Assessment of cell proliferation by Ki-67 staining and flow cytometry in fine needle aspirates (FNAs) of reactive lymphadenitis and non-Hodgkin''s lymphomas

This study was undertaken to assess cell proliferation in FNAs from a series of 57 non-Hodgkin's lymphomas (NHL) and 11 cases of reactive lymphadenitis using Ki-67 staining and flow cytometry (FCM). The results were compared and correlated to the cytomorphological subgrouping according to Kiel classification. The mean percentages of Ki-67 positivity were 16.6% and 61.1% for low and high grade lymphomas, respectively (P < 0.001). The mean S-phase fraction (SPF) determined by FCM was 4.61% for low grade and 12.9% for high grade lymphomas (P < 0.001). The figures for Ki-67 positivity and S-phase fraction in reactive lymphadenitis were 16.8% and 40%, respectively. We observed a strong correlation in low grade lymphomas between Ki-67 and SPF. A good correlation was also found in reactive lymphadenitis. In high grade lymphomas, however, with highly scattered Ki-67 and S-phase values, this correlation was lost. In some cases this discrepancy can be explained by a rich admixture of non-neoplastic, non-proliferating cells in aspirates from diploid tumours. In addition, the existence of a minor aneuploid tumour cell population of high proliferation such as that in Ki-1 lymphomas will not be accurately analysed by FCM but is easily assessed by Ki-67 staining. However, the main reason seems to be a high variability between the fraction of cells in S-phase and the total number of cells in G1, S and G2 in individual tumours.     

Cell proliferation in prostatic carcinoma: comparative analysis of Ki-67, MIB-1 and PCNA

Summary Antibodies to assess the proliferative index of tumours are being increasingly employed together with established markers for prognostic evaluation. This study set out to compare three cell proliferation markers, Ki-67, MIB-1 and PCNA, utilizing a semiquantitative method of assessment, in 20 human prostatic carcinomas. The streptavidin-biotin immunostaining system was used for the monoclonal antibodies MIB-1 and PCNA and an indirect immunoperoxidase assay for the monoclonal antibody Ki-67. Significant correlations were found between the expression of Ki-67 in frozen tissues and MIB-1 in formal saline-fixed wax-embedded tissues (p = 0.0003); between Ki-67 and PCNA expression in Bouin's-fixed tissues (p </ 0.0001); and MIB-1 (formalin-saline-fixed tissues) and PCNA (Bouin's-fixed tissues) (p </ 0.0001). A more intense nuclear staining pattern with less heterogeneity was observed for MIB-1 compared with PCNA, suggesting the antibody of choice, on formal saline-fixed tissues, is MIB-1, which closely correlated with Ki-67, a marker we have previously shown to be of prognostic value in prostatic carcinoma.     

MIB1 proliferation index in breast infiltrating carcinoma: comparison with other proliferative markers and association with new biological prognostic factors

AIMS: In breast invasive carcinoma our objectives were I) to compare cellular proliferation determined by MIB1 index with S-phase fraction (SPF) assessed by flow cytometry and with mitotic index, and II) to examine the association of MIB1 index with classical and with new biological prognostic factors [bcl-2, p53, c-erbB-2 and cathepsin D (CD)]. METHODS AND RESULTS: From 102 cases of breast invasive carcinoma, 5-microm thick serial sections were cut from formalin-fixed, paraffin-embedded tissue blocks, and processed for detection of CD, c-erbB-2, p53, bcl-2, Ki-67 antigen MIB-1 and estrogen receptors (ER) and progesterone receptors (PR). SPF was measured by flow cytometry in fresh-frozen tissue samples taken from the carcinoma in each patient. MIB1 index was correlated with SPF (rho=0.45, p<0.0001) and with mitotic index (rho=0.42, p<0.0001). The MIB-1 index was positively associated with the histological grade (p=0.001), tumor size (p=0.04) and the presence of metastases in axillary lymph nodes (p=0.01). MIB1 was associated directly with p53 (p=0.045) and inversely with bcl-2 (p=0.0002). The MIB-1 index was not statistically associated with c-erbB-2. There was a weak association between MIBI index and stromal cell CD. The median MIB1 index was higher in tumors with moderate to strong CD staining of stromal cell, but the difference did not reach statistical significance (p=0.09). CONCLUSIONS: MIB1 index correlates with well established methods for assessing tumor proliferation and with parameters of an aggressive phenotype of tumor. MIB1 index is an effective and readily accessible method for assessing tumor proliferation in breast carcinoma.     

