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Primary vitreoretinal lymphoma (PVRL) is a high-grade lymphoma affecting the vitreous and/or the retina. The vast majority of cases are histopathologically classified as diffuse large B-cell lymphoma (DLBCL) and considered a subtype of primary central nervous system lymphoma (PCNSL). To obtain more insight into the ontogenetic relationship between PVRL and PCNSL, we adopted an immunogenetic perspective and explored the respective immunoglobulin gene repertoire profiles from 55 PVRL cases and 48 PCNSL cases. In addition, considering that both entities are predominantly related to activated B-cell (ABC) DLBCL, we compared their repertoire with that of publicly available 262 immunoglobulin heavy variable domain gene rearrangement sequences from systemic ABC-type DLBCLs. PVRL displayed a strikingly biased repertoire, with the IGHV4-34 gene being used in 63.6% of cases, which was significantly higher than in PCNSL (34.7%) or in DLBCL (30.2%). Further repertoire bias was evident by (1) restricted associations of IGHV4-34 expressing heavy chains, with κ light chains utilizing the IGKV3-20/IGKJ1 gene pair, including 5 cases with quasi-identical sequences, and (2) the presence of a subset of stereotyped IGHV3-7 rearrangements. All PVRL IGHV sequences were highly mutated, with evidence of antigen selection and ongoing mutations. Finally, half of PVRL and PCNSL cases carried the MYD88 L265P mutation, which was present in all 4 PVRL cases with stereotyped IGHV3-7 rearrangements. In conclusion, the massive bias in the immunoglobulin gene repertoire of PVRL delineates it from PCNSL and points to antigen selection as a major driving force in their development., (© 2020 by The American Society of Hematology.)

B-cell prolymphocytic leukemia (B-PLL) is a rare hematological disorder whose underlying oncogenic mechanisms are poorly understood. Our cytogenetic and molecular assessments of 34 patients with B-PLL revealed several disease-specific features and potential therapeutic targets. The karyotype was complex (≥3 abnormalities) in 73% of the patients and highly complex (≥5 abnormalities) in 45%. The most frequent chromosomal aberrations were translocations involving MYC [t(MYC)] (62%), deletion (del)17p (38%), trisomy (tri)18 (30%), del13q (29%), tri3 (24%), tri12 (24%), and del8p (23%). Twenty-six (76%) of the 34 patients exhibited an MYC aberration, resulting from mutually exclusive translocations or gains. Whole-exome sequencing revealed frequent mutations in TP53, MYD88, BCOR, MYC, SF3B1, SETD2, CHD2, CXCR4, and BCLAF1. The majority of B-PLL used the IGHV3 or IGHV4 subgroups (89%) and displayed significantly mutated IGHV genes (79%). We identified 3 distinct cytogenetic risk groups: low risk (no MYC aberration), intermediate risk (MYC aberration but no del17p), and high risk (MYC aberration and del17p) (P = .0006). In vitro drug response profiling revealed that the combination of a B-cell receptor or BCL2 inhibitor with OTX015 (a bromodomain and extra-terminal motif inhibitor targeting MYC) was associated with significantly lower viability of B-PLL cells harboring a t(MYC). We concluded that cytogenetic analysis is a useful diagnostic and prognostic tool in B-PLL. Targeting MYC may be a useful treatment option in this disease., (© 2019 by The American Society of Hematology.)

Trisomy 12 (tri12) is the second most frequent chromosomal aberration (15%-20%) in chronic lymphocytic leukemia (CLL). Tri12 confers an intermediate prognosis but is a heterogeneous entity. We examined whether additional mutational or chromosomal alterations might impact tri12 patient outcomes. This retrospective study, carried out by the French Innovative Leukemia Organization, included 188 tri12 patients with comprehensive information on immunoglobulin heavy chain (IGHV) gene status, karyotypic/FISH abnormalities, and NOTCH1, TP53, SF3B1, and MYD88 mutations. The main cytogenetic abnormalities associated with tri12 were del(13q) (25%), additional trisomies (14%) (including tri19 (10%) and tri18 (4%)), 14q32 translocations (10%), del(17p) (6.5%), del(14q) (4%), and del(11q) (4%). Unmutated (UM) IGHV, NOTCH1, and TP53, mutations were identified in respectively 66%, 25%, and 8.5% of cases. Multivariate analyses showed that additional trisomies (HR = 0.43, 95% CI = 0.23-0.78, P = .01) were associated with a significantly longer time to first treatment in Binet stage A patients and with a lower risk of relapse (HR = 0.37, 95% CI = 0.15-0.9, P = .03) in the overall tri12 population. Binet stage B/C, TP53 disruption, and UM IGHV status were associated with a shorter time to next treatment, while Binet stage B/C (HR = 4, 95% CI = 1.6-4.9, P = .002) and TP53 disruption (HR = 5, 95% CI = 1.94-12.66, P = .001) conferred shorter overall survival in multivariate comparisons. These data indicate that additional cytogenetic and mutational abnormalities, and particularly additional trisomies, IGHV status, and TP53 disruption, influence tri12 patient outcomes and could improve risk stratification in this population., (© 2018 Wiley Periodicals, Inc.)

Chronic lymphocytic leukemia (CLL) with 17p deletion (17p-) is associated with a lack of response to standard treatment and thus the worst possible clinical outcome. Various chromosomal abnormalities (including unbalanced translocations, deletions, ring chromosomes and isochromosomes) result in the loss of 17p and one copy of the TP53 gene. The objective of the present study was to determine whether the type of chromosomal abnormality leading to 17p- and the additional aberrations influenced the prognosis in a series of 195 patients with 17p-CLL. Loss of 17p resulted primarily from an unbalanced translocation (70%) with several chromosome partners (the most frequent being chromosome 18q), followed by deletion 17p (23%), monosomy 17 (8%), isochromosome 17q [i(17q)] (5%) and a ring chromosome 17 (2%). In a univariate analysis, monosomy 17, a highly complex karyotype (≥5 abnormalities), and 8q24 gain were associated with poor treatment-free survival, and i(17q) (P = .04), unbalanced translocations (P = .03) and 8q24 gain (P = .001) were significantly associated with poor overall survival. In a multivariate analysis, 8q24 gain remained a significant predictor of poor overall survival. We conclude that 17p deletion and 8q24 gain have a synergistic impact on outcome, and so patients with this "double-hit" CLL have a particularly poor prognosis. Systematic, targeting screening for 8q24 gain should therefore be considered in cases of 17p- CLL., (© 2017 Wiley Periodicals, Inc.)

Deletions of the long arm of chromosome 14 [del(14q)] are rare but recurrently observed in mature B-cell neoplasms, particularly in chronic lymphocytic leukemia (CLL). To further characterize this aberration, we studied 81 cases with del(14q): 54 of CLL and 27 of small lymphocytic lymphoma (SLL), the largest reported series to date. Using karyotype and fluorescence in situ hybridization (FISH), the most frequent additional abnormality was trisomy 12 (tri12), observed in 28/79 (35%) cases, followed by del13q14 (12/79, 15%), delTP53 (11/80, 14%) delATM (5/79, 6%), and del6q21 (3/76, 4%). IGHV genes were unmutated in 41/53 (77%) patients, with a high frequency of IGHV1-69 (21/52, 40%). NOTCH1 gene was mutated in 14/45 (31%) patients. There was no significant difference in cytogenetic and molecular abnormalities between CLL and SLL. Investigations using FISH and SNP-array demonstrated the heterogeneous size of the 14q deletions. However, a group with the same del(14)(q24.1q32.33) was identified in 48% of cases. In this group, tri12 (P = 0.004) and NOTCH1 mutations (P = 0.02) were significantly more frequent than in the other patients. In CLL patients with del(14q), median treatment-free survival (TFS) was 27 months. In conclusion, del(14q) is associated with tri12 and with pejorative prognostic factors: unmutated IGHV genes (with over-representation of the IGHV1-69 repertoire), NOTCH1 mutations, and a short TFS., (© 2014 Wiley Periodicals, Inc.)

