Interlaboratory reproducibility of liquid-based equivocal cervical cytology within a randomized controlled trial framework.

Within a multicentre controlled trial framework, an external quality control (EQC) was scheduled to evaluate the interlaboratory reproducibility of liquid-based cytology. In particular, this EQC intended to evaluate the reproducibility of the ASCUS diagnosis.A selected set of 30 slides (4 within normal limit cases, 16 atypical squamous cells of undetermined significance; 4 low-grade squamous intraepithelial lesions and 6 high-grade squamous intraepithelial lesions) circulated among the 13 laboratories involved in the trial.Kappa values were obtained from the comparison between individual laboratory diagnoses and majority diagnoses with target diagnoses. Specific kappa values resulted moderate to high for HSIL and low to moderate for LSIL and WNL. Meanwhile, the specific kappa for ASCUS was below 0.4 in 12 of 13 participating laboratories. The lack of reproducibility for ASCUS was not a result of the introduction of this new technology but rather to the low reproducibility of the ASCUS category itself stemming from intrinsic uncertainties in the reporting criteria.

p21 RNA and protein expression in non-small cell lung carcinomas: evidence of p53-independent expression and association with tumoral differentiation.

p21, the product of the WAF1/CIP1/SDI1/mda-6 gene, is an inhibitor of cyclin-dependent kinases. In cell cultures p21 is induced by p53-dependent and p53-independent pathways by DNA damage and induction of differentiation. We investigated p21 RNA and immunohistochemical expression in 43 non-small cell lung carcinomas and corresponding normal lung samples previously investigated for p53 and WAF1 gene status and p53 protein expression. p21 RNA and protein expression in normal and neoplastic tissues were strictly associated (p-0.0001). In normal tissue p21 RNA was expressed at low levels and p21 immunoreactivity was seen in scattered differentiated bronchial, alveolar and stromal cells. In the majority of neoplasms p21 protein and RNA were expressed at higher levels than in the corresponding normal tissues: p21 overexpression was seen in 27 (63%) and 28 (65%) cases respectively. p21 was expressed independently from p53 gene/protein alterations. p21 overexpression was more frequent in well differentiated tumors (P=0.01 and P=0.022 for RNA and protein respectively), and p21 immunoreactivity was usually seen in foci of more pronounced differentiation. We conclude that p21 expression is related to tumor differentiation, and that p53-independent p21 expression is a common feature of in vivo neoplasms.

Tumor microvessel density, p53 expression, tumor size, and peritumoral lymphatic vessel invasion are relevant prognostic markers in node-negative breast carcinoma.

PURPOSE: To determine the absolute and relative value of microvessel density (MVD), p53 and c-erbB-2 protein expression, peritumoral lymphatic vessel invasion (PLVI), and conventional prognosticators in predicting relapse-free (RFS) and overall survival (OS) rates in patients with node-negative breast carcinoma (NNBC). PATIENTS AND METHODS: We monitored 254 consecutive patients with NNBC for a median of 62 months. Intratumoral MVD was measured after microvessels were immunostained using anti-CD31 antibody. p53 and c-erbB-2 protein and hormone receptors were also determined immunocytochemically. Results were analyzed by both univariate and multivariate statistical analysis. RESULTS: Univariate analysis showed that MVD was significantly predictive of both RFS (odds ratio [OR], 8.30; P = .0001) and OS (OR, 4.50; P = .012) when tested as a continuous or dichotomous variable. Likewise, tumor size (OR, 3.16; P = .0012), PLVI (OR, 4.36; P = .0009), estrogen receptor (ER) status (OR, 2.35; P = .016), progesterone receptor (PR) status (OR, 2.00; P = .017), and expression of p53 protein (OR, 2.82; P = .004) were significantly associated with RFS. Tumor size (OR, 3.80; P = .0038) and expression of p53 protein (OR, 2.58; P = .024) were significantly associated with OS by univariate analysis. Multivariate analysis showed that MVD (P = .0004), p53 protein expression (P = .0063), tumor size (P = .0144), and PLVI (P = .0033) were all significant and independent prognostic factors for RFS. However, only tumor size (P = .004) and MVD (P = .047) were independent predictors for OS. c-erbB2 expression was not associated with outcome by either univariate or multivariate analysis. CONCLUSION: MVD, p53 expression, PLVI, and tumor size are independent prognostic indicators of recurrence, which are useful in selection of high-risk NNBC patients who may be eligible to receive adjuvant therapies.

c-erbB-3 and c-erbB-2 protein expression in node-negative breast carcinoma--an immunocytochemical study.