Immunoreactivity to MIB-1 in breast cancer: methodological assessment and comparison with other proliferation indices

The recent availability of the monoclonal antibody MIB-1 (which is able to detect the human nuclear cell proliferation-associated antigen Ki-67 even on formalin-fixed, paraffin-embedded sections, microwave-treated and routinely processed for immunohistochemistry) could open new avenues for validation of the clinical role of tumour cell proliferation on large, consecutive and unselected series of human tumours. However, the routine use of such a marker requires a methodological standardization as well as the comparative assessment of some technical and biological aspects. The MIB-1 index was determined in parallel samples from 50 consecutive invasive breast carcinomas processed with different fixatives for different times. The median values of MIB-1 indices following 2, 6 and 24 h of formalin fixation were similar (29.4%, 30.6% and 29.7%, respectively) and consistent with those reported in the literature; squared linear regression coefficients were 0.99. The median values of MIB-1 indices were markedly lower in Bouin-fixed, paraffin-embedded, and in frozen samples (20.0% and 19.8%, respectively), with a poor correlation coefficient with the values detected following formalin fixation ( R ²=0.456). Moderate and poor correlations were observed between Ki-67 index and MIB-1 detected on frozen ( r _s, 0.78) or formalin-fixed, paraffin-embedded samples ( r _s, 0.47) and a minimal concordance was observed between TLI and MIB-1 or Ki-67 ( r _s, 0.25 and 0.22, respectively). Our results indicate interference of the fixative type on immunoreactivity to MIB-1 and also suggest that Ki-67 and MIB-1 reacted with different epitopes of the same antigen.     

p53 Protein expression and proliferative activity in ductal breast carcinomas

The immunocytochemical expression of p53 protein and Ki-67 labelling index in tumour cells of 100 ductal breast carcinomas of different histological grade and stage was evaluated in cytological material. In order to investigate p53 expression and Ki-67 expression an avidin-extravidin immunocytochemical technique was applied to imprints. Monoclonal antibody (MoAb) DO-p53 and proliferating cell monoclonal antibody were used as primary antibodies. A statistically significant difference was observed between p53 protein expression and grade of malignancy and clinical stage (P = 0.001, P < 0.001, respectively). A statistically significant difference was also observed between Ki-67 LI and histological grade and stage of the tumours (P < 0.001, P < 0.001 correspondingly). A correlation was observed between p53 protein expression and Ki-67 LI (P < 0.001). The immunocytochemical study of p53 protein and Ki-67 expression in cytological material represents a simple method which can be applied in routine cytological laboratories for the investigation of potential malignancy of ductal breast cancer.     

A new scoring system using multiple immunohistochemical markers for diagnosis of uterine smooth muscle tumors

The diagnosis of uterine smooth muscle neoplasms by light microscopy is difficult. Multiple classification schemes have been proposed based on mitotic rate, nuclear atypia, and the presence or absence of necrosis. None of these classification systems has been entirely successful. This study was undertaken to evaluate the use of selected immunohistochemical and histochemical markers in differentiating these tumors, in addition to accepted morphologic criteria. Ten cases of each of the following: leiomyosarcomas (LMS), atypical leiomyomas (AL), cellular leiomyomas (CL) and usual leiomyomas (UL), were classically evaluated for histological diagnosis and were stained for Ki-67 (MIB-1), bcl- 2 and p53 using monoclonal antibodies and the avidin-biotin peroxidase method, and argyrophilic nucleolar organizer region (AgNORs). The number of stained cells was counted in the most positively stained region in a 4 mm2 square cover glass mounted on each slide. The mean value was calculated for each group of tumors. The data for Ki-67 (MIB- 1), bcl-2, p53 and AgNOR staining respectively, were significantly higher in LMS by comparison to UL, CL or AL. Because many singular cases had superimposed data being difficult to diagnose, a new scoring system for pathological evaluation was created. The results obtained by this scoring system suggest that immunohistochemical markers Ki-67 (MIB-1), bcl-2, p53 together with the AgNOR staining could be useful, by the scoring system, as an adjunct to the current accepted morphologic criteria in differentiating smooth muscle tumors of the uterus.     