Aims: We aimed to study epidermal growth factor receptor (EGFR) expression in surgically resected pancreatic ductal adenocarcinomas (PDACs) by immunohistochemistry and their relationship to clinicopathological features, cell proliferation and cell adhesion protein expression., Methods: A total of 99 PDACs were analysed on tissue microarrays for EGFR, E-cadherin and β-catenin expression patterns in tumour cells. The percentage of cells expressing the three proteins (membrane, cytoplasm or nuclear pattern) and of Ki67-positive tumour cells was assessed. Tumour protein expression was studied with regard to clinicomorphological features, Ki67 index and for postsurgical survival., Results: Membrane tumour EGFR correlated with histological poor differentiation (dedifferentiation), increased number of mitoses and severe tumour cell atypia (pleiomorphism) as well as with aberrant adhesion protein expression such as nuclear β-catenin and cytoplasmic E-cadherin. Cytoplasmic tumour E-cadherin correlated with an increased Ki67-positive tumour cell component, whereas nuclear E-cadherin correlated with a shorter postsurgical overall survival, as well as with tumour necrosis and an abundant clear cell component., Conclusions: In conclusion, the results of our study suggest a complex role for EGFR in PDAC carcinogenesis, tumour expression of this protein being associated with tumour dedifferentiation, mitotic activity or pleiomorphism, as well as with aberrant tumour cell adhesion protein expression.

Whether sex chromosome loss (SCL) is an age-related phenomenon or a cytogenetic marker of hematological disease is unclear. To address this issue in chronic lymphocytic leukemia (CLL), we investigated 20 cases with X or Y chromosome loss detected by conventional cytogenetics (CC). The frequency of SCL was low in CLL (2.3%). It was the sole abnormality, as detected by CC, in 10/20 (50%) patients. Fluorescence in situ hybridization (FISH) analyses confirmed SCL in all patients tested, present in 5-88% of cells (median: 68%). Deletions of 13q were observed by FISH in 16/20 (80%) patients. Compared with CLL without SCL, SCL was significantly associated with 13q deletion, especially when bi-allelic (P = 0.04). Co-hybridization analyses showed that SCL could be a concomitant, primary or secondary change, or be present in an independent clone. FISH analyses were performed on blood sub-populations isolated by Ficoll or flow cytometry. Comparing mononuclear cells (including CLL cells) and polynuclear cells separated by Ficoll, a maximum of 2% of polynuclear cells were found with SCL, whereas mononuclear cells exhibited a significantly higher loss frequency (range: 6-87%) (P = 0.03). Comparing B-cells (including CLL cells) and T-cells sorted by flow cytometry, the proportion of B-CD19+ cells with SCL was significantly higher (range: 88-96%) than that observed in T-CD3+ cells (range: 2-6%) (P = 0.008). We conclude that SCL has to be considered as a clonal aberration in CLL that may participate in the oncogenic process., (Copyright © 2013 Wiley Periodicals, Inc.)

Objectives: The effect of ionic low osmolar contrast media (ICM) and nonionic iso-osmolar CM (NICM) on acute thrombotic complications of percutaneous coronary intervention (PCI) is subject to controversies possibly related to a potential interaction with anticoagulation regimens. We sought to compare physical and morphological properties of fibrin clots made in the presence of ioxaglate (ICM), iodixanol (NICM) versus control and to evaluate the effect of four anticoagulants used in PCI., Methods and Results: Maximum platelet aggregation (MPA%), maximum elastic modulus (EM, dyne/cm(2) ) fiber density (n/10(-5) /μm(2) ), and lysis front velocity (nm/sec) of fibrin rich clot (FRC) were measured simultaneously using peripheral blood from 12 patients undergoing elective PCI. We compared the effects of adding iodixanol or ioxaglate or saline (control) to blood with enoxaparin, unfractionated heparin, fondaparinux, and bivalirudin. Iodixanol and ioxaglate led to nonsignificant reduction in MPA compared to control (33.6% ± 16.9%, 28.2% ± 18.9%, and 40.7% ± 13.9%, respectively, P = ns). Fibrin formed with iodixanol was stiffer (42.7 ± 41.9, 18.7 ± 3.7, and 15.9 ± 9 dyne/cm(2) , P < 0.01) and displayed more fibrin fibers (1089 ± 175, 260 ± 108, and 456 ± 131 n/10(-5) /μm(2) , respectively, P < 0.01) than with ioxaglate or control. This resulted in a profound reduction in the lysis front velocity (191 ± 95, 261 ± 112, and 360 ± 153 nm/sec). None of the four anticoagulants displayed any significant interaction on the effect of contrast media., Conclusions: The prothrombogenic effect of iodixanol is related primarily to an increase in fibrin stiffness with subsequent delayed fibrinolysis, something not seen with ioxaglate. Anticoagulation does not appear to have any impact on this fibrin clot abnormalities., (Copyright © 2011 Wiley-Liss, Inc.)

Objectives: The aims of this study were to study the biological and clinical significance of 3 main proteins of the mitogen-activated protein kinase (MAPK) signaling pathway, ERK1/2, P38, and MKK4, in a series of patients having pancreatic adenocarcinomas treated by surgery., Methods: We examined the immunohistochemical expression of 3 MAPK proteins, ERK1/2, P38, and MKK4 in 99 surgically resected pancreatic ductal adenocarcinomas. Tumor protein expression was studied with regard to pathological characteristics and to postsurgical recurrence-free and overall survivals., Results: MKK4 expression was related to tumor cell proliferation, evaluated by the Ki67 index (P < 0.01). ERK1/2 expression was related to a shorter recurrence-free survival on both univariate and multivariate analysis (P < 0.01; odds ratio, 8.39; 95% confidence interval, 2.68-26.26) independently of lymph node metastases and tumor size, and to a shorter overall survival (P = 0.01) on univariate analysis. In patients without postsurgical treatment, both ERK1/2 and P38 tumor expression correlated with a shorter recurrence-free survival (P < 0.01 and P = 0.02, respectively)., Conclusions: The results of our study suggest that in pancreatic ductal adenocarcinomas, the MKK4 protein was directly related to high cell proliferation, and that tumor ERK1/2 and P38 expression correlated to shorter postsurgical recurrence-free and overall survivals.

Background: Chromosomal translocations are usually analyzed as a single entity, and are associated with a poor outcome in chronic lymphocytic leukemia. Translocations involving immunoglobulin genes are recurrent, but uncommon (<5%), and their individual prognosis is not clear. The two most frequent partners are BCL2 (18q21) and BCL3 (19q13)., Designs and Methods: Herein, 75 cases are reported of chronic lymphocytic leukemia and t(14;18) (BCL2-CLLs). Our series benefits from morphological, immunological and cytogenetical reviews. The IGHV status analyses were performed by referring laboratories. Comparison was made with our previously published series of chronic lymphocytic leukemia patients with t(14;19) (BCL3-CLLs, n=29)., Results: Compared with BCL3-CLLs, lymphocytosis was lower in BCL2-CLLs (p<0.008), and splenomegaly was less frequent (p<0.0001). There were more "typical" morphologies (p<0.005) and Matutes scores >4 (p<0.001) in the BCL2-CLLs group, and less CD38 expression (p<0.04). More variant BCL2-translocations were observed (t(18;22), n=11; 2t(2;18), n=2; p<0.02), and BCL2-translocation was frequently single (p<0.002). Complex karyotypes (p<0.02), trisomy 12 (p<0.03), 6q deletion (p<0.002) and TP53 deletion (p<0.02) were less frequent in BCL2-CLLs, whereas 13q deletion was more frequent (p<0.005). The IGHV gene was frequently mutated in BCL2-CLLs (p<0.0001). Treatment-free survival was longer in BCL2-CLLs (p<0.0001)., Conclusions: BCL2-CLL.S express CD5 and lack expression of CD38, and have a Matutes score ≥4, frequent trisomy 12, no ATM and 6q deletions, and a mutated IGHV status. Compared to BCL3-CLLs, BCL2-CLLs are much less aggressive; indicating that identifying individual translocations and cytogenetic partners would allow improved patient stratification.

