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Prepublished online as a Blood First Edition Paper on July 5, 2002; DOI 10.1182/blood-2002-01-0084.
NEOPLASIA
From the Departments of Hematology/Oncology, and
Anatomy and Histopathology, Azienda Ospedaliera
"Pugliese-Ciaccio," Catanzaro, Italy; Department of Biomedical
Sciences and Human Oncology, Section of Internal Medicine and Clinical
Oncology, and Department of Human Anatomy and Histology, University of
Bari Medical School, Bari, Italy; Institute of Hematology, University
of Ferrara, Ferrara, Italy; and Clinical Pathology Service, Institute
"Regina Elena" IRCCS, Rome, Italy.
Because tumor progression is angiogenesis-dependent,
angiogenesis density was investigated by immunohistochemistry and
computed image analysis in bone marrow (BM) biopsies of 45 newly
diagnosed patients with Binet stage A B-cell chronic lymphocytic
leukemia (BCLL) and correlated to upstaging and
progression-free survival during a 40-month follow-up period. Their
microvessel areas and counts were significantly higher than those of
patients with anemia due to iron or vitamin B12
deficiencies. A cutoff value of 0.90 mm2 × 10 Tumor progression in the form of growth,
invasion, and metastasis depends on angiogenesis,1 whose
increase is thus indicative of poor prognosis in solid
tumors.2 The expression of several angiogenic growth
factors, including vascular endothelial growth factor (VEGF) and
fibroblast growth factor-2 (FGF-2), is of comparable prognostic
value.3 Knowledge of these relations in hematologic tumors, however, is circumstantial. A tumor's microvessel density predicts a risk of progression in multiple myeloma.4 It is correlated with progression stages in both B-cell non-Hodgkin lymphomas5 (NHL) and mycosis fungoides,6 and
agrees with the growth of acute leukemias7 and
myelodysplastic syndromes.8,9
Angiogenesis is involved in the pathogenesis of B-cell chronic
lymphocytic leukemia (BCLL).10,11 VEGF, present in the
patient's serum and leukemic cells,12-14 is related to
poor prognosis. FGF-2 expression by leukemic cells is associated with
advanced disease and resistance to fludarabine.15 Studies
on 2 series of BCLL bone marrow (BM) angiogenesis carried out in
limited and heterogeneous groups of patients provided conflicting
results on its density and no prognostic information.11,16
This paper presents results of an investigation of microvessel density
and VEGF expression in the BM of a homogeneous series of 45 patients
with BCLL with early disease. Relationships between angiogenesis, VEGF,
and FGF-2 serum levels and other markers of disease activity were
assessed and clinical and prognostic implications were sought.
Patients
Simultaneously with Binet stage, patients were substaged according to
Rai et al19: stage 0, 27 patients (60%); stage I, 5 patients (11%); stage II, 13 patients (29%). BM biopsies were performed at diagnosis, and the histology pattern (nondiffuse or
diffuse) was evaluated according to Rozman et al.20
Lymphocyte doubling time was assessed in 38 patients (84%) according
to Montserrat et al.21 Control subjects were 7 men and 5 women, aged 48 to 82 years (median, 67 years), with anemia due to iron
or vitamin B12 deficiencies.
The study was approved by the local ethics committee and all patients
gave their informed consent.
Measurement of BM angiogenesis
The area occupied by microvessels was estimated by using the direct
planimetric method of "point counting"22 with slight modifications for the computed image analysis (same software) as
described,23 according to which the microvessel area
equals the sum of point areas that hit microvessels. Because cellular areas are vascularized and noncellular areas (fat, dense connective tissue, necrotic and hemorrhagic foci, bone lamellae) are not, and
because the latter hampered comparison between sections, they were
always omitted from the reference area. Thus, the point areas that hit
noncellular areas were subtracted from the reference area. Residual
point areas defined the cellular area only and the microvessel area was
measured inside it. Basically, the measurement of the microvessel and
cellular areas fitted the following equations: microvessel area
[y] = sum of points that hit microvessels
[x] · 72.15 µm2, and cellular
area = 12.5 × 10 Values of the microvessel area were normalized to those of the cellular
area by the equation: [x]/(484 Microvessels were counted at × 250 within a computed square reticle
of 12.5 × 10 Analysis of serial sections (n = 6-10) from 3 biopsy samples revealed an intrabiopsy variability of 10% or less (± 1.8%) in both the microvessel area and number. The variability between the investigators checking neovessels separately was 5.0% or less (± 3.2% ) for both the area and number. The area and number assessments were highly intercorrelated (Pearson r = 0.95; P < .0001). The microvessel area and number were expressed as mean ± 1 SD for each section and biopsy and groups of biopsies. To screen for patients who likely could progress, cutoffs of microvessel area and number corresponding to the highest Youden index24 were chosen. The index combines information on sensitivity and specificity, giving equal weight to each, and measures the percentage gain in certainty of predicting the risk of progression. If microvessel area at a definite cutoff has an index of 0, it has no predictive power; if the cutoff has an index of 100%, progression is perfectly predicted. Microvessel area cutoffs set at the 25th, 50th, and 75th percentiles gave an index of 10%, 13%, and 45%, respectively. Thus, the highest cutoff was set. The microvessel number cutoff was set accordingly, because the 25th, 50th, and 75th percentiles paralleled an index of 0%, 0%, and 19%, respectively. The decision to peak 75th percentile was arbitrary. The Youden index has already been applied in patients with BCLL to compare CD38 expression with the risk of progression.25 Immunohistochemical staining of VEGF The BM immunoreactivity to VEGF was investigated in 11 patients with a rabbit anti-VEGF antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and the immunoperoxidase staining just described. Positive controls were anti- plus anti- chains and anti-CD20 rabbit
antisera (used for immunophenotyping), and the negative control was a
rabbit preimmune serum (all from Santa Cruz Biotechnology). Two 6-µm
sections per biopsy, adjacent to those examined for vascularity, were
stained with each reagent by the same operator (L.T.).