Lower proliferation rate in metastatic effusion mesothelial cells than in benign effusions

OBJECTIVE: To determine the proliferation rates of mesothelial cells in metastatic and benign effusions. STUDY DESIGN: Immunohistochemistry was performed on formalin-fixed pellets from 16 malignant and 9 benign clinical effusions. Dual staining with antibodies against Ki-67 (MIB-1) and desmin was applied to all effusions to differentiate between benign mesothelial cells and malignant cells, and the proportions of desmin+/Ki-67+ and desmin+/Ki-67- cells were calculated. RESULTS: In 7 malignant effusions no proliferating mesothelial cells were found, whereas some rate of proliferation could always be demonstrated in mesothelial cells in the benign effusions. Further, the median proportions of proliferating cells, malignant 2% vs. benign 11%, differed significantly. CONCLUSIONS: To our knowledge this finding has not been previously described, and it may have implications for both cytologic diagnosis and the understanding of tumor biology and the interaction between tumor cells and mesothelial cells.     

Biological characterisation of superficial bladder cancer by bivariate cytokeratin 7/DNA analysis, flow cytometric assessment of MIB- 1, and an immunohistochemical study.

A total of 238 cases of bladder carcinoma stages Ta, Tis, T1 were submitted prospectively to multiparameter flow cytometry and immunohistochemical study in order to determine the biological aggressiveness of the tumour. DNA index (DI), S-phase fraction (SPF) obtained by bivariate cytokeratin 7/DNA analyses, and the immunohistochemical evaluation of p53 and MIB-1 were studied in relation to the traditional prognostic factors in bladder cancer (stage and grade). the variance analysis results showed that DNA aneuploidy was significantly associated with high stage (p = 0.0001), high grade (p = 0.0001), high SPF value > or = 5.5% (p = 0.0001), MIB-1 positivity > or = 31% (p = 0.0001) and high expression of p53 (staining involving > 50% of cells, p = 0.0001). Even if there was no statistical significance the hypotetraploid class (1.70 < DI < 1.89) showed poor prognostic biomarkers more frequently than the other aneuploid classes. Out of 238 cases, 101 were also submitted to flow cytometric measurement of MIB-1 (fMIB-1) to study the correlation between cell proliferation and DNA content. Data obtained from fresh, 3:1 methanol/acetone fixed samples were compared with values obtained from both cell cycle analysis methods and routine application of the MIB-1 immunostaining in histological sections. fMIB-1 values were positively correlated with SPF values (r = 0.801, p < 0.01) and S+G2M fraction (percentage of cells in S and in G2M phases) (r = 0.763, p < 0.01) but no correlation with paraffin sections was found. A fMIB-1 value > 7% was strongly associated with aneuploidy (p = 0.0001). The determination of DNA content coupled with the study of the epithelial (cytokeratin 7) and proliferative (MIB-1) markers could be useful in providing important information on the biological behaviour of superficial bladder tumours.     

Comparison of cell proliferation in breast carcinoma using image analysis (Ki-67) and flow cytometric systems.

Cellular proliferation has been implicated as an important predictor of biologic behavior in breast cancer. Cellular proliferation of 95 breast carcinomas was evaluated by comparing Ki-67 immunoreactivity in frozen sections quantitated by image analysis with S-phase and S + G2/M phase fraction determined by flow cytometry on nuclei extracted from fixed, paraffin-embedded sections (modified Hedley's technique). These parameters were correlated with traditional morphologic features of histologic grade, including mitotic count. Ki-67 immunoreactivity correlated with S-phase fraction determined by flow cytometry (r = .41, P = .001) and with S + G2/M phase fraction determined by flow cytometry (r = .29, P = .008). There was also a correlation between histologic grade and Ki-67 immunoreactivity (r = .30, P = .004) and between histologic grade and S-phase fraction (r = .42, P = .0001). Mitotic count correlated with Ki-67 immunoreactivity (r = .25, P = .015) and with S-phase fraction (r = .35, P = .001). Image and flow cytometric analysis systems provide comparable measurements of cellular proliferation; their measurements correlate with histologic grade and mitotic count in breast cancer.     