Evolution to myelofibrosis (MF), acute myeloid leukemia or myelodysplastic syndrome (AML/MDS) may occur over time in myeloproliferative neoplasms (MPN) patients most likely due to the acquisition of additional mutations. The Groupe Francophone de cytogenetique hematologique (GFCH) has collected and reviewed 82 patients with transformation of MPN (66 AML/MDS and 16 MF). JAK2V617F and TET2 mutations were searched for in 40 and 32 patients, respectively. Significantly more -7/del(7q) (P = 0.004) and -5/del(5q) (P = 0.03) were found in AML/MDS with a higher incidence of dup1q (P = 0.01) in MF. Some specific chromosomal abnormalities occurred together, for example -5/del(5q) and -17/del(17p) (P = 0.0007). In multivariate analysis, two factors were independently associated with an inferior overall survival (OS); AML/MDS transformation (P < 0.0001) and -5/del(5q) abnormality (P = 0.02). Although both giving rise to loss of 7q, der(1;7) differed from other 7q deletions in terms of distribution (lower frequency of AML/MDS, P = 0.02), association with chromosomal abnormalities (absence of -5/del(5q), P = 0.003; increased del(20q), P = 0.05), and longer OS (P = 0.0007). We detected 24/40 (60%) JAK2V617F and 8/25 (32%) TET2 mutations in samples following transformation, ranging from wild-type to mutated forms of both genes. The mutated and wild-type forms of the genes were not found to be associated with a specific chromosomal abnormality. There was no evidence that JAK2 or TET2 mutations were associated with the type of MPN transformation, whereas the type of cytogenetic abnormalities were strongly linked, perhaps indicating that they play a specific role in the transformation process.

Objective: To use a purely geometric data-unravelling method to identify, without a priori, direct links of neoangiogenesis with known clinicobiologic variables in 23 untreated chronic lymphocytic leukemia (CLL) patients at the time of marrow sampling., Study Design: We measured neovascular surface density (CD34 endothelial marker) in the 10 most labeled fields (hVA), cellularity (CA) and the percentage of avascular fields (AVF) in bone marrow biopsy specimens from 34 patients with CLL. Vascular endothelial growth factor (VEGF) was assayed in thawed serum samples from 8 of the 23 untreated patients (12 Rai stage 0, 6 Rai stage I-II, 5 Binet stage B-C)., Results: Neoangiogenesis hVA was lower in the 12 stage O patients (p < 0.039) and in the 6 patients with nodular interstitial mixed infiltration (p = 0.058). The link between hVA and serum VEGF (sVEGF) was negative in 8 patients (R = -0.49, -0.70 for a given age). Age was linked to moderate interstitial infiltration (p < 0.02), AVF (R = 0.37) and lower sVEGF levels (R = -0.676). The blood platelet count showed numerous correlations, including links with AVF (R = 0.40), low hVA (R = -0.38), female sex (p = 0.068), absence of splenomegaly (p < 0.001), mixed-type infiltration (p < 0.01) and sVEGF (R = 0.38, 0.61 for a given age)., Conclusion: The links revealed by the iconography of correlations were described statistically. Surprisingly, negative links of hVA with the sVEGF level, 2 known prognostic factors, on the one hand, and with the blood platelet count, on the other hand, were found. The direct link between disease progression and the amount of avascular hematopoietic tissue (NS), not previously described, will require further study. Age should be taken into account.

Using array-based CGH, we identified 2p gain in 22/78 (28%) untreated Binet stages B/C CLL, which was the second most frequent copy number change after 13q deletion. It never occurred as a sole abnormality and was associated with other changes (6q deletion; 1p gain). The region of 2p gain frequently included two oncogenes, REL and MYCN. All patients with gain of REL were unmutated for IGHV (p=0.03). Gain of MYCN was associated with increased mRNA expression (p=0.005), suggesting a pathogenic role for MYCN. Gain of 2p appears to be a marker of progression and may contribute to the poor prognosis., (2009 Elsevier Ltd. All rights reserved.)

Several prognostic factors can predict the rapid progression in chronic lymphocytic leukaemia (CLL), including IGHV mutational status, cytogenetic abnormalities and, more recently, LPL/ADAM29 expression. In contrast, few studies have been devoted to the influence of these factors on clinical outcome in responding patients after therapy. We here propose to analyse the impact of IGHV gene status, LPL and ADAM29 gene expression on disease-free survival (DFS) and overall survival (OS) in 41 stage B or C CLL patients in remission after oral fludarabine plus cyclophosphamide. The median follow-up was of 64 (16-74) months. Sequencing of IGHV showed mutated (M) VH genes in 16 of 41 cases and unmutated (UM) in 25 cases. Analysis of LPL and ADAM29 expression in 35 of 41 cases showed overexpression of ADAM29 in 17 cases (14 M and three UM) and LPL in 18 cases (all UM). Patients expressing UM IGHV and LPL had shorter DFS and OS when compared to patients expressing M IGHV and/or ADAM29. Furthermore, blood minimal residual disease (MRD) evaluation using four-colour flow cytometry was performed in 33 out the 41 patients. We showed that patients who achieved phenotypic remission displayed longer DFS than those with MRD(+). Our results support the use of LPL and ADAM29 gene expression associated to IGHV mutational status for predicting the clinical outcome of patients treated by oral fludarabine + cyclophosphamide and could be considered for treatment strategies.

Objective: Hypofibrinolysis promotes atherosclerosis progression and recurrent ischemic events in premature coronary artery disease. We investigated the role of fibrin physical properties in this particular setting., Methods and Results: Biomarkers of recurrent thrombosis and premature coronary artery disease (CAD) were measured in 33 young post-myocardial infarction patients with angiographic-proven CAD and in 33 healthy volunteers matched for age and sex. Ex vivo plasma fibrin physical properties were assessed by measuring fibrin rigidity and fibrin morphological properties using a torsion pendulum and optical confocal microscopy. The fibrinolysis rate was derived from continuous monitoring of the viscoelastic properties after addition of lytic enzymes. Young CAD patients had a significant increase in plasma concentration of fibrinogen, von Willebrand factor, plasminogen activator inhibitor type 1, and lipoprotein(a) as compared with controls (P<0.05). Fibrin of young CAD patients was stiffer (P=0.002), made of numerous (P=0.002) and shorter fibers (P=0.04), and lysed at a slower rate than that of controls (P=0.03). Fibrin stiffness was an independent predictor for both premature CAD and hypofibrinolysis., Conclusions: This first detailed study of clot properties in such a group of patients demonstrated that abnormal plasma fibrin architecture is an important feature of both premature CAD and fibrinolysis rate. The determinants of this particular phenotype warrant further investigation.

Extra-cellular signal-related kinase (ERK) can modulate P27 in several ways. ERK is phosphorylated in a subset of salivary gland mucoepidermoid carcinoma (MEC). We determined immunohistochemical expression of P27, SKP2, cyclin A, Ki67, phospho-RB, and phospho-ERK in 43 MEC. SKP2 correlated with tumour size, microscopic grade, and a worse prognosis. Cyclin A and Ki67 also predicted prognosis, and were correlated with SKP2. P27 did not predict prognosis. P27 had no inverse relationship with SKP2, and correlated with neither Ki67 nor cyclin A. Instead, P27 high expressers had higher levels of phospho-ERK and phospho-RB. When highly expressed, P27 co-localized with cyclin D1 in the nuclei. Relationships of P27 with ERK and RB and its nuclear co-localization with cyclin D1 favour the hypothesis that P27 is in complexes with cyclin D1. This may explain why P27 in contrast to SKP2 and cyclin A does not correlate with tumour cell proliferation and prognosis.

