Comparative analysis of DNA content estimated by flow and image cytometry in breast tumor samples.

The DNA ploidy status of 186 fresh primary breast tumors was analyzed in a comparative study of flow cytometric (FCM) and image (IA) analyses. Tumor size, histology and nodal status were also taken into account. The same piece of fresh tissue was used for touch imprints (IA) and for DNA analysis by FCM. Both methods provided concordant DI values in 158 (85%) tumors (r = 0.7490). The tumor grade and lymph node status significantly correlate with ploidy estimated by FCM and IA. We conclude that FCM and IA provide comparable results of DNA content although occasional discrepancies occur. IA seems to be a more sensitive method especially for diploid cases detected by FCM.

Current problems in clinico-morphological assessment of soft tissue tumors.

Soft tissue tumors differ in their histology and biological behavior. Irrespectively of changing classification, different grading and staging systems of these lesions are based on similar characteristics and are of comparable clinical value. Electron microscopy, immunohistochemistry and particularly molecular biology facilitate their diagnosis, identification of new tumor entities and provide indicators for their prognosis and strategy of treatment. The new treatment modalities demand tumor characterisation before surgery, whereby sampling of tumor material is of special importance. Fine needle aspiration biopsy, apart of its limitations, is of special value in pre-treatment assessment of soft tissue tumors. Diagnosis and treatment of soft tissue tumors should be performed in cooperation between clinicians and representatives of diagnostic disciplines.

Calretinin and other mesothelioma markers in synovial sarcoma: analysis of antigenic similarities and differences with malignant mesothelioma.

Synovial sarcoma (SS) is a mesenchymal neoplasm that typically shows epithelial differentiation. SS commonly metastasizes to lung and pleura, and has also been reported as the primary in these locations. The histologic distinction of SS from mesothelioma may be difficult because of the combination of epithelioid and spindle cells, potentially shared locations, and antigenic expression. In this study the authors examined 103 well-documented SSs including 41 biphasic, 44 monophasic, and 18 poorly differentiated SSs in comparison with 23 epithelioid and seven sarcomatous mesotheliomas. Most biphasic SSs (29 of 41, 71%) had fields or foci of calretinin-positive tumor cells. The spindle cell components were more often positive (55%), whereas 14% of tumors had positive epithelial cells. The monophasic and poorly differentiated SSs commonly had foci of calretinin-positive cells (in 52% and 56% of cases respectively). In comparison, all 23 epithelioid mesotheliomas (EM) were extensively calretinin positive and seven sarcomatoid mesotheliomas were variably calretinin positive. HBME-1 positivity was similarly detected in biphasic SS and EM (100% and 87% respectively). Among the other sarcomas, two of 15 malignant peripheral nerve sheath tumors were focally calretinin positive, whereas 16 epithelioid sarcomas, 20 leiomyosarcomas, 20 gastrointestinal stromal tumors, and 20 angiosarcomas were negative. Biphasic SSs differed from mesotheliomas by their more common BerEp4 positivity (90%) whereas EMs showed focal reactivity in 13% cases. Marked CD15 reactivity was rare in both. Wilms tumor protein-1 (WT1) was not detected in SS, but was present in 12 of 17 EMs. CD141 was rare in SS, limited to spindle cell components, whereas EMs typically showed prominent membrane staining in epithelial cells. Simple epithelial keratins were present in all epithelial cells of biphasic SS and mesothelioma (keratin 7[K7], K19), but were only focal in monophasic and poorly differentiated SS. Biphasic SSs were extensively K14 positive (89% of cases), whereas epithelial and sarcomatoid mesotheliomas typically showed only scattered positive cells. The potentially shared calretinin patterns in SS and mesothelioma require the use of other markers. The discriminating features include extensive BerEp4 positivity, rarity of CD141, and lack of WT1 in SS. Global expression of K7 and K19 in mesotheliomas versus focal expression in monophasic and poorly differentiated SSs, and differential patterns of K14 expression may also be helpful.

