Björn Risberg

DNA CONTENT AS A PROGNOSTIC MARKER IN PATIENTS WITH ORAL LEUKOPLAKIA

BACKGROUND Oral leukoplakia may develop into squamous-cell carcinoma, which has a poor prognosis. Risk factors for oral carcinoma have been identified, but there are no reliable predictors of the outcome in individual patients with oral leukoplakia. METHODS We identified 150 patients with oral leukoplakia that was classified as epithelial dysplasia and measured the nuclear DNA content (ploidy) of the lesions to determine whether DNA ploidy could be used to predict the clinical outcome. Biopsy specimens obtained at annual follow-up visits were graded histologically and classified with respect to DNA content in a blinded fashion. Disease-free survival was assessed in relation to DNA ploidy and the histologic grade. The mean duration of follow-up was 103 months (range, 4 to 165). RESULTS Among 150 patients with verified epithelial dysplasia, a carcinoma developed in 36 (24 percent). Of the 150 patients, 105 (70 percent) had diploid (normal) lesions, 20 (13 percent) had tetraploid (intermediate) lesions, and 25 (17 percent) had aneuploid (abnormal) lesions at the time of the initial diagnosis. A carcinoma developed in 3 of the 105 patients with diploid lesions (3 percent), as compared with 21 of the 25 patients with aneuploid lesions (84 percent), yielding a negative predictive value of 97 percent with respect to the diploid lesions and a positive predictive value of 84 percent with respect to the aneuploid lesions. Carcinoma developed in 12 of 20 patients with tetraploid lesions (60 percent). The mean time from the initial assessment of the DNA content to the development of a carcinoma was 35 months (range, 4 to 57) in the group with aneuploid lesions and 49 months (range, 8 to 78) in the group with tetraploid lesions (P=0.02). The cumulative disease-free survival rate was 97 percent among the group with diploid lesions, 40 percent among the group with tetraploid lesions, and 16 percent among the group with aneuploid lesions (P<0.001). CONCLUSIONS The DNA content in cells of oral leukoplakia can be used to predict the risk of oral carcinoma.

EGFR family expression in breast carcinomas. c-erbB-2 and c-erbB-4 receptors have different effects on survival

One hundred patients with breast carcinoma followed for 7–11 years were included in the present study of EGFR family members, using immunohistochemistry and RT‐PCR. By immunohistochemistry, 36%, 27%, 26%, and 82% of the tumours were positive for EGFR, c‐erbB‐2, c‐erbB‐3, and c‐erbB‐4. All the immunoreactive tumours were confirmed positive by RT‐PCR. Tumour size, histological grade, lymph node status, S‐phase fraction, and stage were confirmed to be significantly associated with both disease‐free and cancer‐specific survival in the present study. Methods of treatment, histological type, and ploidy had no significant effect on survival. Statistical analysis of EGFR family members in these tumours showed a significant association between c‐erbB‐2 expression and reduced disease‐free and cancer‐specific survival. c‐erbB‐4 expression was associated with a more favourable outcome. Co‐expression of c‐erbB‐2 and EGFR was associated with a worse prognosis. c‐erbB‐4 expression, however, showed an antagonistic effect on the clinical influence of c‐erbB‐2 expression. In conclusion, c‐erbB‐2 expression in breast carcinomas is associated with an unfavourable clinical course and EGFR expression has a synergistic effect. However, c‐erbB‐4 antagonizes the c‐erbB‐2 effect on clinical course in breast carcinomas. To achieve best results with immunotherapy against the c‐erbB‐2 receptor, clarifying the status of c‐erbB‐4 expression may be of significance. Copyright

The role of desmin and N-cadherin in effusion cytology: A comparative study using established markers of mesothelial and epithelial cells