The type I growth factor receptor family has been found to play an important role in the control of normal growth and differentiation. Moreover, the epidermal growth factor receptor and the c-erbB-2 oncogene seem to be implicated in the pathogenesis and behaviour of several cancers, including breast cancer. c-erbB-3 is a new member of the type I receptor family for which there is currently little information available on its expression in neoplastic tissues, and on its possible prognostic significance. This study was undertaken to define the prognostic value of c-erbB-3 expression in a series of node-negative breast cancer (NNBC) patients when compared, by multivariate analysis, with expression of the c-erbB-2 protein and conventional clinicopathological features. cerbB-3 was recognised by the novel monoclonal antibody RTJ1, whereas c-erbB-2 was detected by the polyclonal antibody 21N, using immunocytochemical methods. We found that overexpression of c-erbB-3 occurs frequently in NNBC. Overall, 138 of 212 carcinomas (65%) had some degree of membrane RTJ1 staining, and 28 (13%) showed strong and generalised positivity ( ). Twenty-four per cent of carcinomas had membrane 21N staining, and 12% presented strong and generalised positivity ( ). c-erbB-3 protein expression was significantly associated only with that of c-erbB-2 (P = 0.05), whereas 21N positivity was significantly associated with small tumour size (P = 0.02) and ductal histotype (P = 0.04). No significant correlation between expression of either receptor proteins or relapse-free survival was observed after a median follow-up of 63 months. Applying multivariate analysis, only tumour size approached significance. Our results indicate that analysis of expression of c-erbB-3 and c-erbB-2 alone do not seem to be useful in identifying patients with NNBC at different risk of relapse or death.

Prognostic value of estrogen receptor status can be improved by combined evaluation of p53, Bcl2 and PgR expression: an immunohistochemical study on breast carcinoma with long-term follow-up.

Steroid receptor analysis is the only widely accepted prognostic/predictive marker in breast cancer (BC) treatment. In the present study we evaluated the prognostic role of ER/PgR with p53 and Bcl2, in a series of 277 BC (153 pN1/2, 122 pNO, 2 pNx) with a long-term follow-up (67 months for DFS, 75 months for OS). Our results, besides confirming the usefulness of ER immunohistochemical expression as a prognostic marker, showed that PgR expression alone had a borderline/not significant prognostic value in the whole series (p=0.08 for DFS and p=0.09 for OS), while showed to be prognostic in N+ cases (p=0.02 for DFS and p=0.03 for OS). PgR prognostic value, however, was not independent at the multivariate analysis. By combining ER with PgR, p53 and Bcl2, we showed that ER/p53 and ER/Bcl2 phenotypes had a better discriminant role than ER/PgR phenotype. The ER+/p53+ phenotype was at higher risk of relapse/death as compared with ER+/p53- phenotype. Conversely ER-/p53+ phenotype showed the most unfavourable prognosis. Similar results could be observed concerning ER/Bcl2 phenotypes. Our study showed that the combined evaluation of ER/PgR weakly enhanced the prognostic/predictive power of ER status alone. On the contrary, the combined evaluation of ER/p53 and ER/Bcl2, improved this prognostic/predictive capability and allowed the separation of ER positive and ER negative cases into subgroups with different prognosis.

Bcl-2 and p53 expression in node-negative breast carcinoma: a study with long-term follow-up.