Background: A few studies have suggested that von Willebrand factor (vWF) or plasminogen activator inhibitor-1 (PAI-1) can be associated with outcomes of acute coronary syndromes. The present study was designed to assess the acute release of these markers in ST-segment elevation myocardial infarction (STEMI) and their relations to death., Methods and Results: In 153 consecutive patients with STEMI, vWF and PAI-1 antigens were measured on admission (H0) and 24 hours later (H24). At 30 days, the death rate was 7.2%. Heart failure (Killip stage > or =3) on admission was present in 13.7% of patients. The acute release of PAI-1 (H24-H0, in ng/mL) and of vWF (H24-H0, in %) was dramatically higher in patients who died than in those who survived (46.9+/-26.3 versus -0.6+/-2.8 ng/mL, P=0.0001 and 65.8+/-20.0% versus 10.0+/-5.1%, P=0.004 for PAI-1 and vWF, respectively) and in patients developing heart failure compared with those without (24.8+/-10.1 versus -1.1+/-3.3 ng/mL, P=0.004 and 47.3+/-11.0% versus 8.1+/-5.6%, P=0.005 for PAI-1 and vWF, respectively). The release of PAI-1 correlated weakly with the left ventricular ejection fraction (R=-0.195, P=0.01) and the peak of troponin (R=0.149, P=0.045). Postangioplasty TIMI-3 flow and the acute release of PAI-1 were the only 2 independent predictors of death at 30 days., Conclusions: The acute release of vWF and PAI-1 over the first 24 hours of STEMI is associated with death and heart failure. The acute rise of PAI-1 is also a strong independent predictor of death at 30 days.

Clotting and fibrinolysis are initiated simultaneously in vivo, and fibrinolysis usually occurs without any individualized lysis front (intrinsic fibrinolysis). We have developed a novel model to assess whether morphological changes resulting from intrinsic fibrinolysis are similar to those previously reported at the lysis front using externally applied lytic agents. Fibrin assembly and fibrinolysis were followed in real-time by confocal microscopy using gold-labeled fibrinogen molecules. An increase in fiber absorbance (30%, p < 0.01) and a decrease in fiber diameter (60%, p < 0.01) due to the ongoing accumulation and packing of fibrin molecules were the most significant detectable features occurring during fibrin assembly. Similar features with a similar magnitude were observed during fibrin dissolution, but in the reverse order and with a 3-fold increase in duration. Then, lysing fibers were progressively transected laterally, and thinner fibers were cleaved at a 2.5-fold faster rate than thicker fibers (p < 0.001). Frayed lysing fibers were seen to interact progressively with adjoining fibers (agglomeration), leading to a 76 and 88% increase in the network pore diameter (p < 0.05) and fiber diameter (p < 0.01), respectively. At the maximum decrease in fiber absorbance (46%, p < 0.05), the network suddenly collapsed with the release of large fragments that gradually vanished. Morphological changes of fibrin that occur during intrinsic fibrinolysis are similar as those observed next to the lysis front, although they are not restricted spatially to the clot/surrounding milieu interface but are observed through the entire clot.

Objectives: In the present study, we describe the characteristics and examine the anticoagulation levels and safety of subcutaneous enoxaparin in unstable angina (UA)/non-ST-segment elevation myocardial infarction (NSTEMI) patients who would not have been eligible in the Efficacy Safety Subcutaneous Enoxaparin in Non-Q-wave Coronary Events (ESSENCE) and Thrombolysis In Myocardial Infarction (TIMI)-11B trials., Background: It is not known whether the benefit shown with enoxaparin in the selected population of pivotal trials can be extended to the real world., Methods: In our center, all patients with UA/NSTEMI are anticoagulated with subcutaneous enoxaparin adjusted to creatinine clearance. Among 515 consecutive patients, we identified 174 who would not have been eligible for ESSENCE or TIMI-11B ("EP" group for excluded patients). We evaluated cardiovascular death or non-fatal myocardial infarction (MI), as well as major and minor bleeding events, at 30 days in the EP group and in patients without any of the exclusion criteria ("NEP" group for non-excluded patients)., Results: This EP group was older, had a higher female/male ratio, and more frequently had a history of MI or a diagnosis of non-Q MI on admission than the NEP group. The distribution of the anti-Xa activity was similar in both groups. The bleeding rates (major and minor) at 30 days were similar in the EP and NEP groups (2.3% vs. 2.9%, respectively, P = NS). On multivariate analysis, the use of glycoprotein IIb/IIIa inhibitors and the presence of hypertension were the only independent predictors of bleeding found in the whole population. Compared with the NEP group, the EP group had a fourfold increased rate of death or MI at 30 days (15.5% vs. 4.1%, p < 0.01). On multivariate analysis, the independent predictors of death or MI at 30 days were NSTEMI on admission, creatinine clearance, and heart failure., Conclusions: Patients who do not fit the enrollment criteria of ESSENCE/TIMI-11B have higher risk baseline characteristics for both bleeding and ischemic events. In these patients, enoxaparin with dose adjustment to creatinine clearance provides adequate anti-Xa levels and no excess of bleeding.

Glycoprotein IIb/IIIa (GP IIb/IIIa) inhibitors were shown recently to facilitate the rate and the extent of pharmacological thrombolysis. However, their synergistic potential with rtPA in dissolving thrombotic vaso-occlusions is not fully understood. We have therefore developed a dynamic and structural approach for analysis of fibrinolysis to assess the inhibiting effect of platelets and the facilitating effect of GPIIb/IIIa inhibitors in dissolving platelet-rich clots (PRCs). Fluorescent rtPA was used to study the architecture of PRCs, to follow the progression of the rtPA binding front, and to measure the lysis-front velocity using confocal microscopy. Fibrinolysis resistance of PRCs was related to a reduction of both rtPA binding and lysis-front velocities of platelet-rich areas compared with platelet-poor areas (2.4 +/- 0.2 versus 3.5 +/- 0.4 microm/min for rtPA binding velocity, P=0.04, and 1.2 +/- 0.6 versus 2.8 +/- 0.2 microm/min for lysis-front velocity, P=0.008, in platelet-rich and platelet-poor areas, respectively). Fibrinolysis appeared heterogeneous, leaving platelet-rich areas un-lysed. Adding pharmacological concentrations of abciximab (0.068 micromol/L) or eptifibatide (1 micromol/L) before clotting decreased the average surface of platelet-rich areas by 64% (P=0.0005) and 72% (P=0.0007), respectively. The resulting equalization of rtPA binding rate and rtPA binding-front velocity between platelet-rich and platelet-poor areas led to a 3-fold increase of the lysis-front velocity in platelet-rich areas of either abciximab-PRC (P=0.006) or eptifibatide-PRC (P=0.03). The overall lysis rate of treated-PRC was increased by 74% compared with control-PRC (P<0.01). These results demonstrate that fibrinolysis resistance of PRCs is related primarily to the heterogeneity in the clot structure between platelet-rich and platelet-poor areas. GP IIb/IIIa inhibitors facilitate the rate and the extent of fibrinolysis by improving rtPA binding velocity and, subsequently, the lysis rate in platelet-rich areas. These findings provide new insights on the synergistic potential of GP IIb/IIIa inhibitors and fibrinolytic agents.

Through a global analysis of diagnostic features, the aim was to profile CLL patients with circulating cleaved lymphocytes at diagnosis, a controversial prognostic factor. Although some of them could have been considered today as having Non-Hodgkin's lymphoma, all 106 patients of our retrospective series have had CLL treatments. Slide review distinguished seven lymphocyte morphotypes. With minimal a priori assumptions, excluding in particular clinical staging systems, forty-five diagnostic features were analyzed in 37 patients. CORICO (Correlations Iconography), a purely geometric method, deciphered the multidimensional structure of the raw data. Probabilistic monoparametric tests were made on the 106 patients. In ten patients (Binet stages: 3A, 6B, 1C), at least 8% of the lymphocytes were cleaved. Unrelated to the prolymphocytes, this morphotype had neither links with the CD5+CD23+ (9/10 vs 80/86), FMC7+ (5/10 vs 22/62), CD38 (1/7 vs 7/64) markers nor with any major CLL laboratory values; only three links characterized it: no cases of mixed marrow infiltrate (nodular: 1, interstitial: 6, diffuse: 3; ns), a lower percentage of eosinophils (ns), and predominance of CD11c (7/10 vs 20/66, p < 0.02). In conclusion, in contrast to the PLL morphotype, or to the lactic dehydrogenase (LDH) activity, which was a strong prognostic factor in this series, an independent detrimental value of the cleaved morphotype has not yet been found. Our study shows that free of modeling constraints, this method makes possible a rapid and objective insight into variable interrelations. If further explored in a prospective study, this approach may contribute to the understanding of discrepancies in the literature.

