Pathological non-response to chemotherapy in a neoadjuvant setting of breast cancer: an inter-institutional study.

To identify markers of non-response to neoadjuvant chemotherapy (NAC) that could be used in the adjuvant setting. Sixteen pathologists of the European Working Group for Breast Screening Pathology reviewed the core biopsies of breast cancers treated with NAC and recorded the clinico-pathological findings (histological type and grade; estrogen, progesterone receptors, and HER2 status; Ki67; mitotic count; tumor-infiltrating lymphocytes; necrosis) and data regarding the pathological response in corresponding surgical resection specimens. Analyses were carried out in a cohort of 490 cases by comparing the groups of patients showing pathological complete response (pCR) and partial response (pPR) with the group of non-responders (pathological non-response: pNR). Among other parameters, the lobular histotype and the absence of inflammation were significantly more common in pNR (p < 0.001). By ROC curve analyses, cut-off values of 9 mitosis/2 mm(2) and 18% of Ki67-positive cells best discriminated the pNR and pCR + pPR categories (p = 0.018 and < 0.001, respectively). By multivariable analysis, only the cut-off value of 9 mitosis discriminated the different response categories (p = 0.036) in the entire cohort. In the Luminal B/HER2- subgroup, a mitotic count <9, although not statistically significant, showed an OR of 2.7 of pNR. A lobular histotype and the absence of inflammation were independent predictors of pNR (p = 0.024 and <0.001, respectively). Classical morphological parameters, such as lobular histotype and inflammation, confirmed their predictive value in response to NAC, particularly in the Luminal B/HER2- subgroup, which is a challenging breast cancer subtype from a therapeutic point of view. Mitotic count could represent an additional marker but has a poor positive predictive value.

A simple and reproducible prognostic index in luminal ER-positive breast cancers.

BACKGROUND: The group of estrogen receptor (ER)-positive breast cancers (both luminal-A and -B) behaves differently from the ER-negative group. At least in early follow-up, ER expression influences positively patients' prognosis. This low aggressive biology flattens out the differences of clinical management. Thus we aimed to produce a prognostic index specific for ER-positive (ERPI) cancers that could be of aid for clinical decision. PATIENTS AND METHODS: The test set comprised 495 consecutive ER-positive breast cancers. Tumor size, number of metastatic lymph nodes and androgen receptor expression were the only independent variables related to disease-specific survival. These variables were used to create the ERPI, which was applied to the entire test set and to selected subpopulations (grade 2 (G2)-tumors, luminal-A and -B breast cancers). A series of 581 ER-positive breast cancers, collected from another hospital, was used to validate ERPI. RESULTS: In the test population, 96.9% of patients classified as ERPI-good showed a good prognosis compared with 79.6% classified as ERPI-poor (P < 0.001). ERPI effectively discriminated outcome in luminal-A and luminal-B and in G2-tumors. In the validation series, the ERPI maintained its value. CONCLUSION: ERPI is a practical tool in refining the prediction of outcome of patients with ER-positive breast cancer.

A novel biomarker-based analysis reliably predicts nodal metastases in anal carcinoma: preliminary evidence of therapeutic impact.

AIM: Routine prophylactic inguinal irradiation in anal cancer may cause significant toxicity associated with overtreatment bias. The aim of this study was to determine the risk of regional node metastases in anal carcinoma by identifying predictive molecular biomarkers. METHOD: Clinicohistopathological data from 50 pretreatment anal carcinoma biopsies were collected. Immunohistochemical analyses with antibodies against Ki67, p53, epidermal growth factor receptor (EGFR) and YKL-40 were performed. Statistical correlations between biomarkers and clinicopathological features and outcomes were studied. Sentinel lymph node biopsy was performed in a subset of 36 patients. RESULTS: All patients had undergone synchronous radiochemotherapy; tumour recurrence had developed in 26%, and 16% had died. YKL-40 tumour expression correlated with lymph node metastasis, whereas no inguinal node metastases were found in any of the (14%) patients presenting with a YKL-40/EGFR-negative tumour. YKL-40 expression and node metastasis were both significantly associated with shorter overall and disease-free survival. Tumour grade significantly correlated with disease-free survival only. HIV, tumour histological type, Ki67, p53 and EGFR were not associated with outcome. CONCLUSION: YKL-40 expression in anal carcinoma is correlated with a poor outcome and can predict lymph node metastases. The combined absence of YKL-40 and EGFR expression in a first biopsy of anal carcinoma reliably selects a subset of patients without inguinal metastases. Such patients could be spared sentinel lymph node biopsy and/or inguinal radiotherapy.