Bcl-2 and p53 gene products (Bcl-2, p53) are important regulators of apoptosis and cell proliferation, and their immunohistochemical expression may help to identify high-risk breast cancer patients. The authors evaluated p53 and Bcl-2 immunoreactivity in 178 node-negative breast cancers (NNBC) with long-term follow-up (median, 60 months). Bcl-2 was seen in 111 (62%) cases, and was significantly associated with small tumor size, nonductal morphology, low tumor grade, estrogen-receptor (ER) positivity, and p53 negativity. p53 overexpression (ie, > 15% reactive nuclei) was observed in 31 (17%) cases, and was associated with lower age, large tumor size, ductal morphology, high tumor grade, negative ER status, and lack of Bcl-2 immunoreactivity. In univariate analysis, the variables associated with short relapse-free survival (RFS) were large tumor size (P = .002), high histological grade (P = .01), high mitotic count (P = .03), and high Nottingham prognostic index (NPI) (P = .0002). In multivariate analysis (final model), only the NPI was of independent prognostic value concerning RFS.

p53 and c-erbB-2 protein expression in breast carcinomas. An immunohistochemical study including correlations with receptor status, proliferation markers, and clinical stage in human breast cancer.

Receptor status, proliferative activity, loss of differentiation, inactivation of tumor suppressor genes, and overexpression of oncogenes are related events that may affect the prognosis of patients with breast cancer. Ninety-seven unselected breast carcinomas were immunostained for estrogen and progesterone receptors, Ki-67 proliferation-associated antigen, p53 tumor suppressor gene product (p53), and c-erbB-2 protein. Immunohistochemical results and clinical data were compared. Altered p53 expression (regarded as indirect indication of inactivating gene alterations) was found in 25.8% of cases and was associated with a high Ki-67 labeling index, high mitotic count, and high histologic grade, with c-erbB-2 overexpression, and with negative estrogen and progesterone receptor status. p53 immunostaining could be found also in cytologic samples and correlated with p53 immunoreactivity on frozen sections of the corresponding tumors. c-erbB-2 protein overexpression was seen in 24.7% of cases and was associated with p53 altered expression and negative receptor status. Double immunohistochemical staining showed p53 and c-erbB-2 immunoreactivity in the same cells. Median and mean +/- standard deviation Ki-67 labeling index values were 15 and 16.32 +/- 10.05, respectively. Ki-67 labeling index was correlated with high mitotic count and was positively associated with histologic grade, negative progesterone receptor status, and p53 expression. Estrogen receptor status was not associated with any histologic or clinical parameters, whereas progesterone receptor status was associated with grading. The direct relation of p53 protein alterations with c-erbB-2 overexpression may be interpreted in light of the multistep model of tumor progression. Cases with altered expression of both p53 and c-erbB-2 proteins could be interpreted as having lost one inhibitory control mechanism of cell proliferation and having gained one activator of the malignant potential. However, in comparing cases with the p53 + c-erbB-2 + phenotype with cases showing positivity for only one of these gene products, no association with higher stages was seen. Detection of p53 altered expression on cytologic samples of malignant tumors may have diagnostic relevance, and p53 immunostaining may prove to be an additional diagnostic criterion in cytologic diagnosis.

Robotic telepathology for intraoperative remote diagnosis using a still-imaging-based system.

The aim of the present study was to assess whether a telemicroscopy system based on static imaging could provide a remote intraoperative frozen section service. Three pathologists evaluated 70 consecutive frozen section cases (for a total of 210 diagnoses) using a static telemicroscopy system (STeMiSy) and light microscopy (LM). STeMiSy uses a robotic microscope, enabling full remote control by consultant pathologists in a near real-time manner. Clinically important concordance between STeMiSy and LM was 98.6% (95.2% overall concordance), indicating very good agreement. The rates of deferred diagnoses given by STeMiSy and LM were comparable (11.0% and 9.5%, respectively). Compared with the consensus diagnosis, the diagnostic accuracy of STeMiSy and LM was 95.2% and 96.2%. The mean viewing time per slide was 3.6 minutes, and the overall time to make a diagnosis by STeMiSy was 6.2 minutes, conforming to intraoperative practice requirements. Our study demonstrates that a static imaging active telepathology system is comparable to dynamic telepathology systems and can provide a routine frozen section service.

Quantitative growth fraction evaluation with MIB1 and Ki67 antibodies in breast carcinomas.