Background: Abciximab plus aspirin improves the TIMI 3 flow rate of the infarct-related artery in patients treated with either percutaneous coronary intervention or thrombolysis. The present study investigated whether the reperfusion efficacy of abciximab relates to modifications of clot architecture in patients admitted for acute myocardial infarction (AMI)., Methods and Results: A total of 23 AMI patients in the Abciximab before Direct angioplasty and stenting in Myocardial Infarction Regarding Acute and Long term follow-up (ADMIRAL) trial received, in a double-blind fashion, either abciximab (n=13) or placebo (n=10) before primary stenting. Viscoelastic (G' in dyne/cm(2)) and morphological (mean platelet aggregate surface area [SAG] in micrometer(2)) indexes of ex vivo platelet-rich clots (PRC) were assessed in a double-blind fashion before and after the bolus administration of abciximab or placebo. G' and SAG reflect the mechanical and morphological impact of activated platelets on the PRC fibrin network, respectively. Abciximab administration reduced G' by 63% (P=0.0001) and SAG by 65% (P=0.0007), and no effect was seen in the placebo group. These abciximab-related changes increased fibrin exposure as a consequence of the platelet-aggregate surface reduction and may have improved endogenous fibrinolysis. These effects were identified in all patients, independent of previous heparin administration., Conclusions: Abciximab dramatically reduces platelet aggregate size and increases the fibrin accessibility of ex vivo PRC in AMI patients. These modifications could participate in the better coronary artery patency observed with abciximab.

The glycoprotein IIb/IIIa receptor inhibitor abciximab has been shown to facilitate the rate and the extent of pharmacological thrombolysis with recombinant tissue plasminogen activator (rtPA) in patients with acute myocardial infarction. However, the underlying mechanisms remain not fully determined. We sought to demonstrate that this facilitating effect of abciximab could be related to its potential to modify the clot architecture and the clot physical properties. Compared with fibrin-rich clots, platelets dramatically modified the in vitro properties of the fibrin network, leading to a significant increase of the permeability (K(s)) and the viscoelasticity (G') indexes but also leading to the appearance of platelet aggregates (surface area [S.ag]). These modifications resulted in a 2.6-fold decrease of the fibrinolysis rate when rtPA (1 nmol/L) was added before the initiation of clotting. Adding aspirin (100 microgram/mL) or abciximab (0.068 micromol/L) before the clotting of platelet-rich clots (PRCs) lowered K(s) by 50% and 70%, respectively (P<0.01), G' by 41% and 66%, respectively (P<0.01), and S.ag by 32% and 61%, respectively (P<0.01). As a consequence, the lysis speed was increased by 21% with aspirin (P<0.01) and 45% with abciximab (P<0.01). However, unlike aspirin, permeation of preformed PRCs with abciximab (0.068 micromol/L) decreased G' (37%, P<0.01), K(s) (35%, P<0.001) and S.ag (25%, P=NS) and resulted in a 27% (P<0.01) increase of the lysis speed when abciximab and rtPA (0.2 micromol/L) were simultaneously permeated. This effect was found to be time dependent and was observed only with early permeation, starting within the first 10 minutes of clotting. These changes in the physical properties of the PRC architecture suggest that fibrin is removed from the platelet-fibrin aggregates and reexposed into the surrounding fibrin network, increasing rtPA access to fibrin and therefore the fibrinolysis rate. The superiority of abciximab over aspirin in accelerating fibrinolysis of forming and preformed PRCs is related to its ability to modulate the interactions of fibrinogen and fibrin with platelets. These findings provide new mechanistic information on reperfusion therapy.

Abnormal fibrin architecture is thought to be a determinant factor of hypofibrinolysis. However, because of the lack of structural knowledge of the process of fibrin digestion, relationships between fibrin architecture and hypofibrinolysis remain controversial. To elucidate further structural and dynamic changes occurring during fibrinolysis, cross-linked plasma fibrin was labeled with colloidal gold particles, and fibrinolysis was followed by confocal microscopy. Morphological changes were characterized at fibrin network and fiber levels. The observation of a progressive disaggregation of the fibrin fibers emphasizes that fibrinolysis proceeds by transverse cutting rather than by progressive cleavage uniformly around the fiber. Plasma fibrin clots with a tight fibrin conformation made of thin fibers were dissolved at a slower rate than those with a loose fibrin conformation made of thicker (coarse) fibers, although the overall fibrin content remained constant. Unexpectedly, thin fibers were cleaved at a faster rate than thick ones. A dynamic study of FITC-recombinant tissue plasminogen activator distribution within the fibrin matrix during the course of fibrinolysis showed that the binding front was broader in coarse fibrin clots and moved more rapidly than that of fine plasma fibrin clots. These dynamic and structural approaches to fibrin digestion at the network and the fiber levels reveal aspects of the physical process of clot lysis. Furthermore, these results provide a clear explanation for the hypofibrinolysis related to a defective fibrin architecture as described in venous thromboembolism and in premature coronary artery disease.

Porosity, viscoelasticity and morphological properties of plasma fibrin from 16 nephrotic patients and 16 healthy volunteers were compared. Nephrotic patients were characterized by formation of tight and rigid plasma fibrin gels which resulted in a slower rate of fibrin lysis studied either under pressure-driven permeation or diffusional transport of fibrinolytic agents. These latter findings indicated that both abnormal fibrin network conformation and abnormal fibrin fiber structure were involved in hypofibrinolysis. Albumin supplementation up to 40 mg/ml partially restored normal fibrin architecture and increased the rate of fibrinolysis in these patients. Multiparametric analysis showed that nephrotic patients were mainly characterized by a low plasma albumin level (R = -0.85), a low albumin to fibrinogen ratio (R = -0.89) and a high resistance to lysis (R = -0.82). High triglycerides level was the only plasma modification related to the slower fibrin lysis rate (R = -0.54). High fibrin rigidity (G') was the only fibrin parameter simultaneously related to the nephrotic state (R = 0.75) and the lysis resistance (R = -0.71). After eliminating the effects of age, albumin and fibrinogen levels, low fibrin porosity (Ks) and low fiber mass-length ratio (mu) were the main features of the nephrotic state. These findings are discussed in relation to both the pathophysiology of thrombotic complications in nephrotic syndrome and their pharmacological prevention.

This investigation was undertaken to further study cyclosporin A (CsA)-induced gingival overgrowth. Thirty mg/kg/d of vehicle or CsA solutions were given orally to 6-wk-old male Sprague-Dawley rats. After 4, 9, 14 and 19 wk 2 control and 2 experimental rats were anaesthetized, tissues fixed by intracardiac perfusion of fixative solution and jaws processed for Epon inclusion. Histological and ultrastructural studies conducted in a gingival portion (free gingiva) revealed the presence of hyalinization areas and of multinucleated cells (MCs) containing collagen fibrils (connective tissue), of amorphous areas and disorders of keratinization (epithelia). Histomorphometric evaluation indicated that in the CsA rats the mean cross-sectional area of the free gingiva was 2.52-fold increased compared to the controls. The connective tissue comprised 41.43% of this area (instead of 31.49% in controls). Additional histomorphometric evaluation was performed in 3 groups of free gingival portions: control (C group), CsA-non-respondent (CsA-nR) and CsA-respondent (CsA-R). The cross-sectional gingival areas studied were slightly lower than the mean area of all the control sites previously defined (groups C and CsA-nR) or showed the higher degrees of enlargement (CsA-R). In the CsA-R group the mean cross-sectioned area of the vessel profiles was increased and the number of fibroblast profiles decreased. In the CsA-nR group the number of vessel profiles and that of MCs profiles were increased. In the epithelia of the CsA-R group were increased (a) keratinized epithelia: thickness; thickness of the inner and of the outer compartments; surface area of spinous cell profiles; (b) oral gingival epithelium: number of cell layers (inner compartment); (c) oral sulcular epithelium: surface area of granular cell profiles; (d) junctional epithelium: thickness; number of cell layers. These results indicate that (a) the CsA induced modifications are not limited to enlarged gingiva (b) the overgrowth of the GCT is the result of a vasodilatation and of an increase in the volume of the extracellular matrix and (c) the increase of the epithelial thickness is mainly the result of a cell hypertrophy in the keratinized epithelia and of a cell hyperplasia in the junctional epithelium.