Simultaneous flow cytometric analysis of surface markers and nuclear Ki-67 antigen in leukemia and lymphoma

The monoclonal antibody Ki-67 identifies an antigen present during the late G1, S, G2, and M phases of the cell cycle, whereas resting cells do not express this antigen. Immunostaining with Ki-67 provides a simple method with which to determine the growth fraction of a malignant cell population without requiring a laborious procedure or use of radioactive materials. Thus far, detection of Ki-67-positive cells by flow cytometry was limited because of nuclear location of the antigen. In this study, periodate-lysine-paraformaldehyde (PLP) fixation of cells in suspension, labeling with Ki-67, and the subsequent flow cytometric analysis of the tumor growth fraction is described. Fixation with PLP at -10 degrees C for 15 min rendered the plasma membrane permeable without destroying cell surface antigens. Thus double immunofluorescence studies using both a surface marker and Ki-67 could be performed. This offers the additional advantage of being able to define the phenotype of proliferating cells. This method was applied to determine the growth fraction in peripheral blood and bone marrow samples of patients with leukemia and non-Hodgkin's lymphoma. The results of Ki-67 studies in 91 patients are shown. A wide variability of individual Ki-67 values was observed within each entity. Use of this flow cytometric procedure substantially facilitates the quantification of proliferating cells in pathological blood and bone marrow samples.     

Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67

The monoclonal antibody Ki-67 detects a nuclear antigen that is present only in proliferating cells. The aim of the present investigation was to clarify whether the Ki-67 nuclear antigen is restricted in its expression to certain phases of the cell cycle. All experiments consistently showed that the Ki-67 nuclear antigen is present in S, G2, and M phase, but is absent in G0. However, the results concerning Ki-67 antigen expression in G1 phase varied: cells passing the early events of mitogen triggered transition from G0 to G1, i.e., G1T and first G1A, lacked the Ki-67 nuclear antigen, whereas G1 cells after mitosis were constantly Ki-67-positive. This result suggests that after mitosis cells might not follow the same metabolic pathways as G0 cells do when entering G1 for the first time. Therefore, we suggest that the early stages of mitogen stimulation represent initial sequences of proliferation and not parts of the cell cycle. Because our data show that the Ki-67 nuclear antigen is present throughout the cell cycle, immunostaining with monoclonal antibody Ki-67 provides a reliable means of rapidly evaluating the growth fraction of normal and neoplastic human cell populations.     

Comparison between different cell kinetic variables in human breast cancer

Cell kinetics holds a prominent role among biological factors in predicting clinical outcome and response to treatment in neoplastic patients. Different cell kinetic variables are often considered as valid alternatives to each other, but the limited size of case series analysed in several studies and the lack of simultaneous determinations of all the variables on the same tumours do not justify this conclusion. In the present study, the correlation between [³H]thymidine labelling index ([³H]dT LI), flow cytometric S phase cell fraction (FCM-S) and Ki-67 immunoreactivity (Ki-67/MIB-1) was verified and the type of correlation with the most important clinical, pathological and biological patient and tumour characteristics was investigated in a very large series of breast cancer patients. Ki-67/MIB-1, FCM-S and [³H]dT LI were determined in 609, 526 and 485 patients, respectively, and all three cell proliferation indices were evaluated in parallel on the same tumour in a series of 330 breast cancer patients. All the cell kinetic determinations were performed within the context of National Quality Control Programmes. Very poor correlation coefficients (ranging from 0.37 to 0.18) were observed between the different cell kinetic variables determined in parallel on the same series of breast cancers. Moreover, Ki-67/MIB-1 and FCM-S showed a significant relationship with histological type, grade and tumour size, whereas statistically significant correlations were not observed for [³H]dT LI. In conclusion, the results show that the different cell kinetic variables provide different biological information and cannot be considered as alternatives to each other.     