The intensity of VEGF staining related to the degree of antigen expression. It was scored by the KS-300 software in 4 to 6 × 400 fields/section, judged to be representative of the BCLL tissue viewed from several × 160 fields. In all patients, inflammatory mononuclear cells (fibroblasts, macrophages, and polymorphs) also stained with VEGF. These cells were found as few, isolated, or clustered elements throughout the stroma and were clearly distinguishable from tumor cells both on morphologic basis and because they usually displayed a different staining intensity (in plus or minus) from tumor cells. Care was taken to recognize these inflammatory cells and omit them from the evaluation. The red-staining intensity of BCLL tissue due to VEGF was fixed in each pixel, allocated on a gray intensity scale as density unit counts × 105, and expressed as the mean of the 4 to 6 fields/section, and finally as mean of the 2 sections. The background given by the negative control reagent in 4 to 6 × 400 fields/section was also expressed as mean of the 2 sections and subtracted. The resulting value represented the mean gray density unit counts of VEGF intensity per biopsy. The staining technique was performed 3 times, on separate days, for each biopsy, and revealed that the intrabiopsy variability in the staining intensity was 20% or less (± 3.6%) for all reagents. To overcome this variability for VEGF intensity and express it as a sole value, the final value per biopsy was the mean ± 1 SD of the 3 means per section, as described.26 Measurement of serum levels of VEGF (sVEGF) and FGF-2 (sFGF-2) Sera from peripheral venous blood sampled at diagnosis were stored at 80°C. Their sVEGF and sFGF-2 levels were determined in
duplicate by using the sandwich enzyme-linked immunosorbent assay
(ELISA; Quantikine human VEGF and Quantikine human FGF-2; R & D
Systems, Minneapolis, MN), according to the manufacturer's instructions. Its sensitivity was less than 5 pg/mL for VEGF and less
than 3 pg/mL for FGF-2. The coefficients of variation reported by the
manufacturer for interassay and intra-assay determinations vary from
6.2% to 8.8% and from 2% to 9%, respectively, for sVEGF, and from
7.4% to 9.1% and from 3% to 9.7%, respectively, for
sFGF-2.
Patients were compared with 63 healthy blood donors (40 men and 23 women), aged 22 to 61 years (median, 51 years). Because measurement of sVEGF could be overestimated due to VEGF release by platelets during clotting,27 we correlated the sVEGF with the plasma VEGF in 30 randomly chosen patients and found a close interrelation (Pearson r = 0.51, P < .0001) and correlation of both with the platelet counts (sVEGF r = 0.52; plasma VEGF r = 0.44; P < .0001). FISH studies Twenty-eight patients with available cytogenetic pellet at diagnosis were characterized by fluorescence in situ hybridization (FISH), using the 13q14.3 LSI D13S25 probe, the 17p13.1 LSI p53 probe, and the chromosome 12 centromeric probe CEP 12 (Vysis, distributed by Olympus, Milan, Italy), in dual-color experiments with appropriate control probes. The EMBL3 clones 19 and 65, spanning an area of
approximately 40 kb within the middle portion of the ATM
gene, were used to detect 11q22-23 deletions, as previously described.28 At least 200 interphase nuclei with
well-delineated signals were counted in each slide. The FISH procedure
was repeated in those slides with low hybridization efficiency (ie,
< 80% cells with the expected 2 normal signals of the control probe).