Ki67 and MIB1 monoclonal antibodies are directed against different epitopes of the same proliferation-related antigen. Whereas Ki67 works only on frozen sections, MIB1 may be used also on fixed sections. The authors immunostained a series of 40 breast carcinomas with MIB1 and Ki67 antibodies on serial frozen sections and on fixed material. The Ki67 labeling index (LI) was 12.9 +/- 8.9 and 12 (mean +/- SD and median, respectively). MIB1 LI was 21.2 +/- 11.9 and 19.5 on frozen sections and 24 +/- 15.2 and 21.5 on fixed sections (mean +/- SD and median, respectively). Ki67 LI and MIB1 LI on frozen and fixed sections were strictly correlated (P < .001). The results are in keeping with the reported coincidental nuclear staining pattern of Ki67 and MIB1, but the mean and median values of MIB1 LI are almost twice the values of Ki67 LI. The cut-off values to define high and low proliferative activity with the two antibodies are therefore different. The differences in immunolabeling may be due to better survival of the MIB1 epitope in freezing and acetone fixation or to differing accessibilities of the MIB1 and Ki67 epitopes during the cell cycle due to molecular conformational modifications. The MIB1 monoclonal antibody is a reasonable substitute for the Ki67 monoclonal antibody. The advantages of MIB1 immunostaining on paraffin sections include the feasibility of retrospective studies and of obtaining clear morphologic specimens that are optimal for use with computer-assisted image analysis systems. Our image-processing system allows automatic nuclear counting, detects positive nuclei and measures their staining intensity.

Grading of the prostate carcinoma: cyto-histological correlations on 100 cases.

From January 1980 to December 1983, 154 patients underwent prostatic FNAB and histological control. The sensitivity of cytology was 85%, the specificity 68% and the predictive value of positive cases 83%. The cyto-histological correlation of the grading of the 100 prostatic carcinomas histologically confirmed (85 cytologically positive for carcinoma and 15 negative or uncertain) showed a predictive value of positive results of 58%, ranging from 42% for G3, 50% for G1 and 69% for G2. The usefulness of cytological grading is stressed to monitor the follow-up of those patients treated for prostatic carcinoma who, because of the advanced stage or age, were not surgically treated.

The virtual case: a new method to completely digitize cytological and histological slides.

The purpose of this study was to present a new method for handling histological/cytological cases. Thanks to the introduction of information technology in pathology, including the amenities afforded by robotic microscopes and digital imaging, tissue slides can be represented and evaluated using digital techniques in order to construct virtual cases through completely automated procedures. A virtual case (VC) is composed of a collection of digital images representing a histological/cytological slide at all magnification levels together with all relevant clinical data. In the present study, we describe an automated system to manage robotic microscope and image acquisition for the proper construction of VCs. These can then be viewed on a computer by means of an interface ("user-friendly") that allows one to select the more appropriate fields and to examine them at different magnifications, rapidly going from panoramic views to high resolution and vice versa. In comparison with glass slides, VCs have several advantages arising from their digital nature and can be considered a common platform for a wide range of applications such as teleconsultation, education, research, and quality control and proficiency tests.

p53 protein expression in central nervous system neoplasms.

AIMS: To demonstrate, immunohistochemically, p53 protein expression in a selection of central nervous system tumours; to investigate the relation between p53 expression and that of the proliferation related antigen, PCNA. METHODS: Surgical specimens from 86 central nervous system tumours were routinely fixed, paraffin wax embedded, and immunostained with a monoclonal (PAb 1801) and a policlonal antibody (CM1) p53 protein and a monoclonal antibody against PCNA (PC10). Normal brain samples obtained at necropsy and 10 surgically obtained samples of gliotic brain parenchyma were also immunostained. RESULTS: p53 protein expression was observed in 35 of 86 brain tumours, suggesting frequent p53 gene mutation. p53 protein alterations were associated with all grades of malignancy in tumours displaying solely astrocytic differentiation, with the exception of pilocytic astrocytomas. In those showing oligodendroglial or ependymal differentiation they appeared to be restricted almost to only high grade lesions. No p53 immunoreactivity was observed in normal or gliotic brain tissue; p53 altered expression was not related to the percentage of PCNA labelled cells. CONCLUSIONS: The use of sophisticated gene amplification techniques or highly sensitive immunohistochemical methods might be useful in distinguishing between reactive and neoplastic astrocytic lesions, and in the identification of malignant progression in other non-astrocytic glial tumours. Tumours with very similar histogenetic differentiation features might actually be a genetically heterogeneous group with possible different clinical courses.