Correlation between Skp2 expression and nodal metastasis in stage I and II oral squamous cell carcinomas.

PURPOSE: The aim of this study was to investigate the role of S-phase kinase associated protein (Skp2) in the development of nodal metastasis and to assess its influence on prognosis in stage I and II oral squamous cell carcinomas (OSCCs). EXPERIMENTAL DESIGN: Seventy-one patients affected by OSCC (stage I-II) were observed in the period ranging from March 2003 to December 2006. The research was performed using immunohistochemical and histopathological analysis. RESULTS: The overall survival rate was 89.6% at 3 years, 87% at 5 years and 80.7% at 10 years. Patients with vascular or perineural invasion showed no statistically significant survival difference when compared with the ones with no invasion. The tumour depth of invasion did not prove to be related to the metastatic potential. Nine of the seventeen patients with Skp2 positive nuclei (≥20%) developed nodal metastasis. Conversely, only 6 of the 54 patients with a nuclear positivity lower than 20% developed a laterocervical metastasis (P=0.001). When comparing survival curves of Skp≥20% and Skp2<20% OSCCs, no significant P value emerged from the statistical analysis. CONCLUSIONS: This study is the first to report an important correlation between an Skp2 expression lower than 20% and the capability of the tumour not to develop nodal laterocervical metastases (P=0.001).

Solitary pancreatic head metastasis from tibial adamantinoma: a rare indication to pancreaticoduodenectomy.

Pancreatic metastases are rare, <2% of all pancreatic neoplasia. This is the first case of pancreatic metastasis from adamantinoma, a rare, low grade and slow growing tumor which is frequently localized in long bones. We describe a case of a 45-year-old woman presenting with increased bilirubin level. Computed tomography and ecoendoscopic ultra sonography revealed a pancreatic head mass. Fine-needle aspiration biopsy was consistent with metastatic adamantinoma. The patient was submitted to a standard pancreaticoduodenectomy. As in the case presented, standard pancreatic resections are safe and feasible options to treat non-pancreatic primary tumor improving patient's survival and quality of life.

Parameters and outcomes in 525 patients operated on for oral squamous cell carcinoma.

PURPOSE: This report analyzed the outcomes of patients undergoing surgery for oral squamous cell carcinoma (OSCC) to identify the value of prognostic factors. MATERIAL AND METHODS: A total of 525 patients were studied who had undergone surgery for oral squamous cell carcinoma (OSCC) between 2000 and 2011, of whom 222 had received postoperative radiation-therapy (PORT) and or chemoradiation-therapy (PORTC). For each patient, personal data, histological findings, treatment and outcome were recorded and analyzed statistically. Survival curves were calculated using the Kaplan-Meier algorithm, and the difference in survival among subgroups was examined. RESULTS: The overall survival (OS) and disease-specific survival (DSS) 5-year survival rate in the 525 patients were respectively 71.38% and 73.18%. The differences in the overall survival and disease-specific 5-year survival were significant (p < 0.05) for age < 40 years, site of origin, N status, staging, grading, osseous medullar infiltration, and perineural invasion. In patients undergoing radiation therapy, only perineural invasion negatively influenced the survival prognosis. In 150 pT1 cases of tongue and floor-of-mouth cancer, an infiltration depth (ID) > 4 mm was statistically correlated with poorer prognosis. CONCLUSIONS: The results demonstrate an improvement in the 5-year OS and DSS rates during the past decade compared with the previous decade. Univariate analysis revealed that age, tumor staging, and lymph node involvement, extracapsular spread, grading, perineurial invasion, infiltration depth, and osseus medullary invasion were associated significantly with overall survival and disease-specific survival.