Spectral karyotyping reveals 17;22 fusions in a cytogenetically atypical dermatofibrosarcoma protuberans with a large marker chromosome as a sole abnormality.

The presence of an extra ring chromosome containing material from 17q and 22q, or, less frequently, a t(17;22)(q22;q13), is a cytogenetic hallmark of dermatofibrosarcoma protuberans (DFSP). However, occasionally tumors with other, atypical karyotypes are encountered. We describe a case of recurrent DFSP without a ring chromosome or a t(17;22) on standard cytogenetic analysis. In all cells analyzed by G-banding, an additional, large marker chromosome was present as a sole abnormality. This chromosome apparently included chromosome 8 or the 8q arm, but the origin of its remaining part could not be determined with certainty. To characterize further the abnormal chromosome, we applied spectral karyotyping (SKY). SKY confirmed the presence of an extra chromosome 8 or arm 8q in the marker and showed that its remaining part was composed of segments from chromosomes 7, 17, 21, and 22, with two copies of a 17;22 fusion. Our results and the literature data suggest that, in addition to a specific 17;22 fusion, amplification of material from chromosomes 17, 22, 8, 5, 7, and 21 may play a role in DFSP development and/or progression. Furthermore, our case demonstrates the usefulness of SKY in detection of a diagnostically relevant 17;22 fusion in DFSP patients who have unusual karyotypic features.

Prognostic evaluation of proliferative activity and DNA content in the phyllodes tumor of the breast: immunohistochemical and flow cytometric study of 118 cases.

The goal of this study was the prognostic evaluation of histology, mitotic rate, S-phase fraction (SPF) and expression of proliferative antigen Ki67 and p53 protein in phyllodes tumor of the breast. The study was performed in the group of 118 patients with phyllodes tumor treated by surgery from 1952 to 1998. Mitotic rate was assessed on the representative histological specimens. Expressions of Ki67 and p53 were evaluated by immunohistochemistry on a section from the corresponding paraffin blocks which were also used for flow cytometric DNA evaluation. Histologically, 52 tumors were benign (LGM), 24 borderline malignancies (BM) while among 42 malignant tumors, 20 were monomorphous (HGM) and the remaining 22 revealed heterologic elements (HGH). Tumor recurrencies occurred in 17 patients, predominantly during the first three years after surgery, and 13 patients died of the tumor (1 BM, 12 both malignant variants). Multivariate analysis demonstrated mitotic rate, SPF and p53 expression as independent prognostic parameters for the disease-free survival. Histological tumor type and expression of Ki67 influenced independently the overall survival. In conclusion, the histological type of tumor phyllodes forms the basis for the prognosis of clinical outcome, but the indicators of the proliferative activity, especially Ki67 index, are valuable prognostic factors among patients with malignant variant of phyllodes tumor of the breast. Expression of the p53 protein in tumor cells could be also useful when the percentage of cells and intensity of expression are considered.

Patterns of keratin polypeptides in 110 biphasic, monophasic, and poorly differentiated synovial sarcomas.

Synovial sarcoma is a mesenchymal neoplasm of unknown histogenesis that shows various degrees of epithelial differentiation. It is known to contain simple epithelial keratins, and the possibility of complex epithelial keratin expression has been suggested. In this study, we immunohistochemically examined 110 well-documented synovial sarcomas including 44 biphasic, 48 monophasic, and 18 poorly differentiated (undifferentiated, highly mitotically active) tumors for 11 different keratin (K) polypeptides of the Moll catalogue. The epithelia of biphasic synovial sarcomas showed consistent, extensive reactivity for K7, K8, K14, K18, and K19. Other keratins seen in the epithelia of biphasic tumors included K17 (variable, in 77%), K13 (25%), K16 (23%), and K6 (24%) in the minority of biphasic tumors, predominantly in stratified-appearing epithelia. K10 was detected only focally in one case that showed keratinizing squamous differentiation. Focal expression of K20 was seen in 27% of cases. Monophasic synovial sarcomas had a more limited keratin repertory. Simple epithelial keratin positivity was detected, usually focally for K7 (79%), K19 (60%), K8 (45%), and K18 (46%). Two cases showed more extensive keratin positivity in the spindle cells. The monophasic tumors showed limited positivity for complex epithelial keratins: K14 (28%) and K17 (10%). K20 was detected focally in 6% of the monophasic tumors; other keratins were not detected. The poorly differentiated synovial sarcomas showed limited simple epithelial keratin reactivity, usually limited to scattered cells: K19 (61%), K7 (50%), K18 (47%), K8 (33%), but five cases showed more extensive positivity. Complex epithelial keratins were scant: K14 in one case and K17 in two cases. The immunoreactivity of capillary endothelia seen for K7 and K18 (but not for K8 and K19 with the antibodies used) is a potential diagnostic pitfall, and may cause overdiagnosis of synovial sarcoma if not properly recognized. In summary, we show complex patterns of keratins in synovial sarcoma, especially in the biphasic tumors. Such patterns establish a baseline in differential diagnostic considerations, and give an insight into the complex epithelial differentiation of this enigmatic mesenchymal tumor.