The objective of the present study was to analyze the role of the mesothelial markers desmin and N-cadherin in the diagnostic panel of serous effusions. A total of 181 pleural and peritoneal effusions consisted of 101 cases cytologically diagnosed as malignant (89 carcinomas, 12 mesotheliomas), 78 benign, and 2 inconclusive specimens. All specimens were immunostained using 11 antibodies, against epithelial membrane antigen, Ber-EP4, carcinoembryonic antigen, E-cadherin, CA 125, N-cadherin, desmin, calretinin, p53, vimentin, and CD45. After evaluation of immunocytochemistry results, 110 specimens were diagnosed as malignant (98 carcinomas, 12 mesotheliomas) and 71 as benign (56 cellular, 15 paucicellular). The presence of desmin was detected in benign mesothelial cells in 47 of 56 (84%) reactive cellular specimens compared with 1 of 12 (8%) malignant mesotheliomas and 2 of 98 (2%) carcinomas. N-cadherin was expressed in 48 of 56 (86%) reactive cases, 12 of 12 (100%) mesotheliomas, and 47 of 98 (48%) carcinomas. In carcinomas, N-cadherin expression was most often seen in ovarian carcinoma but was also found in other carcinomas. Calretinin, an established marker of mesothelial cells, was detected in 52 of 56 (93%) reactive specimens, 11 of 12 (93%) mesotheliomas, and 3 of 98 (3%) carcinomas. Evaluation of staining results led to reclassification of six malignant specimens as benign, whereas 17 cases diagnosed as benign and the two diagnosed as inconclusive were classified as malignant. In conclusion, desmin appears to be a promising marker for the distinction between reactive mesothelium and malignant epithelial cells in terms of both specificity and sensitivity, and its complementary use with calretinin is recommended. Unlike calretinin, it may also prove valuable for the distinction between benign and malignant mesothelial cells. N-cadherin does not have a role in the distinction between mesothelial and epithelial cells. However, it may prove useful in the characterization of carcinomas of unknown origin. As has previously been shown, a significant number of diagnoses that are based on morphologic examination alone are modified after the use of a broad antibody panel.

Expression of Ephb2 and Ephb4 in breast carcinoma

Eph receptor tyrosine kinases and their cell-surfacebound ligands, the ephrins, play key roles in diverse biological processes. Eph receptors comprise the largest family of receptor tyrosine kinases consisting of eight EphA receptors (with five corresponding ephrinA ligands) and six EphB receptors (with three corresponding transmembrane ephrinB ligands). Originally identified as neuronal pathfinding molecules, EphB receptors and ephrinB ligands are later proved to be crucial regulators of vasculogenesis and embryogenesis. More studies indicate that Eph receptors are involved in angiogenesis and tumorigenesis. This study aimed to investigate the expression of EphB2 and EphB4 in breast carcinomas. Semiquantitative RT-PCR and immunohistochemistry were used to examine the expression patterns of EphB2 and EphB4. Clinicopathological and survival correlations were statistically analyzed in a series of 94 breast carcinomas, 9 normal specimens and 4 breast carcinoma cell lines. 1(1%), 16(17%), 29(31%), 48(51%) of the 94 tumors were negative, weak, moderate and strong EphB2 protein expression, respectively. 6(6%), 27(29%), 28(30%), 33(35%) of the tumors were negative, weak, moderate and strong EphB4 expression, respectively. Both EphB2 and EphB4 RT-PCR products could be detected in all specimens. Increased EphB2 protein expression was negatively associated with overall survival, and there was a trend that increased EphB2 protein expression was correlated with shorter disease free survival, while EphB4 protein expression was associated with histological grade and stage. EphB4 membrane staining was increased with S phase fraction and associated with DNA aneuploidy. These findings indicate that both EphB2 and EphB4 are involved in the development of breast cancer and that both molecules could be potential predictive markers.

NK- and B-Cell Infiltration Correlates With Worse Outcome in Metastatic Ovarian Carcinoma