Oncogenes and male breast carcinoma: c-erbB-2 and p53 coexpression predicts a poor survival.

PURPOSE: To investigate the prognostic value of biomarkers in male breast carcinoma (MBC). PATIENTS AND METHODS: Fifty patients (mean age, 62.2 years) with invasive ductal carcinoma were retrospectively studied. All patients received surgery; 35 had adjuvant postoperative therapy. The median follow-up was 59 months (range, 1 to 230 months). c-myc, c-erbB-2, p53, and bcl-2 proteins were immunohistochemically detected on sections from formalin-fixed, paraffin-embedded tissues using 9E11, CB11, DO7, and bcl-2 124 monoclonal antibodies (mAbs). Estrogen, progesterone, and androgen receptors were detected using specific mAbs. Cell proliferation was assessed by MIB-1 mAb. RESULTS: In univariate analysis, c-myc, c-erbB-2, and p53 protein overexpression was significantly correlated with prognosis. The median survival was 107 months for c-myc-negative and 52 months for c-myc-positive patients (P =.01), 96 months for c-erbB-2-negative and 39 months for c-erbB-2-positive patients (P =.02), and 100 months for p53-negative and 33 months for p53-positive patients (P =.0008). Tumor histologic grade (P =.01), tumor size (P =.02), patient age at diagnosis (P =.03), and MIB-1 scores (P =.0004) also had prognostic value. In multivariate analysis, only c-erbB-2 and p53 immunoreactivity retained independent prognostic significance. All nine patients who did not express c-erbB-2 and p53 proteins were alive after 58 months, whereas none of the 14 patients expressing both proteins survived at 61 months follow-up (P =.0002). CONCLUSION: Overexpression of c-myc, c-erbB-2, and p53 proteins may be regarded as an additional prognostic factor in MBC. The combination of c-erbB-2 and p53 immunoreactivity can stratify patients into different risk groups.

Nucleolar organizer region counts predict complete remission, remission duration, and survival in adult acute myelogenous leukemia patients.

PURPOSE: The analysis of the nucleolar organizer regions (AgNORs) was performed in patients with acute myelogenous leukemia (AML) to verify the role of cell proliferation in predicting complete remission (CR) and survival. MATERIALS AND METHODS: Bone marrow biopsies from 40 adult patients with AML were stained with the argyrophilic method. The mean AgNOR number (AgNOR count) was calculated for each case. After induction therapy, patients who achieved CR received intensive consolidation; two underwent autologous and four allogeneic bone marrow transplantations (BMT). RESULTS: The mean AgNOR count for the whole series was 6.6 (SD = 1.35); it was higher in CR patients than in resistant ones (P = .02). The median duration of CR was 26 months for patients with an AgNOR count greater than 6.6, but only 6 months for those with lower counts (P = .01). Sixteen patients who achieved a CR relapsed and 14 reached a second CR; the median duration of second CR was 16 months for patients with AgNOR count greater than 6.6, but only 5 months for those with lower counts (P = .01). The median survival time for the whole series was 14 months, with 30% of patients alive and in continuous CR at 103 months. Survival was longer for patients with an AgNOR count greater than 6.6 (33 months) than for those with lower counts (6 months; P = .0009). In multivariate analysis, when CR was excluded from the model, AgNOR count appeared as an independent prognostic variable (P = .005). CONCLUSION: AgNOR analysis is a suitable method to assess cell proliferation in bone marrow biopsies and can predict CR, remission duration, and survival in AML patients.

Risk groups of myeloma patients by histologic pattern and proliferative activity.