[Tumors of borderline malignancy or atypical proliferative tumors of the ovary?]. / Guzy o granicznej zlosci czy atypowe proliferacyjne guzy jajnika?

Nearly sixty years have passed since Taylor first published the concept of ovarian epithelial semimalignant tumors. After confirmation of this suggestion by FIGO and WHO these tumors became the subject of analyses which helped to separate unfavourable prognostic cases in this group. We would like to present a description of basic and latest papers on borderline ovarian tumors.

DNA analysis of laryngeal carcinoma cells by flow cytometry: the histoclinical factors and their significance.

In patients suffering from several types of malignant tumours, changes in deoxyribonucleic acid (DNA) content are usually associated with poorer survival prognosis. In the present study, DNA content and clinical and histopathologic features were analyzed in patients suffering from laryngeal carcinoma, with a view to establishing the crucial prognostic factors. In the 5-year follow-up study, flow cytometry was used to analyze DNA content in the paraffin-embedded samples of laryngeal carcinoma tissue obtained from 90 patients who had undergone surgical treatment in the Department of Otolaryngology, Collegium Medicum, Jagiellonian University, Cracow, Poland, in 1987 and 1988. The group consisted of 59 and 31 patients with T3 and T4 tumours, respectively. In each case, neck dissection was carried out either on one or both sides. Metastases in regional lymph nodes were found in 26 patients. The disease-free 5-year survival rate was 55.6%. Among the investigated cases, there were 14 aneuploid and 76 diploid tumours. The treatment yielded the worst results, when the S-phase fraction (SPF) and proliferative index (PI) were equal to or higher than 15.8% and 16.0%, respectively. The values of SPF and PI index did not correlate, however, with the frequency of regional metastases. Univariate analysis revealed that tumour size (T stage), presence of lymph node metastases, age of patients (< or = 60, > 60), tumour differentiation, tumour front grading (<15 points, > or = 15), mode of infiltration, SPF, and PI were positively correlated with the actual survival rate. Presence of lymph node metastases (p = .0001) and the PI (p = .0067) were found to be the only independent prognostic factors when the Cox multivariate analysis was applied. The assessment of the PI by flow cytometry may effectively facilitate the selection of patients recommended for a more aggressive treatment.

Prognostic significance of telomerase activity in soft tissue sarcomas.

Only few reports on the prognostic significance of telomerase activity in human cancer exist. To find a new prognostic marker in soft tissue tumors, we investigated 60 soft tissue sarcomas of different histology and six benign tumors for telomerase activity. Telomerase activity was measured by using the non-radioactive PCR-based TRAP-assay. PCR products were analyzed on an automated fluorescence sequencer. Tumors of grade-II and grade-III histology showed a significantly poorer prognosis. Both disease-free (p<0.03) and the overall survival (p<0.02) were reduced in the highly malignant sarcoma patients. We found telomerase activity in 38.3% of the cases, there being a correlation with a more aggressive behavior of soft tissue sarcomas. Telomerase activity correlated with the grade of malignancy (p=0.04), but not with sex (p=0.64) or age (p=0. 48) of the patients. The total survival was significantly reduced in patients with telomerase-positive sarcomas (p=0.04). Both of the patients having grade I tumors with telomerase activity died of disease, whereas 10 of 11 patients with telomerase-negative grade I tumors are still alive. Only one of the benign tumors showed telomerase activity. We suggest that telomerase activity is a potential prognostic factor in malignant soft tissue tumors. Despite the histological heterogeneity of soft tissue tumors, single entities should be assessed for telomerase activity.