Clinical studies and disease progression evaluation The degree of BM angiogenesis was correlated with main clinical and hematologic variables, namely, Rai substages, BM histology, absolute PB lymphocytosis, lymphocyte doubling time, LDH, 2-m, cytogenetics, and with sVEGF and sFGF-2. The 75th
percentile of the microvessel area and number were chosen as cutoffs,
which could act as predictors of clinical outcome by using an end-point disease progression, defined as the appearance of Binet
upstaging17 during the treatment-free period. The
significant impact of this end-point on the overall survival of Binet A
patients29,30 enables it to replace overall survival as a
prognostic parameter and shortens their clinical studies. The cutoffs
were chosen according to the Youden index just described.
Prognostic value of enhanced BM angiogenesis Table 1 shows the BM microvessel area and number and the cellular area in patients and control subjects. The patients' microvessel area was significantly higher. The counting gave similar results. Because of the large bias of microvessel area and number within the patient group, the 75th percentile of the area (0.90 mm2 × 10 2) and number (6/12.5
mm2 × 102 fields) were selected as cutoffs
to discriminate between highly and poorly vascularized BM, and related
to progression.
After a median follow-up of 13 months (range, 2-40 months), 18 of 45 patients (40%) progressed to a more advanced Binet stage, the risk of
progression being 55.5% at 36 months. As shown in Figure
1, the median duration of
progression-free survival was 19 months for 12 patients with the
microvessel area at least in the 75th percentile (or
Histologically, the 12 patients with an area of at least 0.90 mm2 × 10
The risk of progression as progression-free survival was evaluated
according to known and putative prognostic parameters. As shown in
Table 2, parameters found to be
significant predictors of the progression-free survival by univariate
analysis were the microvessel area, BM histology, LDH,
Although the microvessel area was not an independent prognostic factor
in multivariate analysis (Table 2), it might be incorporated into the
Rai substaging of Binet stage A patients to allow a better assessment
of their risk of progression. As shown in Figure
3, when Rai 0 patients were split by the
cutoff area, 6 of them with at least 0.90 mm2 × 10
Serial studies were performed in 4 Rai 0 and 3 Rai I-II patients and
showed a trend for the microvessel area to increase in step with
progression. Specifically, 2 Rai 0 patients (of the 6 with area
Another approach was to correlate the microvessel area cutoff
with the prognostic parameters representative of tumor mass (Rai
substages, absolute PB lymphocytosis, BM histology, LDH,
VEGF expression of the BCLL BM and sVEGF levels Expression of VEGF evaluated immunohistochemically as an arbitrary intensity unit in the 11 BM samples increased in proportion with both microvessel area and number (Figures 2 and 4). We were unable to correlate the BM histology pattern with the intensity of VEGF expression because only one sample (the unique in our series) was diffuse and the remaining nondiffuse. The diffuse sample displayed neither the highest nor the lowest intensity, but was allocated on 4 × 105 intensity units, similarly to what was seen in 2 nondiffuse samples, which displayed 3.3 and 3.7 × 105 units, respectively. A higher number of diffuse samples is thus needed to reach safe conclusions.
The sVEGF was significantly higher in patients (median = 218 pg/mL,
range = 9-2000 pg/mL) than controls (median = 142 pg/mL, range = 40-487 pg/mL; P < .02; Mann-Whitney test). As
shown in Table 2, adoption of the median value of 218 pg/mL as a cutoff showed that sVEGF was a significant prognostic factor by univariate analysis. Because a high correlation between sVEGF and platelet counts
had been previously observed (Pearson r = 0.52;
P < .0001), we wondered whether the counts also had
prognostic power. No association was found between the counts and the
risk of disease progression (P = .09). Thus, sVEGF levels
are high in Binet stage A BCLL patients and higher ( Serial evaluation of sVEGF in 6 patients showed full agreement
with outcome. Three patients whose average value at diagnosis was 412, 305, and 615 pg/mL displayed 741 (+45%), 455 (+53%), and 825 (+26%)
after 22, 25, and 18 months when they progressed. The others who had
278, 145, and 340 at diagnosis gave 180 ( Karyotype and angiogenesis Because genomic aberrations are independent predictors of disease progression in early CLL,32 their correlation with microvessel area was sought. Twenty-eight patients were available for comparison. Thirteen patients (46.4%) had normal karyotype, whereas 15 (53.6%) had aberrations. Of these, 8 patients displayed 13q (6 had deletion as a sole aberration, and 2 had 13q plus 12q trisomy), 6 patients displayed 12q trisomy as a sole aberration, and 2 patients displayed 11q or 17p deletions. Patients were stratified into 4 groups according to major cytogenetic categories (normal karyotype, 13q as a sole aberration, 12q trisomy, 11q or 17p deletion) and aberrations were compared to microvessel area. No correlation was found (Figure 5 and Table 3).