We investigated the histologic pattern and the cell proliferative activity of myeloma cells by the analysis of the nucleolar organizer regions (AgNORs) in bone marrow biopsy specimens from 150 multiple myelomas at diagnosis. The objective was an attempt to define risk groups of myeloma patients. On univariate analysis, the percentage of bone marrow plasma cells (BMPC%), the pattern of infiltration, the degree of plasma cell (PC) atypia, the marrow fibrosis, and the number of AgNOR/PC were correlated with survival time. On multivariate analysis, only AgNOR counts and pattern of infiltration retained independent prognostic significance. At 4-year followup, all patients with BMPC% < or = 20, interstitial pattern of invasion, and well-differentiated (G1) PC plus AgNOR/cell < or = 3.32 were alive, while no patient with BMPC% >50, diffuse pattern of infiltration, and poorly differentiated (G3) PC plus AgNOR/cell >5.15 survived (p < 0.0001). In conclusion, the histologic pattern and proliferative activity of myeloma cells, evaluated by AgNOR counts, are reliable predictors of survival in myeloma. Both parameters can be easily assessed in the same biopsy specimen, are reproducible, and permit identification of a group of patients with favourable outcome at 4-year followup. Thus, bone marrow biopsy should always be included in the diagnostic procedures for myeloma patients.

Long-term survival of thymoma patients by histologic pattern and proliferative activity.

We performed DNA flow cytometry and analysis of the argyrophilic nucleolar organizer regions (AgNORs) in formalin-fixed, paraffin-embedded sections from 60 surgically resected thymomas. The results were correlated with histologic pattern, stage, associated clinical features, and survival to assess which parameters could best predict prognosis. On univariate analysis, the 10-year survival rates were 86% for predominantly lymphocytic type but only 42% for predominantly epithelial, mixed lymphoepithelial, or spindle cell thymomas (p = 0.006); survival rates were 85% for noninvasive but only 34% for invasive thymomas (p = 0.0002); 73% for diploid but only 38% for aneuploid cases (p = 0.005); 88% for thymomas with 5.75 AgNORs per cell or fewer but only 34% for thymomas with more than 5.75 AgNORs per cell (p < 0.0001). On multivariate survival analysis, tumor stage (p < 0.001) and AgNOR counts (p = 0.009) retained independent prognostic significance. The 16 patients with predominantly lymphocytic type and 5.75 AgNORs per cell or fewer were all alive at the end of the observation period. In conclusion, the histologic type of the American classification and the proliferative activity evaluated by AgNOR analysis are the best predictors of long-term survival for patients with thymoma. Both predictors can be easily evaluated in the same histologic section, are highly reproducible, and permit identification of a group of patients with a favorable outcome regardless of other clinicopathological features.

Biologic differences between noninvasive papillary urothelial neoplasms of low malignant potential and low-grade (grade 1) papillary carcinomas of the bladder.

We investigated the expression of oncogenes p53, c-erbB-2, and bcl-2 and cell proliferative activity in 62 newly diagnosed superficial pTa papillary bladder tumors. Based on the 1998 World Health Organization/International Society of Urological Pathology (WHO/ISUP) and 1999 WHO classifications, 19 were urothelial neoplasias of low malignant potential (LMP) and 43 low-grade (grade 1) papillary carcinomas. All the patients underwent transurethral resection and were followed up to 97 months; 42 had recurrences. Initial biopsies were tested for p53, c-erbB-2, and bcl-2 proteins using DO7, CB11, and bcl-2 124 monoclonal antibodies. Cell proliferation was assessed by MIB-1 mAb and mitotic count. LMP had significantly lower MIB-1 (p = 0.002) and p53 immunopositivity (p = 0.03), mitotic count (p = 0.006), and recurrence rates (p = 0.04) than did grade 1 cases, whereas no difference was observed for c-erbB-2 and bcl-2 expression. The median disease-free survival for LMP was 76 months but only 15 months for grade 1 cases (p = 0.002). Although the cohort is small, the results indicate that the distinction between LMP and low-grade (grade 1) papillary urothelial neoplasias, as proposed by the 1998 WHO/ISUP and 1999 WHO classifications, reflects different biologic activity and clinical behavior; however, a long-term follow-up is advisable also for patients with LMP.