Prognostic and predictive evaluation of DNA content, S-phase fraction and immunophenotyping in non-Hodgkin's lymphomas in adults. A prospective study.

In non-Hodgkin's lymphoma (NHL) patients, prognostic significance of S-phase fraction (SPF) is well known, however the significance of DNA ploidy status and antigen expressions is still unclear. The purpose of the present study is to evaluate the prognostic and predictive impact of SPF, immunophenotype and DNA ploidy in prospectively analysed NHL patients. The study was performed on lymph node biopsies of 117 nodal NHL patients. The median follow-up of patients was 25 months (range 1-64 months). All histologically verified lesions were considered according to the Working Formulation and Kiel classification. SPF, immunophenotype and ploidy were determined by flow cytometry. The rate of 2-year overall survival was 56%. SPF and DNA ploidy status were found to be independent prognostic factors in low and high grade lymphomas, respectively. Patients with near-tetraploid lymphomas were characterised by unfavourable clinical outcome, whereas hypertetraploidy was a favourable prognostic indicator. Among B-cell lymphomas CD5 expression seems to be of prognostic significance.

Prognostic factors in retroperitoneal sarcomas: ploidy of DNA as a predictor of clinical outcome.

BACKGROUND AND OBJECTIVES: Radical surgery is the best mode of treatment of retroperitoneal sarcomas (RS); however, common recurrences are unpredictable. METHODS: For the better understanding of outcomes and possibilities of treatment retrospective analysis of different factors, including DNA content, was performed based on 70 patients. RESULTS: Leiomyosarcoma and liposarcoma were most common histologic types of classified sarcomas. Different kinds of resection were successfully performed in 51 patients (73%) and 35 of their available DNA specimens were analyzed. The actuarial 5- and 10-year survival rates in the resection group were 53% and 40%, respectively, with the median survival of 57 months. Patients with diploid resected tumors had a better 10-year survival rate (58%), than those patients with aneuploid tumors (25%,)--P<0.005. Those patients with low-grade sarcomas had a significantly longer survival than those with high-grade sarcomas (10-year survival rate: 44% compared to 29%). In the univariate analysis, adjuvant therapy, type of histology, type of surgery, location of tumor, and S-phase fraction had no influence on survival. In the multivariate analysis (Cox), only ploidy was an independent prognostic variable. Relative risk of death was over three times higher for aneuploid than for diploid tumors. CONCLUSION: Tumor ploidy should be analyzed in every case of retroperitoneal sarcoma for better assessment of prognosis and possible indication for adjuvant therapy.

A rare chimeric TLS/FUS-CHOP transcript in a patient with multiple liposarcomas: a case report.

Myxoid liposarcomas harbor a unique and specific t(12;16)(q13,p11) chromosomal translocation. The breakpoint has recently been identified, and involvement of the TLS/FUS gene on chromosome 16 and the CHOP gene on chromosome 12 was demonstrated. We report a case of a 45-year-old woman who developed multiple malignant lipomatous tumors of unknown origin and myxoid/round cell histology at different locations. To examine the diagnostic potential of this translocation and to develop a hypothesis on the origin of the tumors, we used cytogenetic and molecular cytogenetic methods (reverse transcription polymerase chain reaction, RT-PCR). We identified a chimeric RNA transcript in the second recurrence in the thigh/groin, as well as in another tumor in the mediastinum, which has an additional sequence of 33 bp, known as fusion transcript type III. Cytogenetic analysis of another tumor in retroperitoneal space revealed a rare type of unbalanced translocation der(16)t(12;16). We hypothesize that these tumors are metastases rather than multicentric tumors. The detection of the chimeric message in the present case is not only useful for differential diagnosis, but also for analyzing the origin of multiple neoplasms.

