/5/10/11, CK19, and variably CK14. Intermediate cells of ductal structures and solid nests were homogeneously stained by antibodies to CK1 and CK1/5/10/11, whereas CK14 was expressed heterogeneously. The basal or outermost layer of ductal structures and solid nests was reactive with antibodies to CK1/5/10/11, CK5/6, and CK14. With regard to CK expression, the results indicate that syringoma represents a tumor differentiating toward both the uppermost part of the dermal duct and the lower intraepidermal duct (sweat duct ridge) of the eccrine sweat gland.

Polysomies but not Y chromosome losses have prognostic significance in pTa/pT1 urinary bladder cancer

A disturbed cellular DNA content is of potential diagnostic and prognostic relevance in urinary bladder cancer. To evaluate the prognostic significance of individual chromosomal aberrations in superficial bladder cancer, specimens of 105 tumors (67 pTa, 38 pT1) were examined by fluorescence in situ hybridization (FISH). FISH allows quantitation of chromosomes on a cell by cell level. Centromere probes for the chromosomes Y, 1, and 17 were used. There was a strong association between polysomies of the chromosomes 1 (found in 46% of tumors) and 17 (40% of tumors, P < .0001). Polysomies (1 and 17) were significantly more frequent in pT1 than in pTa tumors (P < .0001 each). In pTa tumors, polysomies of both chromosomes were linked to a high risk of recurrences; polysomy 17 was associated with an increased risk of progression (P < .05 each). There was no significant association between polysomies and an unfavorable prognosis in pT1 carcinomas. Previous studies had suggested a prognostic role of Y losses in bladder cancer. However, Y losses were not linked to recurrences or tumor progression in pTa or pT1 tumors of 67 male patients. These data show that marked genetic differences exist between pTa and pT1 carcinomas. They also indicate that polysomies of different chromosomes may have prognostic relevance in pTa urinary bladder cancer.

Significantly different bcl‐2 expression profiles in gastric and non‐gastric primary extranodal high‐grade B‐cell lymphomas

Fifty‐five cases of primary extranodal high‐grade B‐cell non‐Hodgkins lymphoma were investigated for bcl‐2 and p53 protein expression as well as for t(14;18) translocations and p53 mutations. Phenotypic and genotypic profiles were compared between tumours of gastric (27 cases) and non‐gastric (28 cases) origin. bcl‐2 protein expression was significantly lower in gastric (11/27) than in non‐gastric (28/28) lymphomas (p<0.0001), while nuclear p53 protein expression did not differ significantly between these two groups. In the stomach, there were no significant differences in either bcl‐2 or p53 expression profiles between high‐grade lymphomas with (n=14) and without (n=13) evidence of a low‐grade component of MALT type. However, secondary high‐grade lymphomas showed a significant down‐regulation of bcl‐2 protein (p<0.0001) and, conversely, an up‐regulation of p53 protein (p<0.0001) as compared with their low‐grade tumour components. In extranodal high‐grade B‐cell lymphomas, bcl‐2 protein expression was not associated with t(14;18) translocation. Only one gastric lymphoma had a p53 point mutation with potential alteration of the amino acid sequence. These findings indicate that primary gastric high‐grade B‐cell lymphomas are immunohistologically distinct from primary extranodal high‐grade B‐cell lymphomas of an origin other than in the stomach. Copyright