P53 overexpression correlates with proliferative activity and prognosis in carcinomas of the pyriform sinus.

p53 overexpression and proliferative activity were investigated in 28 squamous cell carcinomas of the pyriform sinus of the hypopharynx prior to therapy, using DO1 and MIB-1 monoclonal antibodies in routinely processed biopsies. MIB-1 scores were associated with tumour histological grade (35.4% for grade 3 versus 23.8% for grade 2 cases; p=0.008) and survival (the median of survival was 23 months for cases with MIB-1 scores less than or equal to 33.8% but 11 months only for cases with MIB-1 scores >33.8%; p<0.001). p53 scores were associated with tumour histological grade (56.5% for grade 3 versus 37.1% for grade 2 cases; p=0.02) and survival (median of survival 20 months for cases with p53 scores less than or equal to 56.2% versus 11 months for cases with p53 scores >56.2%; p=0.002). Tumour histological grade was also correlated with prognosis (median of survival 50 months for grade 2 versus 14 months for grade 3 cases; p=0.03). In the multivariate analysis, only MIB-1 (p=0.001) and p53 scores (p=0.003) had an independent prognostic significance. A linear relationship between p53 and MIB-1 scores was observed (r=0.54; p=0.012). With the limitation due to the small number of cases, our findings indicate that p53 overexpression correlates with proliferative activity and survival in squamous cell carcinomas of the pyriform sinus, and suggest the use of p53 and MIB-1 immunostainings in the pretherapeutic assessment of the tumour aggressiveness.

Proliferative activity in the malignant cellular blue nevus.

The proliferative activity of four malignant cellular blue nevi (MCBN) was assessed in routinely fixed, paraffin-embedded material using staining for the argyrophilic nucleolar organizer regions (AgNORs), immunohistochemical staining for proliferating cell nuclear antigen (PCNA [PC10]), and DNA flow cytometry. The objective was to determine whether the evaluation of proliferative activity could represent a useful diagnostic parameter. Four cellular blue nevi (CBN), 10 melanocytic nevi (MN), four common blue nevi (BN), and 10 conventional malignant melanomas (MMs) were selected as controls. In the MCBN the mean AgNOR number, evaluated on the basis of 100 tumor cells, was 8.33 +/- 0.83; NORs were small and dispersed throughout the nucleus; the mean PCNA score was 31.93% +/- 4.4; and two of the cases were aneuploid and two diploid. In the CBN the AgNOR count was 3.69 +/- 0.56; NORs were large and mainly grouped in a central cluster; the mean PCNA score was 3.53% +/- 1.28; and three of the cases were diploid and one aneuploid. The AgNOR counts in the MCBN were significantly different from those in the CBN (P = .0002), MN (3.04; P = .00001), and BN (2.93; P = .00006), whereas they were not significantly different from those in the conventional MMs (7.64; P = .58). The PCNA (PC10) scores in the MCBN were significantly different from those in the CBN (P = .00003), MN (2.05%; P = .00001), and BN (5.06%; P = .00002), whereas they were not significantly different from those in the conventional MMs (28.9%; P = .49). In all the cases a linear relationship between AgNOR counts and PCNA scores was observed (r = .94, P = .00001). Our results indicate that AgNOR analysis and PCNA immunostaining can be regarded as useful additional parameters for the diagnosis of MCBN.

DNA ploidy and p53 expression correlate with survival and cell proliferative activity in male breast carcinoma.