Detection of p53 protein and Ki-67 proliferation antigen in human papillomavirus (HPV)-positive and HPV-negative cervical lesions by immunohistochemical double-staining

In HPV-associated genital lesions, low or absent expression of p53 has been attributed to the rapid degradation of p53 through its binding with HPV E6 protein. In this study, we examined p53 protein expression with two antibodies (CM1 polyclonal and PAb 1801 monoclonal antibodies), and Ki-67 proliferation antigen (monoclonal antibody) using an immunohistochemical (IHC) double-staining technique in 77 HPV-positive cervical lesions (HPV6, HPV11, HPV16, HPV18, HPV31, and HPV33) and in 15 HPV-negative cases. p53 protein expression was detected in 36/92 (39.1%) of the specimens. of the p53-positive cases, 80.6% (29/36) were HPV-positive samples, including 10/23 (43.5%) of HPV16- and 3/10 (30%) of HPV18-positive biopsies. In 52.8% of the p53-positive samples, the expression was found in less than 5% of the basal cells which were also positive for Ki-67.
Ki-67 proliferation marker was found in 91/92 specimens, most intensely in those infected by HPV16. p53 was more abundant in progressive or persistent lesions, but no differences were found between HPV-positive and HPV-negative samples. the positive IHC double-staining of both p53 and Ki-67 proliferation antigen in the same basal (and parabasal) cells indicates that these two normal cell-cycle proteins are being expressed while the cells are entering from the G₁ to the S phase of the cell cycle. Since the latter property is only attributed to the wild-type p53 (but not to mutated p53), the p53 protein detected in HPV lesions by IHC is likely to be the wild-type p53 rather than mutated p53, and the result was also confirmed by using p53 mutant specific antibody PAb 240. Accordingly, the concept of HPV inactivating the wild-type p53 protein should be re-examined, and other mechanisms for HPV-mediated carcinogenesis should be considered.     

Antigen Retrieval Using pH 3.5 Glycine-HCI Buffer or Urea Solution for Immunohistochemical Localization of Ki-67

A new antibody (MIB-1) has been described, permitting the demonstration of Ki-67 proliferation antigen in paraffin sections. However, satisfactory results were obtained only after subjecting tissue sections to microwave based antigen retrieval in citrate buffer solution. Other buffer solutions produce equivalent or better results and also permit use of the original Ki-67 antibody, which hitherto has been considered ineffective for paraffin sections.     

p53 protein expression and proliferative activity in imprints of superficial transitional cell carcinoma of the bladder

OBJECTIVE: To investigate p53 protein expression and proliferative activity in imprints of tumor biopsies from superficial transitional cell carcinoma of the bladder in relation to the histologic grade of malignancy and recurrence status. STUDY DESIGN: The study group consisted of 70 cases of superficial transitional cell carcinoma of the bladder. In order to investigate p53 protein expression and Ki-67 expression, an immunocytochemical avidin-extravidin complex technique was performed using monoclonal antibodies p53 D0-7 and proliferating cells correspondingly. RESULTS: Thirty-seven percent of superficial transitional cell carcinoma cases showed positive expression of p53 protein. No correlation was found between p53 protein expression and grade of malignancy (P = .45). p53 Protein expression was statistically correlated with a high Ki-67 labeling index (LI) (P < .001) and recurrence status (P < .001). Forty-seven percent of cases showed a Ki-67 LI > 25%. No correlation was found between a high Ki-67 LI and grade of malignancy (P = .703). A significant difference in high Ki-67 LI between recurrent and nonrecurrent tumors of the same grade (P < .001) and between recurrent and nonrecurrent tumors was found independently of grade (P < .001). CONCLUSION: These results on cytologic material could provide useful information on the biologic behavior of superficial transitional cell carcinoma of the bladder at the time of diagnosis.     