Here we show a significant increase of BM angiogenesis (evaluated as FVIII-RA+ microvessel area and number) in patients with Binet stage A BCLL, compared with control subjects. Kini et al16 obtained similar results with a CD34 monoclonal antibody (another endothelial cell marker) and found that microvessel number, BM cellularity, and Binet stages were intercorrelated. In contrast, no increase in BM neovascularization and no dependence of vascularity on cellularity or stages were shown by Aguayo et al who used the FVIII-RA and counted in "hot spots."11 A different counting system and a heterogeneous population of patients may account for this discrepancy. We also show that the extension of microvessel area predicts the risk
of progression of the Binet stage A. We calculated a cutoff at the 75th
percentile or more of the area ( When the cutoff was applied to the Rai subclassification, the risk was
greater for both Rai 0 and Rai I-II patients on or above the cutoff.
Although such a finding concerned a relatively small series and in
multivariate analysis the microvessel area was not an independent
prognostic factor (different from serum On the other hand, an accurate prognostic assessment of BCLL patients
with early disease is mandatory to optimize the timing of therapy and
avoid exposure to the toxicity of treatment unlikely to be
useful.34 One can also hypothesize that patients of this type who have enhanced BM angiogenesis (microvessel area Overall, the data could be interpreted as pointing to a threshold of BM neovascularization above which the risk of progression increases. This association is consistent with that found in solid tumors,2 and agrees with the stimulatory effect of neovessels on tumor growth, because they convey oxygen and metabolites, and endothelial cells secrete growth factors for tumor cells,1 including those of B-cell lineage.38 The level of BM angiogenesis in hematologic cancers (as in other tumors) is a complex process related to an interaction of an array of angiogenic and antiangiogenic factors released into the microenvironment and of cell populations of tumor and host stromal origin with angiogenic and antiangiogenic activities.39 It is the final product of these interactions. Our data showing that BCLL expression of VEGF was correlated with microvessel area and number suggest that tumor VEGF prevails over antiangiogenic factors/activities and is a substantial angiogenic factor in BM of BCLL. The expression of VEGF by BCLL due to activation of some oncogenes including c-myc, c-fos, ets-140-42 may switch the avascular to the vascular phase and thus contribute to disease progression and a poor prognosis. Also, some inflammatory stromal cells that stained with VEGF might be synergistic with BCLL in the induction of the vascular phase. Studies on the correlation between tumor angiogenesis and sVEGF are circumstantial.3 We found no correlation, in agreement with studies on multiple myeloma.43 Therefore, different from the tumor expression of VEGF in the BM, sVEGF levels cannot be a marker of BM angiogenesis. The presence of many cell sources of sVEGF, such as stromal cells, platelets, and other endothelia, may account for this observation. However, much in the same way as the microvessel area, the sVEGF had prognostic significance. sFGF-2 was not of prognostic interest. Although genomic aberrations as evaluated by the FISH technique retain prognostic value in early CLL,32 we found no correlation with the microvessel area. We consider that this correlation is probably lacking because of the limited number of patients investigated. However, it may well be that aberrations and enhanced angiogenesis reflect different biologic events that are not interrelated, thus providing a biologic basis of clinical variability of early CLL as to the disease progression. In conclusion, our study tentatively suggests that angiogenesis is both a sizable component of the BM in BCLL and a marker of the risk of progression in early disease. Longitudinal studies are warranted to know whether the biologic evolution of early BCLL is prevascular and vascular. This will provide a rationale for the fast use of conventional therapy or antiangiogenic agents in patients with early BCLL who are likely to progress and finally die of their disease.
Submitted January 11, 2002; accepted June 6, 2002.
Prepublished online as Blood First Edition Paper, July 5, 2002; DOI 10.1182/blood-2002-01-0084.
Supported in part by grants from Associazione Italiana per la Ricerca sul Cancro (AIRC, Milan) and from Ministero dell'Istruzione, Università e Ricerca (MIUR, cofinanced and C03 funds, Rome).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Stefano Molica, Department of Hematology/Oncology, Azienda Ospedaliera "Pugliese-Ciaccio," Viale Pio X, I-88100 Catanzaro (CZ), Italy; e-mail: smolica{at}libero.it.
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Top
Abstract
Introduction
10 µm)
and the sufficient distance between 2 adjacent points (10.7 µm) meant
that a point could be occupied by only one microvessel.
0.90
mm2 × 10