DNA flow cytometry and the monoclonal antibody DO7 were applied in formalin-fixed, paraffin-embedded specimens from 34 primary male breast carcinomas to verify whether DNA ploidy and p53 expression were associated with survival and proliferative activity. They were compared with tumor clinicopathologic features, sex steroid hormone receptors and cell proliferative activity, assessed by the counts of the argyrophilic nucleolar organizer regions (AgNORs), the monoclonal antibody PC10 against the proliferating cell nuclear antigen and the monoclonal antibody MIB-1. A significant correlation was found between survival and tumor ploidy (median survival, 77 months for diploid but only 38 months for aneuploid cases; P = .03) and p53 expression (median survival, 95 months for cases with p53 scores < or = 14.06% versus 33 for cases with P53 scores > 14.06%; P = .0004; median survival, 99 months for p53 negative vs 39 for positive cases; P = .007). Tumor histological grade (P = .006), AgNOR counts (P = .0001), PC10 scores (P = .002), and MIB-1 scores (P = .001) were also associated with prognosis. In the multivariate analysis, only p53 scores (P = .001) or p53 immunopositivity (P = .003) and AgNOR counts (P = .022) retained an independent prognostic significance. Aneuploid tumors had higher AgNOR counts (P = .002), PC10 (P = .007), MIB-1 (P = .006), and p53 scores (P = .01) than diploid cases. A linear relationship was observed between p53 scores and AgNOR counts (r = .41; P = .014), PC10 (r = .46; P = .005), and MIB-1 scores (r = .44; P = .011). These results indicate that DNA ploidy and p53 expression are associated with survival and cell proliferative activity in male breast carcinoma. Quantitative parameters, such as DNA ploidy, p53 scores, AgNOR counts, PC10, and MIB-1 scores substantially improve the prognostic significance of the traditional parameters in male breast carcinoma.

Argyrophilic nucleolar organizer region counts and proliferating cell nuclear antigen scores are two reliable indicators of survival in pharyngeal carcinoma.

The proliferative activity of pharyngeal carcinoma has been investigated by means of monoclonal antibody PC10 against proliferating cell nuclear antigen (PCNA/cyclin) and argyrophilic nucleolar organizer region (AgNOR) analysis in formalin-fixed, paraffin-embedded biopsies from 45 primary squamous and undifferentiated carcinomas, prior to therapy. The correlation between AgNOR counts and PCNA(PC10) scores was highly significant (r = 0.73; P < 0.0001) as determined by Pearson's correlation coefficient. Moreover, the univariate Kaplan-Meier survival analysis showed a significant correlation between 3- and 5-year survival rates and the mean AgNOR number per tumour cell (P = 0.0003) or the percentage of PCNA(PC10)-positive cells (P = 0.0001). Our results indicate that both AgNOR counts and PCNA(PC10) scores are reliable markers of the proliferative activity of pharyngeal carcinoma in small, routinely processed biopsies, in which they can allow simultaneous evaluation of the histology and tumour cell kinetics.

Bcl-2 expression in male breast carcinoma.

We have analysed the expression of bcl-2 protein retrospectively in 34 primary male breast carcinomas (MBC), using the monoclonal antibody bcl-2 in formalin-fixed, paraffin-embedded tissues. Bcl-2 expression was compared with tumour clinicopathological features, sex steroid hormone receptors, DNA content, p53 immunoreactivity and cell proliferative activity assessed by counts of the argyrophilic nucleolar organizer regions (AgNORs), the monoclonal antibody PC10 against proliferating cell nuclear antigen and the monoclonal antibody MIB-1. Most (28, or 82.3%) of the 34 cases of MBC were bcl-2 positive. No association was found with clinicopathological features of the tumours, although bcl-2 tended to be more frequently expressed in small tumours (P=0.09) and in cases without necrotic areas (P=0.1). Nor was any association found with hormone receptor status, p53 immunoreactivity, DNA content, cell proliferative activity or patient survival. In multivariate analysis, only proliferative activity (expressed by AgNOR counts) and p53 immunoreactivity had independent prognostic significance. Our results indicate that MBC differs from FBC in that in MBC bcl-2 protein is not related to an oestrogen-dependent transcription pathway and bcl-2 alone is not sufficient to induce increased proliferation. These characteristics, together with the high prognostic value of cell proliferation and the lack of prognostic significance for hormone receptor status, support the hypothesis that MBC is biologically different from FBC.

Cell proliferation indices, morphometry and DNA flow cytometry provide objective criteria for distinguishing low and high grade bladder carcinomas.