The aim of the present investigation was to examine if new cementum (NC) formed during cyclosporin A (CsA) administration was maintained after suspension of the treatment. Thirty mg/kg/d of CsA were given to 3 male Sprague-Dawley rats. Three control rats received oil-based vehicle solution. Nine wk later the drug and vehicle administration were stopped and the rats continued to be fed with the same standard laboratory diet and water ad libitum for 5 months. The rats were anaesthetized, the tissues fixed by intracardiac perfusion of fixative solution and the mandibles processed for Epon inclusion. Histological, histomorphometric and ultrastructural analysis revealed that (a) NC covered extensive areas of the root surfaces; its structural characteristics were identical to those observed in the rats killed during CsA administration. (b) collagen fibres of the adjacent connective tissue were functionally inserted into the NC. (c) In the presence of cervical NC spurs the extent of the apical downgrowth of the junctional epithelium, measured parallel to the cemento-dentinal junction, was decreased (up to 64%) compared to the one occurring in areas devoid of NC deposits. These results suggest that (a) NC deposition and its functional relations with the adjacent connective tissue are not reversible after cessation of CsA treatment and (b) in the presence of cervical NC spurs the amount of connective tissue attachment on the root surfaces is increased.

Cyclosporin A (CsA), a widely used immunosuppressive agent, is known to induce gingival overgrowth; 30 mg/kg/d of CsA were administrated orally in young and adult male Sprague-Dawley rats. The same number of rats received oil-based vehicle solution. After 4, 9, 14 and 19 wk of CsA or vehicle administration 3 control and 3 experimental rats were anaesthetized and tissues fixed by an intracardiac perfusion of fixative solution. Upper and lower jaws were dissected, demineralized and processed for Epon inclusion. Histological examination revealed the presence of large amounts of new cementum (NC) covering extensive areas of the acellular extrinsic fibre cementum (AEFC) in all the root surfaces. NC was particularly abundant at the cervical third of the roots facing the gingival connective tissue, where it occurred as layers, spurs or in both configurations. NC was characterized by its irregular outline, globular body content and infrequent presence of incremental lines. Histomorphometric evaluation by semi-automatic image analysis indicated that the volume and the external surface of NC spurs were 2.86-6.49 and 1.29-1.97-fold increased comparative to those of the AEFC covering the same root areas. Electron microscopy revealed that NC was a functional tissue with insertion of collagen fibres perpendicularly to the long axis of the root. It can be concluded that under some experimental conditions formation of abundant amounts of NC can be achieved and that these results must be taken into account for a new approach in the treatment of periodontal disease.

Cyclosporin A (CsA), a widely used immunosuppressive drug, induces gingival overgrowth and modifications of bone remodelling. The scope of this study was to investigate the possible effect of CsA on dentin. Thirty mg/kg/day of CsA were administered orally to male Sprague-Dawley rats for nineteen weeks. The same number of control rats received oil-based vehicle solution. Rats were anesthetized, and tissues were fixed by an intracardiac perfusion of fixative solution. Mandibles were dissected, demineralized, and processed for Epon embedding. Semi-thin sections of the first molars revealed alterations at the secondary dentin-pulp interface in four out of six experimental animals. The changes consisted of the formation of: 1) osteodentin spurs, in which the volume and interface with the secondary dentin varied from about 25,000 to 75,000 microns 3 and from 1400 to 3530 microns 2, respectively; 2) abnormally shaped and irregularly spaced incremental lines; and 3) numerous globular formations embedded in dentin or free in the pulp. These results indicate that CsA induces abnormal mineralized matrix formation in dentin and in the peripheral part of the pulp in rat molars.

Cytologists have seldom made a quantitative study of the location of the nucleus within the cell. We have mathematically defined three parameters in two dimensions: the number of distinct cell/nucleus boundary contacts (Ncon), the length of such contacts (Lcon), and the degree of nucleus location eccentricity (Ecc), expressed as a function of the greatest distance between cell and nucleus boundaries, and of the nucleus/cytoplasm ratio (N/C). Based on the analysis of 68 circular or elongated cell and nucleus models, we identify 21 topographies, for a range of N/C values between 20% and 70% and for a range of cell surface area values between 2900 and 6700 pixels on a hexagonal raster (256 x 256). Beyond 70%, a maximum of two topographies may be distinguished; below 20%--a figure nearer a nucleolus-nucleus ratio--the relevance and reliability of the approach are subject to some doubt. We conclude that the problem of describing the location of one structure within another remains dependent on N/C. The method has been applied to 59 lymphoid cells on smear photographs recognized internationally by hematologists as characteristic of lymphoid diseases. The results show the robustness of the combination of four criteria in relation to the overall elongation of nucleus or cell profiles, and to significant contour irregularities. The method has demonstrated its ability to describe and quantify the eccentric position of the nucleus within plasmocytic lymphocytes, and provide a further degree of discrimination.

Capillarity, fibre types, fibre area and enzyme activities of different skeletal muscles (pectoralis, extensor digitorum longus), tibialis anterior, plantaris and the myocardium) were compared in Andean coot (Fulica americana peruviana) native to high altitude (Junín, Perú, 4200 m) and the same species nesting at sea level. Numbers of capillaries per square millimeter were higher in all high-altitude muscles when compared with sea-level muscles (P < 0.0001). Moreover, values for capillaries per fibre and capillaries in contact with each fibre were higher in digitorum and tibialis high-altitude muscles. Muscle fibres were classified as Type I, Type IIA or Type IIB on the basis of their myofibrillar ATPase pH lability. Pectoralis muscle of high-altitude and sea-level coots presented only fibres of Type IIA. In contrast, all the leg muscles studied showed a mosaic pattern of the three fibre types. Fibre areas were determined using a Leitz Texture Analysis System. Significant differences in fibre area were observed (P < 0.01) between high-altitude and sea-level muscles. Mean muscle fibre diameters were also lower in the high-altitude group than in the sea-level group. The enzyme activities studied were hexokinase, lactate dehydrogenase, citrate synthase and 3-hydroxyacyl-CoA-dehydrogenase. The oxidative capacity, as reflected by citrate synthetase and hydroxyacyl-CoA-dehydrogenase activities, was greater for myocardial and pectoralis than for leg muscles. However, analysis of maximal enzyme activities showed that there were no significant differences between the glycolytic and oxidative enzyme activities of high-altitude and sea-level coots.(ABSTRACT TRUNCATED AT 250 WORDS)

For over 50 years the received wisdom has been that the shadow cells of Gumprecht otherwise known as basket cells (BC) are in artefact, produced during preparation of films when a drop of blood is spread on a slide. The assumption has been that they are therefore of no significance. They are commonly seen in blood films from patients with lymphoproliferative syndromes and particularly in chronic lymphocytic leukemia (CLL). In 96 patients with CLL a statistically significant correlation existed between the basket cells observed in films and the lymphocytes with dense chromatin (DC) determined by flow-cytometry. There was no statistically significant correlation between the number of BC and DC, and the anatomic-clinical stage of the disease.