Occurrence of malignant non-germ cell components in primary mediastinal germ cell tumours.

METHODS: Thirty-five patients with primary mediastinal germ cell tumours (PMGCT) underwent primary thoracotomy in a 30-year period (1965-1994). Of the 35 patients, 12 had benign teratomas, five pure seminomas and 18 non-seminomatous germ cell tumours. RESULTS: Out of 18 non-seminomatous germ cell tumours, 14 comprised more than one malignant component. In two cases malignant teratomas had an additional malignant non-germ cell component: one a mixed sarcomatous component and the other a neuroendocrinal component. There were different methods of treatment between 1965 and 1994. All but one of patients with seminomas survived for 5 years. Among 18 patients with malignant PMGCT, all but two died within 5 years (mean survival rate was 15 months). CONCLUSIONS: When planning treatment of patients with malignant PMGCT we have to take into account the fact that malignant non-germ-cell components may occur. In this circumstances, surgical resection after initial chemotherapy is recommended.

Prognostic evaluation of cutaneous malignant melanoma: a clinicopathologic and immunohistochemical study.

BACKGROUND AND OBJECTIVES: Depth of invasion and stage of the disease are established prognostic indicators in cutaneous malignant melanoma. The role of other parameters is still an open problem. METHODS: In 93 consecutive patients with cutaneous malignant melanoma, the level of invasion, tumor thickness, ulceration, vascular invasion, lymphoplasmocytic infiltrates, and mitotic index were evaluated by histology. Expression of Ki-67 and PCNA proliferative antigens together with vimentin, S100, and HMB 45 proteins were assessed by immunohistochemistry. RESULTS AND CONCLUSIONS: Disease-free and overall survival were correlated with tumor stage, tumor thickness, level of invasion, macroscopic pattern, ulceration, vascular invasion, expression of HMB 45, PCNA, and Ki-67/MIB1. Stage, HMB 45, and PCNA were independent prognostic factors for disease-free survival, whereas tumor stage, tumor thickness, and expression of both proliferative antigens influenced overall survival independently. The variables studied demonstrated reciprocal correlation; therefore, analysis of many prognostic parameters in malignant melanoma could be recommended.

Telomeric lengths and telomerase activity in liposarcomas.

To assess the role of telomerase in the development of liposarcomas, we measured telomerase activity in 36 malignant and seven benign lipomatous neoplasias from 34 patients. A sensitive polymerase chain reaction-based telomerase assay (the telomeric repeat amplification protocol) was applied. Shortening or elongation of telomeric repeat fragment lengths, as measured by using hybridization with a telomere-specific oligonucleotide probe, was correlated with the presence of telomerase activity. The latter was demonstrable in 69% of malignant tumors. Benign tumors can be distinguished from malignant neoplasias on the basis of telomerase activity. However, telomerase expression seems to be characteristic of poorly differentiated liposarcomas. Myxoid/round cell liposarcomas exhibited a higher telomerase activity level than the classical low-grade variants. Telomerase activity was not correlated with age at the time of diagnosis or with sex. In most cases, telomerase-positive tumors showed higher proliferation indices than did neoplasias lacking telomerase. All eight recurrences expressed telomerase activity, reflecting a close association of telomerase with the biological behavior of liposarcomas. Our findings suggest that telomerase may play a key role in the establishment and progression of malignant lipomatous tumors.

Evaluation of DNA content and proliferative activity in prediction of short-term relapses in mammary carcinoma: a prospective study.