Argyrophilic nucleolar organizer region (Ag-NOR) analysis, proliferating cell nuclear antigen (PC-NA/PC10) and MIB-1 immunohistochemistry, nuclear morphometry and DNA flow cytometry have been performed on formalin-fixed, paraffin-embedded biopsies from 50 patients with transitional cell carcinoma of the urinary bladder. The mean AgNOR count was 6.01 for the 17 grade 1 (G1), 7.59 for the 21 G2 and 13.33 for the 12 G3 carcinomas (p < 0.001). The mean PCNA score was 15.03% for G1, 24.04% for G2 and 40.01% for G3 cases (p < 0.001). The mean MIB-1 score was 11.31% for G1, 17.09% for G2 and 34.47% for G3 carcinomas (p < 0.001). The mean nuclear area was 35.53 microns2 for G1, 38.65 microns2 for G2 and 83.62 microns2 for G3 cases (p < 0.001). Aneuploidy rates were significantly higher (91.7%) in G3 than in G2 (42.9%, p < 0.01) or G1 cases (47.1%, p < 0.05) but not different for G1 versus G2 cases (p = 0.94). While many overlaps of values were seen between G1 and G2 tumours, no overlaps were found between G3 and G1/G2 tumours. Significant differences of values were also found between pTa and invasive tumours (p < 0.0001 for AgNOR count and PCNA score; p < 0.001 for MIB-1 score and mean nuclear area; p < 0.01 for DNA ploidy); however many overlaps were seen. Our findings indicate that the quantitative parameters obtained with different methods are associated with histological grade of bladder urotheliomas and may improve the grading reproducibility. In addition, the absence of overlaps between G3 and G2/G1 carcinomas supports the tendency to classify bladder urotheliomas in only two categories of malignancy.

MIB-1, Ki67, and PCNA scores and DNA flow cytometry in intermediate grade malignant lymphomas.

AIMS: To verify the correlation between MIB-1, Ki67, and proliferating cell nuclear antigen (PCNA-PC10) scores and S-phase fraction in intermediate grade non-Hodgkin's lymphomas (Working Formulation F); and their reliability in differently processed tissues. METHODS: Forty one non-Hodgkin's lymphomas were classified as (F) intermediate grade malignant lymphomas according to the Working Formulation; mitotic counts and percentage of large cells were assessed for each case. Sections from formalin fixed, paraffin wax embedded tissues were stained with anti MIB-1 monoclonal antibody, after microwave oven processing, and anti-PCNA (PC10) monoclonal antibody using an avidin-biotin immunoperoxidase (ABC) method. One thousand cells from 10 representative fields were scored. Frozen sections from surgical specimens were stained with Ki67 monoclonal antibody using the ABC method; the fraction of Ki67 positive cells was calculated scoring 1000 cells. Flow cytometry analysis (FCM) was performed on cell suspensions from fresh tissues. Correlations between data were estimated using linear regression. RESULTS: A linear correlation was found between MIB-1 and Ki67 scores (r = 0.92; p < 0.00001); between MIB-1 and PCNA scores (r = 0.79; p < 0.00001); and between MIB-1 score and S-phase fraction (r = 0.51; p = 0.0006). A linear correlation was also found between Ki67 and PCNA scores (r = 0.85; p < 0.00001); between Ki67 score and S-phase fraction (r = 0.6; p = 0.0002); and between PCNA score and S-phase fraction (r = 0.74; p < 0.00001). A correlation was found between mitotic counts and MIB-1 (r = 0.56; p = 0.0001), PCNA (r = 0.51; p = 0.0007), or Ki67 scores (r = 0.47; p = 0.002); between the percentage of large cells and MIB-1 (r = 0.49; p = 0.0009), PCNA (r = 0.6; p = 0.00003), and Ki67 scores (r = 0.53; p = 0.0003) and S-phase fraction (r = 0.55; p = 0.0002). CONCLUSION: MIB-1, Ki67, and PCNA (PC10) scores and S-phase fraction are highly correlated and equally well represent the proliferative activity of intermediate grade non-Hodgkin's lymphomas in differently processed material. MIB-1 and PCNA stains can be applied even on small biopsy specimens. MIB-1 produces homogenous staining without background; it also strongly stains mitotic figures. It can be performed on routinely processed tissues, permitting the simultaneous evaluation of the morphology and tumour cell kinetics. The wide standard deviations of the proliferative indices found for intermediate grade NHL suggest that this category probably includes various degrees of malignancy.