We studied the clinical role of leukocyte infiltration and chemokine receptor expression in ovarian carcinoma effusions. Expression of leukocyte markers (CD3, CD4, CD8, CD4/CD8 ratio, CD16, CD19, and CD14) and chemokine receptors (CXCR1, CXCR4, CCR2, CCR5, and CCR7) was studied in 73 effusions by using flow cytometry. CXCR4, CCR5, and CCR7 were expressed abundantly on leukocytes, but all receptors were expressed rarely on cancer cells. The presence of natural killer cells (P = .042) and International Federation of Gynecology and Obstetrics (FIGO) stage IV disease (P = .024) predicted worse overall survival (OS). A higher percentage of CD19+ cells (P = .015) and stage IV disease (P = .008) predicted poor survival for patients with postchemotherapy effusions. Only FIGO stage retained significance as a predictor of OS (P = .035) in multivariate analysis. Chemokine receptors are expressed widely on leukocytes but rarely on carcinoma cells in ovarian carcinoma effusions, arguing against an autocrine chemokine pathway in this malignancy. Immune response parameters in ovarian cancer effusions are weak predictors of outcome.

Kallikrein 4 is a Predominantly Nuclear Protein and Is Overexpressed in Prostate Cancer

Kallikreins (KLKs) are highly conserved serine proteases that play key roles in a variety of physiological and pathological processes. KLKs are secreted proteins that have extracellular substrates and function. For example, prostate-specific antigen (or KLK3) is a secreted protein that is widely used as a diagnostic marker for prostate cancer. KLK4 is a recently identified member of the kallikrein family that is regulated by androgens and is highly specific to prostate for expression. Here, we show that the gene product of KLK4, hK4, is the first member of the KLK family that is intracellularly localized. We provide strong evidence that the previously assigned first exon that was predicted to code for a signal peptide that would target hK4 for secretion is not part of the physiologically relevant form of KLK4 mRNA. In addition to detailed mapping of the KLK4 mRNA 5′ end by RT-PCR, this conclusion is supported by predominantly nuclear localization of the hK4 protein in the cell, documented by both immunofluorescence and cell fractionation experiments. Furthermore, in addition to androgens, hK4 expression is regulated by estrogen and progesterone in prostate cancer cells. Finally, in situ hybridization on normal and hyperplastic prostate samples in tissue microarrays indicate that KLK4 is predominantly expressed in the basal cells of the normal prostate gland and overexpressed in prostate cancer. These data suggest that KLK4 has a unique structure and function compared with other members of the KLK family and may have a role in the biology and characterization of prostate cancer.

E‐cadherin and α‐, β‐, and γ‐catenin protein expression is up‐regulated in ovarian carcinoma cells in serous effusions

The aims of this study were firstly, to investigate the expression of E‐cadherin complex proteins in ovarian carcinoma cells in serous effusions and in primary and metastatic lesions; and secondly to study the value of these four proteins and calretinin, a mesothelial marker, in the differential diagnosis of ovarian carcinoma cells from reactive mesothelial cells in effusions. Sixty‐seven malignant effusions and 97 corresponding primary (n=36) and metastatic (n=61) lesions were immunohistochemically stained for E‐cadherin and α‐, β‐, and γ‐catenin. Staining extent and intensity were scored. Effusion specimens were additionally analysed for calretinin immunoreactivity. Membrane immunoreactivity for E‐cadherin and α‐, β‐, and γ‐catenin was detected on carcinoma cells in the majority of the effusions, but rarely on reactive mesothelial cells (p<0.001 for all markers). Calretinin immunoreactivity was confined to mesothelial cells (p<0.001). An association was seen between E‐cadherin and α‐catenin expression, in both effusions and solid tumours, and for β‐catenin in solid tumours (range p<0.001 to p=0.014). Up‐regulation of all four cadherin complex proteins was seen in carcinoma cells in effusions, when compared with corresponding primary tumours (range p<0.001 to p=0.028). As with effusions, metastatic lesions showed up‐regulation of α‐, β‐, and γ‐catenin when compared with primary carcinomas (p=0.002–0.015). Carcinoma cells in effusions showed in addition elevated levels of E‐cadherin when compared with metastatic lesions (p<0.001). Staining results in effusions showed no association with effusion site, tumour type or histological grade. Immunoblotting on 29 malignant effusions confirmed the presence of all four proteins in the majority of samples and co‐precipitation of E‐cadherin and β‐catenin was seen in ten specimens examined. E‐cadherin complex proteins are widely expressed in ovarian carcinoma cells. Together with calretinin, they form a powerful battery of markers for the cytological diagnosis of carcinoma cells in effusions. The up‐regulation of E‐cadherin complex proteins in serous effusions and metastatic lesions may mark an early metastatic phenotype and possibly mediates survival of tumour cells at these sites through the inhibition of apoptosis. Copyright

Detection of cancer cells in effusions from patients diagnosed with gynaecological malignancies. Evaluation of five epithelial markers.