Image sampling in static telepathology for frozen section diagnosis.

BACKGROUND: A frozen section diagnostic service is often not directly available in small rural or mountain hospitals. In these cases, it could be possible to provide frozen section diagnosis through telepathology systems. Telepathology is based on two main methods: static and dynamic. The former is less expensive, but involves the crucial problem of image sampling. AIMS: To characterise the differences in image sampling for static telepathology when undertaken by pathologists with different experience. METHODS: As a test field, a previously studied telepathology method based on multimedia email was adopted. Using this method, three pathologists with different levels of experience sampled images from 155 routine frozen sections and sent them to a distant pathology institute, where diagnoses were made on digital images. After the telepathology diagnoses, the glass slides of both the frozen sections and the definitive sections were sent to the remote pathologists for review. RESULTS: Four of 155 transmissions were considered inadequate by the remote pathologist. In the remaining 151 cases, the telepathology diagnosis agreed with the gold standard in 146 (96.7%). There was no significant divergence between the three pathologists in their sampling of the images. Each case comprised five images on average, acquired in four minutes. The overall time for transmission was about 19 minutes. CONCLUSIONS: The results suggest that in routine frozen section diagnosis an inexperienced pathologist can sample images sufficiently well to permit remote diagnosis. However, as expected, the internet is too unreliable for such a time dependent task. An improvement in the system would involve integrated real time features, so that there could be interaction between the two pathologists.

PCNA and Ki67 expression in breast carcinoma: correlations with clinical and biological variables.

AIMS: To investigate the expression of two cell cycle related antigens (proliferating cell nuclear antigen (PCNA) and Ki67 related antigen) in a series of breast cancers; and the possible correlations between the PCNA and Ki67 labelling indexes (PCNA-LI and Ki67-LI) and their associations with other biological and clinicopathological variables. METHODS: Ninety six ductal and 10 lobular carcinoma specimens were investigated. Samples were fixed in formalin and in Methacarnoy for localisation of PCNA. Ki67 was immunostained on frozen sections. The PCNA-LI and Ki67-LI were evaluated in relation to tumour size, mitotic count, histological grade, nodal state as well as receptor content and altered expression of the p53 gene. RESULTS: PCNA-LI did not correlate with Ki67-LI, nor was it associated with any other variable examined. A high KI67-LI (above the median value of 13.5) was associated with high grade and mitotic count, negative receptor content, and altered expression of the p53 gene, but not with other variables. CONCLUSIONS: The PCNA-LI does not seem to be a substitute for the Ki67-LI in evaluating the growth fraction in breast cancer.

p21 expression in colorectal carcinomas: a study on 103 cases with analysis of p53 gene mutation/expression and clinic-pathological correlations.

The WAF1/CIP1 gene product, p21, an inhibitor of cyclin-dependent kinases, is a critical downstream effector in the p53 pathway. The expression of p21 in human neoplasms is heterogeneous, and may be related to p53 functional status. We evaluated p21 immunoreactivity in 103 colorectal carcinomas (CC) in relation to the p53 gene and protein alterations and clinico-pathologic parameters. High p21 expression (more than 10% reactive cells) was seen in 39% of cases. p21 staining was heterogeneous and often detected in clusters of tumour cells; in some tumours p21 staining was more pronounced in superficial areas. No relation was seen between p21 immunoreactivity and site of the tumours (right vs left), TNM stage and grade. p21 expression was related to p53 status as evaluated with IHC or with SSCP analyses, low p21 expression usually being associated with p53 protein overexpression (P=0.048) and p53 gene alteration (P=0.005). The strongest associations were seen when the combined p53/p21 immunophenotype was compared with p53 gene alterations (P=0.0002). These data support the hypothesis that p21 expression in CC is mainly related to p53 functional status, suggesting that p21 expression could be an interesting adjunct in the evaluation of the functional status of the p53 pathway in CC.

Histoprognostic evaluation of papillary bladder tumors.