DNA content according to six various ploidy classes was analysed by flow cytometry on fresh tumor tissue in 701 unselected, consecutive breast cancers. Age, menopausal status, tumor size, histology and in particular nodal status were also taken into consideration. Diploid and nondiploid mammary carcinomas differed significantly in values of proliferative indicators. In node positive patients nondiploid tumors were more frequent. In node negative and node positive breast cancer patients tumor grade strongly influenced disease free survival and in the last group hypertriploidy (1.6 < DI < or = 1.8) was also an independent prognostic factor. The combination of tumor grade and hypertriploidy permitted differentiation of three groups of node positive patients, differing in the risk of relapses.

The prognostic value of proliferating cell nuclear antigen (PCNA) in the advanced cancer of larynx.

It was retrospectively examined whether the tumor growth fraction determined by PCNA score in the advanced cancer of larynx could be used as a prognostic factor. There was used immunohistochemical method to evaluate the PCNA score of positive cells in paraffin embedded tissues of laryngeal carcinoma obtained from 90 patients. They were treated by surgery in the Department of Otolaryngology at the Medical University in Cracow, Poland in 1987 and 1988. The follow-up period was not shorter than 5 years. In the examined material there were 59 patients with tumor T3 and 31 with T4. Neck dissection on one or on both sides was performed on patients. The metastases in regional lymph nodes was found in 26 patients. Histologic grading (G1 = 18, G2 = 52 and G3 = 20 patients) and number of mitosis was assessed. The PCNA score was defined by counting positive cells and presented as percentage of a cells. It ranged from 2.1 to 73.0% with the average value of 34.5%. The PCNA score below this level was defined as a low PCNA score (49 patients) and above this level as a high PCNA score (41 patients). There was a correlation between PCNA score and survival of the patients. 35 patients (71.4%) with low PCNA score survived and 15 (36.6%) with high PCNA score (P < 0.05). Similarly the high PCNA score influenced the number of metastases in lymph nodes. They occurred in low and high PCNA score respectively at nine (18.4%) and 17 patients (41.5%) (P < 0.05). There was not found any correlation between PCNA score and tumor differentiation, its size and mitosis number. The PCNA may be a prognostic factor for the patients with advanced cancer of larynx.

Prognostic assessment of histologic parameters in breast carcinomas: a prospective study.

A group of 350 unselected breast cancer patients, treated at the Center of Oncology in Cracow, Poland, between January 1992 and December 1994, was analyzed. The following reciprocally interrelated histologic characteristics were evaluated: 1) histologic tumor type (considered in 3 categories of aggressivity), 2) tumor grade (according to Scarf-Bloom-Richardson), 3) constituent of in-situ carcinoma in invasive cancers and characterization of breast lobuli, 4) tumor growth pattern (microfocal, macrofocal or mixed), 5) invasion of nerves, 6) vascular invasion by cancer cells in tumor surroundings, 7) extensiveness of tumor necrosis, 8) involvement of the breast distant from the tumor mass by cancer cells, 9) status of axillary lymph nodes, 10) invasion of metastatic lymph node surroundings. Metastases in axillary lymph nodes were independently influenced by vascular invasion in tumor surroundings and tumor diameter. The disease-free survival was independently influenced by tumor diameter, necrosis and stage of the disease (pTNM), whereas total survival related to tumor diameter, nodal status, microfocal pattern of tumor growth, vascular invasion and involvement of breast by cancer distant from the tumor mass was independently influenced only by tumor stage (pTNM).

Sarcomatoid carcinoma (carcinosarcoma) of the gallbladder.

We report a case of carcinoma of the gallbladder in a 67-year-old woman. The description comprises the histological, immunohistochemical, ultrastructural and cytogenetical picture of the tumor. The ultrastructural features as well as chromosomal changes may denote the epithelial derivation of the tumor studied.

Parachordoma: a rare sarcoma with clonal chromosomal changes.

This is the first report on clonal chromosomal aberrations in a metastatic parachordoma lesion. All neoplastic cells had the karyotype 33-47,X,der(X)t(X;3) (p11;p11),der(3)t(3;6)(p11;q13), del(6)(q13), -9, -13,r(13), +mar. The presence of t(X;3) with a breakpoint at band Xp11 as in synovial sarcoma may suggest a common histological origin of the two tumor types.