Total Ki-67 stained area percentage was studied in 32 B and 46 T malignant lymphomas (ML) using two different image analyser systems (TAS, Leitz; SAMBA TM 2005, TITN) respectively. The total Ki-67 area percentage was highly correlated to the number of Ki-67 positive cellular profiles (B-ML, r = 0.93; T-ML, r = 0.88), indicating that area percentage is a reliable alternative method to the manual cell counting. Image analysis allows quicker measurements, appropriate to large and strictly lymphomatous regions. The cell image processor (SAMBA TM 2005, TITN) linked to a color video camera was more suitable for immunohistochemical sections and allowed more automated and faster measurements than the texture analyser (TAS, Leitz) linked with a black and white camera. Alkaline phosphatase technique with fast red as chromogen was more suitable for the detection of Ki-67 stained area by thresholding than peroxidase technique with aminoethylcarbazol or with diaminobenzidine as chromogens. Significant differences were found between low and high grade in B and T ML according to the Kiel classification (mean values +/- SD of 7.7 +/- 3.8% and 16.6 +/- 6.2% in B-ML and of 10.2 +/- 7.9% and 25.6 +/- 16.3% in T-ML respectively). In follicular B-ML, considering follicular areas only, values were comparable to high grade ML; angioimmunoblastic-lymphadenopathy-like (AILD-type) T-ML belonging to low grade ML showed similar values to pleomorphic T-ML with medium and/or large cells belonging to high grade ML.(ABSTRACT TRUNCATED AT 250 WORDS)

The purpose of this paper is to analyze a method allowing selection of the best morphometric criterion for quantifying AgNOR proteins under conventional observation conditions by light microscopy. We determined 50 parameters for 32 cases of primary breast carcinoma. For each case, three 100-nucleus samples (tumor center and periphery on a Giemsa-stained, 3-microns tumor section, periphery on a silver-stained section) were quantified. Distribution-free multidimensional data analysis was used to explore the geometric significance of the coefficient of correlation. This analysis revealed a clinical message linked with the largest "nucleolar structure" (nucleolus or AgNOR clump) per nucleus on the tumor periphery. Tumor recurrence or death correlated only with the coefficient of variation of the "nucleolar/nuclear" ratio of the greatest clump of AgNORs per nucleus periphery (CVRSa). The prognostic value of CVRSa is independent of that of conventional criteria, such as age, tumor size, Scarff-Bloom-Richardson grading and lymph node status. The largest AgNOR clump per nucleus may prove to be a further practical prognostic predictor.

In sections from 32 B malignant lymphomas (ML), the total KI-67 stained area was compared to the number of KI-67 positive cells in order to demonstrate the reliability of using image analysis to quantify the proliferative activity. The total KI-67 area percentage correlated highly with the number of KI-67 positive cellular profiles (r = .93). Significant differences were found between low- and high-grade ML according to the Kiel classification (mean values +/- SD, respectively, of 7.7 +/- 3.81% and 16.6 +/- 6.23%), and between low-, or intermediate- and high-grade ML only, according to the International Working Formulation. Within the Working Formulation, the statistical analysis grouped the diffuse large cell subtype of intermediate grade with the immunoblastic high-grade subtype. A wide range of KI-67 area percentage values was noted, particularly in follicular ML; for these follicular ML, considering follicular areas only, values were comparable to high-grade ML (14.8 +/- 6.60%). In conclusion, the KI-67 area percentage is a reliable alternative method to manual cell counting, and image analysis allows quicker measurements appropriate to large and strictly lymphomatous areas, using a greater number of cells than in manual cell counting.

A quantitative study of dermal collagen as a function of age was carried out by computerized digital image analysis. Fast Green-Syrius Red stained sections were obtained of skin biopsies taken from the upper inner arm of 33 healthy women and 38 healthy men. The Leitz texture Analysis System (Leitz-TAS) and mathematical morphology (Serra, 1982) were used for the evaluation of the data. Collagen was studied in the superficial dermis and also in the reticular dermis using the same program. There were significant correlations, firstly between the percentage of collagen measured by the morphometric method and the concentration of collagen analysed biochemically (microg/mm(2) of tissue section) (r = 0.79, p < 0.001) and secondly between the decreased concentration of collagen and age (r = 0.58, p < 0.05). The morphometrical measurements have shown that the relative percentage of collagen bundles (surface of collagen fibers as a function of the dermal area analyzed) was 93.35% in the superficial dermis and 89.2% in the reticular dermis. Although this value is higher than the chemically determined ratio of collagen to other proteins (over 70%), this may be due to the relatively uniform distribution pattern of (type I and III) collagen through the dermis covering most other components of the skin. As the collagen fiber density per unit dermal surface did not change with age, the decrease in collagen content of the skin may be ascribed to the loss of about 6% of dermal mass per decade (Branchet, 1990), although large individual variations exist. The histogram of the diameter distribution of collagen fiber bundles of the reticular dermis showed thinner diameters in persons between 20 and 40 years of age than in older persons. The histogram of the distribution of interfiber spaces did not show any variation with age in the superficial dermis, while in the reticular dermis there was a predominance of smaller interspaces in persons older than 50 years.

The position of the nucleolus within a near-convex nuclear profile was defined, without taking stereology into account, in terms of two distances, P and G, where P = pi p2/SN, G = pi 2/SN, p and g being, respectively, the smallest and greatest distances between the center of the nucleolus (considered as a disk) and the superficial nuclear membrane, and SN being the surface area of the nucleus. Nuclear elongation (ND) was defined by the ratio ND = SN/A, where A is the area of the largest inscribed disk. The nucleolus-nucleus ratio RS = Sn/SN (i.e., the surface of the nucleolus over that of the nucleus) describes the size of the nucleolus. A four-class classification of nucleolar topography was then developed from the model of an ellipsoid nuclear profile (1 less than ND less than 2.5), for which the probability that a randomly located nucleolus (with RS less than 0.23) will be classified as central (G less than 2.5 and 0.40 less than or equal to P less than 1) is less than 0.14, as paracentral (G less than 2.5 and 0.23 less than or equal to P less than 0.40) is less than 0.13, as "transversely eccentric" (G less than 2.5 and P less than 0.23) is less than 0.10 and as "longitudinally eccentric" (G greater than or equal to 2.5) is greater than 0.66. This theoretical distribution into four classes was compared to that of nuclear profiles in four cases of breast cancer and in typical cases of immunoblastic, centroblastic and Burkitt's non-Hodgkin's lymphomas. The findings illustrate the ability of this nonparametric method to indicate characteristic nucleolar locations in relation to the number of nucleoli, their size and their nuclear profile elongation.

A quantitative study of dermal elastic fibers was carried out by computerized digital image analysis. Sections of skin biopsies taken from the upper inner arm of 33 healthy women and 38 healthy men were stained by a selective procedure for elastic fibers. The Leitz texture analysis system (Leitz-Tas) and mathematical morphology were used for the evaluation of the data. Distinct programs were used for the vertical superficial elastic fibers and for the mature elastic fibers of the reticular dermis. For elastic fibers of the superficial dermis there was no significant variation with age or sex for total area occupied by the fibers or for the total number of fibers per unit skin area or total fiber length. The distribution of the relative fiber number as a function of their length showed that about 80% of the fibers had a length of 0-20 microm, with no detectable age- or sex-dependent variation. The distribution of fiber number frequency as a function of fiber diameter did not show any significant variation with sex or age, either. The relative area of mature elastic fibers increased significantly with age after the sixth decade of life. The total number (Nt) of these fibers did not show however any significant variation with age or sex: Nt per mm(2) of skin surface was 3220+/-660 (S.D.). We found for all age groups a significant increase of total fiber length by comparing persons between 20 and 30 years with those above 60 years. There was, however, no significant variations according to age or sex for the frequency distribution of mature elastic fiber length and diameter. As a significant decrease with age of the dermal thickness was also found in males and females, the loss of elastic fibers with age is mainly due to a loss of skin thickness (skin volume) with age. This loss is about 30% at 50 years and nearly 50% at 80 years. Loss of skin elasticity with age can be due to loss of cells (fibroblasts), to a decrease of their biosynthetic activity and to qualitative modifications of extracellular matrix macromolecules.