AgNORs and myeloma prognosis.

The argyrophilic nucleolar organizer regions (AgNORs) were analysed in bone marrow biopsies from 80 patients with multiple myeloma (MM) at presentation. The mean AgNOR number per MM cell (AgNOR counts) and their distribution within the nucleus (configuration) were assessed. AgNOR counts were significantly associated with several recognized prognostic factors: Durie and Salmon clinical staging system (p = 0.02), percentage of plasma cells (PCs) in aspirates (p = 0.01) and in bone marrow biopsies (p = 0.0000), pattern of bone marrow involvement (p = 0.0003), calcaemia (p = 0.0005) and creatininaemia (p = 0.0003). AgNOR counts were also associated with the degree of PC differentiation (p = 0.0000). A single central cluster of 2-3 large-sized AgNORs (configuration A) was evident in most G1 MM; one cluster of 4-5 medium-sized dots or two clusters of 2-4 dots (configuration B) were seen in most G2 MM; many small-sized, scattered dots were present in G3 MM (configuration C). AgNOR counts and configuration were related to the prognosis: in the univariate analysis, the 5 year survival rate was 7% for cases with > 4.5 AgNORs/cell and 46% for cases with < or = 4.5 AgNORs/cell (p = 0.01), 53% for configuration A, 12% for configuration B and 0% for configuration C (p = 0.0000). AgNOR counts (p = 0.02) and configuration (p = 0.000) were independent prognostic variables in the multivariate analysis. The AgNOR counts were significantly higher in "fulminant myeloma" than in less aggressive cases (p = 0.002). The plasma cell labelling index (LI%), evaluated in 44 MM patients, showed significant correlation with prognosis: the 5 year survival rate was 51% for LI% < or = 1 and 17% for LI% > 1 (p = 0.02). More than 70% of patients with low LI% had low AgNOR counts and more than 70% of patients with high LI% had high AgNOR counts (p = 0.007). AgNOR counts and configuration reflect the myeloma cell mass, the degree of differentiation and the kinetics of the myeloma cells. They offer an exact evaluation of the tumour characteristics and can be useful additional parameters for MM prognosis.

Biological heterogeneity of diffuse mixed small and large cell non-Hodgkin's lymphomas assessed by DNA flow cytometry and Ki67.

The cell proliferative activity of the clinico-pathologically heterogeneous non-Hodgkin's lymphomas (NHL) included in the intermediate grade F category of the Working Formulation (WF) was investigated. S-phase fraction with flow cytometry on cell suspensions, and Ki67 on frozen tissue sections were performed in 42 F NHL. An avidin-biotin immunocomplex method was used and 1000 cells from 10 representative fields were counted. DNA content, S-phase and Ki67 were also detected in 194 NHL covering the whole spectrum of the WF. DNA content anomalies were found in 52 of 194 NHL. Their incidence, like that of S-phase fraction and Ki67 positive cells, progressively increased from low- to high-grade. A linear correlation was found between Ki67 and S-phase (r = .59). Using the median value of proliferating cells obtained with both procedures as a cut off, two very different groups of lymphomas could be distinguished within a series of 42 F-intermediate NHL: with low and high proliferative cell activity (p < .0001) that were termed F(low) and F(high), respectively. A intermediate group was placed between them. It differed significantly from the others if Ki67 was used but only from the F(high) group if the S-phase fraction analysis was applied. No significant differences were seen when comparing F(low) with the single categories of low-grade NHL and F(high) with H high-grade NHL; no significant differences were found between F(high) and G, and between G and H categories. The existence of distinct groups of NHL in the F category, as defined by biological parameters assessing the cell proliferative activity, indicates that this category includes biologically heterogeneous lymphoma subtypes with different grades of aggressiveness. The results also indicate that the G intermediate category displays proliferation indices similar to those of H high grade category.