c-myc mRNA expression in non-Hodgkin’s lymphomas

Steady state c-myc mRNA levels determined by Northern blot analysis were examined in non-Hodgkin's lymphomas (NHL) of both high (n = 29) and low malignancy (n = 18), and in non-specific chronic lymphadenitis (n = 6). High grade NHL, classified according to the updated Kiel classification, revealed significantly larger amounts of c-myc mRNA compared with low grade NHL and lymphadenitis. mRNA levels in non-specific lymphadenitis were lower than in low grade NHL, but the differences were not statistically significant. No correlation between c-myc mRNA levels and the immunologic phenotype was discernible. Growth fractions of the NHL were determined by immunostaining with the monoclonal antibody Ki-67. Significant correlations between the percentages of Ki-67-positive cells, as well as the amounts of c-myc mRNA, and classification into high or low grade NHL were found. However, the percentage of Ki-67 positive cells and c-myc mRNA levels in individual cases and in the various histologic entities of NHL did not correlate. Our results indicate the overexpression of the c-myc gene in NHL, and a highly significant correlation of steady state c-myc mRNA levels with the prognosis-related histomorphologic Kiel classification of NHL into different subgroups of low and high grade malignancy.     

MIB-1 and S-phase cell fraction predict survival in non-Hodgkin's lymphomas

The monoclonal antibody anti-Ki67 is used to detect proliferating cells, but its main limitation is the requirement of fresh-frozen material. On a series of patients with non-Hodgkin's lymphoma, we used a Ki67 equivalent monoclonal antibody, the recently proposed MIB-1, on formalin-fixed histopathological material using microwave antigen retrieval. MIB-1 expression was analysed in relation to other proliferation indices, such as autoradiographic ³H-thymidine labelling index (³HTL1) and flow cytometric S-phase cell fraction (FCM-S) and to pathological status. Moreover, the prognostic relevance of the cell kinetic indices was defined in uni- and multivariate analyses including histology and tumour stage. The relationship between MIB-1 index and the other proliferation indices was statistically significant even though the correlation coefficient was around 0.6. The MIB-1 index was also related to the REAL (Revised European American Lymphoma) classification, but not to the Ann Arbor stage classification. Univariate analysis showed that the MIB-1 index was a significant predictor of 6-year survival in the overall series and in distinctly analysed low-grade and high-grade lymphoma subgroups. With regard to S-phase indices, ³HTLI was a powerful prognosticator in patients with high-grade histologies and FCM-S in patients with low-grade histologies. Multivariate analyses revealed that MIB-1 indiex, ³HTLI and FCM-S retained their prognostic significance independent of histology. In conclusion, the MIB-1 antibody provides prognostic information in non-Hodgkin's lymphomas and has the main advantage that it can be used in formalin-fixed, paraffin-embedded specimens.     

Direct comparison of bromodeoxyuridine and Ki-67 labelling indices in human tumours

Direct comparison of bromodeoxyuridine (BrdUrd) and Ki-67 labelling indices was achieved by selecting similar areas from serial sections of human tumours. Fifteen patients were selected who had been administered BrdUrd in vivo and both proliferation markers were assessed by immunohistochemistry. The data show a good correlation between both BrdUrd LI and MIB-1 LI and T_pot (calculated using the flow cytometry derived duration of S phase) and MIB-1 LI. The contribution of BrdUrd LI to growth fraction varied as a function of proliferation characteristics. In tumours with a high LI, the number of DNA synthesizing cells represented half the growth fraction, whilst in tumours with lower LI's (<10%) the ratio of DNA precursor labelled cells as a function of growth fraction fell to between 10% and 20%. T_pot showed a linear correlation with MIB-1/BrdUrd ratio with a slope approaching unity. It was apparent that both intra- and interpatient variation in proliferation index was greater for BrdUrd labelling than for MIB-1 expression.     

PCNA independence of Ki-67 expression in HPV infection.

12 Infectious Wart lesions were stained using the streptavidin-biotin immunoperoxidase method for PCNA10, MIB-1 (Ki-67 equivalent antigen) and Human Papilloma Virus antigen to study the effect of HPV presence on epidermal proliferation. Using strict methods to avoid observer bias, Ki-67 antigen was found in a high proportion of nuclei in the suprabasal layers together with HPV antigens, in the absence of PCNA staining. This finding indicates that DNA synthesis related, Ki-67 antigen bearing structures can be raised in the human nucleus in the absence of induction of PCNA bearing structures, suggesting also structural independence between the antigen bearing molecules.