Survivals of two series of CLL patients (99 from a retrospective series and 196 from a prospective series) were studied separately. The three main staging systems (Rai, Binet, Rundles) agreed well, but as far as survival is concerned, too many stages are defined. The authors performed a Cox multivariate analysis of survival in order to isolate important prognostic factors at diagnosis and to use them to build a simple three-stage classification. Thrombopenia and anemia appeared as the most important risk factors. Among the nonanemic and nonthrombopenic patients, the number of involved areas was clearly related to prognosis in the authors' two series. This study allowed the authors to propose a new classification in three prognostic groups. Group C: anemia (Hb less than 10 g) and/or thrombopenia (platelets less than 100,000/mm3); about 15% of the patients; median of 2 years. Group B: no anemia, no thrombopenia, three or more involved areas (counting as one each of the following: axillary, cervical, inguinal, lymph nodes, whether unilateral or bilateral, spleen and liver); about 30% of patients; median of 7 years. Group A: no anemia, no thrombopenia, less than three involved areas; about 55% of patients; the survival of this group does not seem different from that of the French population of the same age and sex distribution. This three-stage classification only requires clinical examination and routine hemogram, has a good prognostic value which was confirmed on the series of Montserrat and Rozman (146 patients), and should therefore be helpful in planning new clinical trials.

A Coulter ZB was used to measure electronically the size of circulating platelets collected from normal subjects. EDTA or ACD were used for collection. Three mathematical models of volume histograms were tested. The lognormal law model is clearly unacceptable if all particles are used, but is applicable in the particle range of 3-15 mu 3. The model of a mixture of two lognormal populations closely approximates observed volume distribution. The model of three lognormal populations also approximates well when the largest volume population is divided into two subpopulations. The contribution of these results to an understanding of heterogeneity in platelet size is discussed.

In an attempt to determine the importance of perivenular fibrosis (PVF) in alcoholic liver disease, we studied 71 liver biopsies using histological grading and a morphometric method. The histological grading used 7 variables which allowed us to classify the patients into 7 groups: controls, patients without alcoholic hepatitis but with steatosis, steato-fibrosis, portal fibrosis and patients with mild, moderate, or severe alcoholic hepatitis. The quantitative analysis examined 3 parameters: (1) The inner diameter of the terminal hepatic veins (THV). (2) The thickness of the THV rims, related to perivenular fibrosis (PVF). (3) Centrolobular fibrosis (CLF) which represented the association of perivenular and perisinusoidal centrolobular fibrosis. No changes in the inner diameter of the terminal hepatic veins was observed for the different groups except in the case of severe alcoholic hepatitis. This fact indicated the absence of veno-occlusive lesions in early stages of mild and moderate alcoholic disease. In severe alcoholic hepatitis, THV were destroyed by centrolobular scars and most of them were indistinguishable and unmeasurable. Of the 26 cases with steatosis (with or without portal fibrosis) only two cases with steatofibrosis showed perivenular fibrosis. In contrast, a significant increase in PVF and in CLF appeared in patients with alcoholic hepatitis. CLF is easier to quantify and more significative than PVF. Thus, it seems to us that CLF is a better indicator of the intensity of sclerosis and of the risk of developing cirrhosis than PVF alone.

Parallel morphometric studies were conducted in the same cases of ossifying fibroma and fibrous dysplasia to confirm differences suggested by the previous study between the two bone diseases.

During the period from 1982 to 1987, 19 patients with advanced Hodgkin's disease, clinical stages IIIB and IV underwent treatment with combination chemotherapy and radiotherapy. Alternating chemotherapy (3 cycles of CVCPP/3 cycles of ABVD) was sandwiched with low-dose total nodal irradiation including the splenic area; at the end of the chemotherapy, a supplemental 20 Gy radiotherapy was sometimes delivered to bulky previously involved sites. The complete remission rate was 84.2% (16/19 patients) and actuarial survival was 85% at 48 months. These preliminary results show that the alternating technique for combining chemotherapy and sandwiched irradiation is feasible and improves the outcome in advanced Hodgkin's disease. Patients with mediastinal masses associated with pulmonary and/or pleural involvement may need more aggressive chemotherapy at an earlier phase of the treatment program.

In addition to the nuclear area and a form factor, four morphometric parameters of nuclear shape (ID, R1, R2 and ND), obtained by the application of the principles of mathematical morphology, were used to characterize the nuclear contours in non-Hodgkin's malignant lymphomas. The values for each parameter were determined in 58 cases of non-Hodgkin's lymphoma categorized according to the Kiel and National Cancer Institute classifications. Small-cell, mixed and large-cell lymphomas could be distinguished on the basis of the mean nuclear area. The shape parameters R1, R2 and ID were efficient discriminators of the large centrocytic (cleaved-cell) lymphomas. Neither size nor shape factors could distinguish between centroblastic and immunoblastic tumors. The good correlation between the morphometric findings and the histopathologic categories suggest that morphometry may provide a quantitative and objective method for grading lymphomas.

Using principles from the theory of mathematical morphology, a semiautomatic analysis of the size and shape of cell nuclei on tissue sections was carried out on a Leitz Texture Analysis System (Leitz-TAS). The four parameters proposed here are more discriminatory than conventional shape evaluation by the form factor (FF), which is based on the ratio of perimeter squared to area. The parameters quantified, respectively, nuclear elongation (ND), narrow (R1) and wide (R2) irregularities, and the distribution of R1 and R2 along the nuclear contour (ID). The properties of these parameters were tested nucleus-by-nucleus on 24 nuclear models. The methodology was then illustrated by a study of lymph node nuclei in non-Hodgkin's lymphoma (NHL). Prior to analysis, 45 lymphomas were classified into five categories of nuclear size and shape according to the International Working Formulation (IWF). Two hundred nuclei were measured on each lymph node section. Statistical interpretation was based upon an analysis of the nuclear surface area on sections and upon the mean values of R1, R2, and ND, the standard deviations of R1 and R2, and the percentage of cleaved nuclei detected by ID. The mean value of R2 discriminated best between the two sets of populations with regular and irregular nuclear contours, respectively. Parameters R1, ND, and ID permitted the distinction of certain NHL cases among populations with irregular nuclei. Nuclear invaginations decreased in depth as the nuclear area became greater. The median surface area was well correlated to the IWF, and the skewness coefficient (third statistical moment of the nuclear surface area distribution) was related to the number of nuclear size or shape subpopulations.

We quantitated the skin elastic fibers of the papillary and reticular dermis using specific staining procedure followed by automated computerized image analysis. Fifty skin biopsies of patients consulting for cardiovascular risk factors were studied. The following parameters were estimated: surface area (% of total surface), length, and number of fibers as a function of age. The evaluation of the skin elastic fiber system showed no significant trend with age in the number of elastic fibers per unit area in the superficial or deep dermis. We found, however, a continuous increase with age in the relative surface area, and the length of the elastic fiber system. These results can best be interpreted as a continuous apposition of elastic-type material to the preexisting fibers by skin fibroblasts. This material may well be of a different composition than young elastin, for instance enriched in structural glycoproteins (microfibrils), lipids and calcium as found in the elastic fibers of aorta and lung. Such quantitative modifications may explain the decrease in skin elasticity, and the continuous increase in stained fibers.

The modal volume of peripheral blood lymphocytes from 23 patients with chronic lymphoid leukemia (CLL) was measured by a Coulter ZBI linked to a channalyzer C 1000, varied from 140 to 262 mu3. A statistical correlation between the modal volume and anatomo-clinical forms of the disease was determined: a modal volume below 190 mu3 was statistically found among early forms while a volume over this threshold corresponded to advanced forms. Since Sipe et al. postulated that cell volume might be relayed to the cell kinetics, we attempted to determine the volume distribution law of lymphocytes from 12 CLL cases by applying the chi-square distance test. The best adjustment was observed with the two log-normal populations model, but this adjustment was acceptable for only five cases over twelve. The instability in the determination of the percentage of the second population made it impossible + relate it with the proliferative cell fraction. Moreover, this model did not provide more significant information than the single modal volume.