Abstract The detection of malignant cells in pleural, peritoneal, and pericardial fluids of cancer patients marks the presence of metastatic disease and is associated with a grave prognosis. We evaluated five epithelial markers for the detection of cancer cells in 94 fresh pleural, peritoneal and pericardial effusions. Eighty-four of the samples were regarded as adequate for analysis after evaluation of cytological smears, including 61 samples from patients known to have gynaecological neoplasms. The other 23 samples were from patients with various non-gynaecological malignancies or tumours of unknown origin. Our control cases were 10 fallopian tubes not affected by any malignancy and 12 malignant mesotheliomas. Cell blocks from all cases were stained for CA-125, BerEP4, carcinoembryonic antigen (CEA), BG8 (Lewis Y blood antigen), and B72.3 (TAG-72). Fifty-one of 84 cases were diagnosed as malignant or suggestive of malignancy in cytological smears and/or cell block sections. However, staining for epithelial markers highlighted the presence of malignant cells in 7 additional cases. When membrane staining was evaluated, the sensitivity of the markers studied in detecting malignant cells was as follows: CA-125: 88%, BerEP4: 78%, CEA: 26%, BG8: 86%, B72.3: 79%. Membrane positivity for CEA, B72.3 and BerEP4 was not detected in reactive mesothelial cells. However, membranous staining in mesothelial cells was evident in 13% and 31% of cases with the use of BG8 and CA-125, respectively. Weak cytoplasmic staining for CEA was observed in mesothelial cells in 2 cases. When Ber-EP4, B72.3, and BG8 staining results in cancer cells were combined, the following sensitivity levels were observed: BG8+B72.3: 91%; BG8+Ber-EP4: 90%; B72.3+Ber-EP4: 93%; BG8+ Ber-EP4+B72.3: 95%. The detection of malignant cells in effusions is facilitated by the use of immunocytochemistry using a wide panel of antibodies. BerEP4 and B72.3 appear to be the best markers when both sensitivity and specificity are considered, followed by BG8, while CEA and CA-125 have a limited role in the detection of metastases from gynaecological tumours owing to the low sensitivity of the former and the low specificity of the latter. Analysis of all staining results should be based on a thorough morphological examination.

Granulin-Epithelin Precursor Is a Novel Prognostic Marker in Epithelial Ovarian Carcinoma

The granulin‐epithelin precursor (GEP) was preferentially expressed in invasive ovarian tumor epithelium specimens compared with specimens of borderline ovarian tumors. The objective of the current study was to evaluate the anatomic site‐related and cellular expression of GEP and its association with clinicopathologic parameters and survival in patients with advanced‐stage ovarian carcinoma.

Flow cytometric measurements of DNA index and S-phase on paraffin-embedded early stage endometrial cancer: An important prognostic indicator

Paraffin-embedded curettage material from 120 patients with stage I and II endometrial cancer were analyzed by flow cytometry. The follow-up time in all cases was at least 5 years or until death. It was possible to determine DNA ploidy in 111 cases and S-phase was also evaluated in 92/111 cases. DNA ploidy and S-phase were then compared with the FIGO grading and clinical outcome. DNA ploidy results showed that 11% of grade 1 tumors were aneuploid, 14% of grade 2, 42% of grade 3, and 85% of the cases of uterine papillary serous carcinoma (UPSC) were aneuploid. Patients with tumors that showed an S-phase fraction below 5% had a cumulative 5-year cancer mortality of 7% whereas 49% of the patients with tumors having S-phase fraction above 10% died of their cancer. The prognostic significance of DNA and S-phase correlated fairly well with the FIGO grading as determined by a pathologist with special interest in gynecologic oncology and the DNA parameters added prognostic information independent of the FIGO grading.