A total of 799 cases of papillary bladder tumors were reviewed using both Franksson and Koss classifications. We report the results of a comparative analysis of the two classifications. No correlation between the histologic gradings in the two classifications emerges, especially regarding papilloma (Franksson, 39.3 per cent; Koss, 2.3 per cent). In both classifications recurrences are more frequent as the degree of malignancy increases, although recurrence cannot be excluded even in the lowest grades. As a result of our study, it appears that cellular anaplasia rather than invasion is a better criterion for reliable histoprognostic evaluation of papillary bladder tumors.

Clinical value of pathologic changes after intravesical BCG therapy of superficial bladder cancer.

Bladder pathologic features related to intravesical bacillus Calmette-Guerin (BCG) therapy in superficial bladder cancer (Ta, T1, Tis) were evaluated and related to clinical outcome. A total of 105 patients were treated with 75 mg Pasteur BCG weekly for six consecutive weeks. When tumor was not demonstrated a maintenance course was given. An additional six-week course was given when tumor recurrence or persistence, without progression, was observed after the induction course. An inflammatory change in the bladder was the most common pathologic finding. Granuloma was the only specific BCG-related feature and did not appear to be a prognostic factor because of low incidence (24%) and lack of correlation with clinical course. Dysplasia occurred more frequently (57%) in nonresponder patients and (26%) in responder patients, often heralding recurrence of tumor. All patients showing concurrent squamous and/or glandular metaplasia were unresponsive to BCG therapy. Histology and cytology did not correlate perfectly: cytology was ineffective in low-grade tumors and improved diagnostic accuracy, particularly when dysplasia was histologically evident.

p53 Protein expression in nevi and melanomas.

p53 Protein immunohistochemical expression is a wide-spread feature of the malignant phenotype; most melanomas are reported as p53 positive, while nevi are reported as p53 negative. We investigate a series of 75 benign nevi and 47 melanomas (40 primary and seven metastatic) to evaluate their pattern of p53 immunoreactivity with a panel of specific antibodies (PAb1801, PAb240, DO7, and CM1) in view of a possible diagnostic role of p53 immunostaining. Our results demonstrate that 15% of nevi show p53 immunoreactive nuclei (usually in less than 1% of the cells) and that 30% of melanomas show p53 immunoreactive nuclei (one case with 20% immunoreactive cells, six cases with 1% to 5% positive cells, and four cases with less than 1% positive nuclei). p53 Positivity was seen also in basal and suprabasal keratinocytes. p53 Positivity in nevi is at variance with literature data supporting that nevi are p53 negative. p53 Positivity in nevi and in epidermis may be related to mechanisms of DNA repair, apoptosis, or to a specific phase of the cell cycle. In our series, p53 expression in melanomas is not as frequent as reported in the literature. Population-based differences or differences in case selection and sample handling may account for the above discrepancies. The demonstration of p53 positivity in benign skin lesions greatly hinders the possibility of a diagnostic use of p53 immunostaining in dermatopathology.

Detection of nodal micrometastases using immunohistochemistry and PCR in melanoma of the arm and trunk.

Sentinel node (SN) mapping and biopsy seems at present the best way to assess the nodal status in cutaneous melanoma without removing the lymphatic chain. The procedure is minimally invasive, safe and low cost, and allows selection of patients who can benefit from elective node dissection. From March 1997 up to July 1999 we examined 112 SNs excised after lymphatic mapping from 95 patients (48 males and 47 females) with stage I cutaneous melanoma affecting the trunk or limbs. Of these, 88 SNs from 74 patients were submitted to polymerase chain reaction (PCR) in order to detect tyrosinase mRNA. A new antibody (anti-tyrosinase, Clone T311, IgG2a type, Lab Vision Corporation) was used to detect nodal micrometastases. The search for micrometastases was histologically positive in 15 SNs and negative in 97. The 88 SNs examined using molecular biology were positive in 40 cases and negative in 48. In 28 only the PCR was positive. The new antibody used to detect micrometastases was shown to be very useful. Cases positive on both conventional histology and PCR were Clark level II or more and were thicker than 0.6 mm. No difference with regard to site or sex was observed. Lymphoedema and hypersensitivity reactions, nor the inability to work, did not occur. Only patients with histologically proven micrometastases underwent elective node dissection. Cases positive only on molecular biology were submitted to close follow-up.