Bone marrow angiogenesis in multiple myeloma and its correlation with clinicopathological factors
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- Rana, C., Sharma, S., Agrawal, V. et al. Ann Hematol (2010) 89: 789. doi:10.1007/s00277-010-0919-z
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Increased angiogenesis has been found to be an adverse prognostic factor in solid tumors but evidences show that angiogenesis also plays an important role in hematological malignancies including multiple myeloma (MM). In this report we studied the various angiogenesis parameters like microvessel density (MVD) and total vascular area (TVA), on bone marrow biopsies in 50 newly diagnosed cases of MM. The aim was to study bone marrow angiogenesis in MM using light microscopy (MVD-A) and computerized image analyzer (MVD-B and TVA) and correlate it with clinical features, laboratory findings, histological features, and response to treatment on follow-up. Bone marrow biopsies of test cases (n = 50) were immunohistochemically stained with CD34 for visualization of microvessels. MVD-A (range 8–80; mean 50.4; SD 17.5), MVD-B (5.2–33.2; mean 16.3; SD 5.1), and TVA in percentage (range 0.42–7.20; mean 2.8; SD 1.5) were measured. Ten age- and sex-matched controls were studied and their parameters were taken as grade I. There was a significant correlation between these angiogenesis parameters (MVD-A vs MVD-B, Pearson’s correlation coefficient (pcc) = 0.724; MVD-A vs TVA, pcc = 0.370; MVD-B vs TVA, pcc = 0.406). The angiogenesis was significantly higher in cases as compared to controls. Patients with residual disease had a higher MVD as compared to the complete responders. High tumor burden and diffuse pattern of infiltration were also associated with grade III MVD and TVA. Hence, it can be concluded that angiogenesis correlates with other histological features associated with prognosis and is also a good predictor for complete response in patients with multiple myeloma.
KeywordsMultiple myelomaAngiogenesisClinicopathological factorsMicrovessel densityComputerized image analysis
Angiogenesis is the formation of new blood vessel from pre-existing vasculature which occurs in either pathological or physiological conditions. It occurs physiologically during embryonal growth, wound healing, and in the female genital tract during menstrual cycle as well as in several pathological conditions such as neoplasia. It is obligatory in the enhancement of progression, i.e., growth, invasion, and metastasis of solid tumors. In absence of angiogenesis, tumor cannot grow beyond 1 to 2 mm3 in size .
In recent years, bone marrow (BM) angiogenesis is indicated to be involved in the pathogenesis and progression of certain hematological malignancies like acute lymphoid leukemia, acute myeloid leukemia, myelodysplastic syndrome, Hodgkin’s lymphoma, low/high grade non-Hodgkin’s lymphomas, and multiple myeloma .
Multiple myeloma (MM) is a B-cell malignancy that accounts for 1% of all cancers and about 10% of all malignant hematological neoplasms. Clinically, it is characterized by a pentad of: (a) anemia, (b) elevated serum and urine monoclonal paraprotein levels, (c) abnormal bone radiographs and bone pains, (d) hypercalcemia, and (e) renal insufficiency . MM was the first hematological malignancy in which a prognostic relevance of angiogenesis was demonstrated. Previous studies have suggested that microvessel density was significantly increased in MM compared to monoclonal gammopathy of unknown significance (MGUS) and moreover active versus non-active myeloma . Hence, possibility of using anti-angiogenic agents in patients with poor outcome after conventional therapy might be a novel strategy for treatment.
Different techniques have been developed for visualizing and estimating the number of microvessels in various tissues. Immunohistochemical staining for substances present in the blood vessels such as CD34, Von willebrand factor, CD31, CD105, and others, allow excellent visualization of microvessels. Quantification of microvessel density (MVD) can be done either in terms of microvessel count per field or by calculating the percentage of the surface area which is covered by microvessels. Different approaches have been adopted for estimating the degree of angiogenesis or MVD, including visual grading [5, 6], vessel count per defined area, use of grids for counting, and computerized image analysis (CIA) .
Few studies have been done to study angiogenesis in multiple myeloma and most of them used light microscopic analysis of BM biopsies. There are very few studies in literature that used CIA for calculating microvessel density. The aim of the present study was to evaluate various angiogenesis parameters in BM biopsies of newly diagnosed patients of MM by light microscopy and computerized image analyzer in newly diagnosed patients of MM and correlated them with various clinicopathological factors associated with prognosis.
Patients and methods
Ten age- and sex-matched bone marrow biopsies performed for non-malignant conditions were taken as control for grading the angiogenesis parameters (Fig. 1a). Lymph node sections were used as positive controls for immunohistochemical staining.
Bone marrow biopsies processing and histological staining
Bone marrow biopsies were collected in B5 fixative solution followed by decalcification using 5% trichloroacetic acid in normal saline and then processed overnight for paraffin embedding. Sections were cut at 3 to 5 μm and stained with hematoxylin and eosin. Reticulin staining was done when required
Marrow aspirations were performed at posterior superior iliac spine by modified Jamshidi needle and multiple smears were made and stained with leishman stain.
Immunohistochemical staining for angiogenesis was performed in bone marrow biopsies of all the 50 patients of MM as well as ten controls, using streptavidin biotin method. Antigen retrieval was done by microwave method. Endogenous peroxidase activity was blocked by treating sections with 3% hydrogen peroxide in methanol in the dark for 30 min. Then these sections were incubated overnight at 4°C in humid chamber with the monoclonal antibody QBEND10 to CD34 (Dako, Denmark) at 1:25 dilution.
Histological staging and grading
All slides (n = 50; H&E) were evaluated for confirmation of original diagnosis, staged, and graded histologically as described by Bartl et al. .
Estimation of angiogenesis parameters
Light microscopy (manually at ×40; MVD-A)
Computerized image analysis
Grading of MVD-B and TVA for test cases and control was done in a manner similar to that of the MVD-A grading, i.e., taking all control as Grade I. MVD-B was graded as follows: Grade I ≤10 microvessels; Grade II, >10 and ≤20 microvessels; and Grade III, >20 microvessels, per 50,000µmm2 . TVA percentage was graded as follows: Grade I ≤1%; Grade II >1% but ≤3%; and Grade III >3% vascular area per total marrow area.
Treatment and follow-up
The patients were treated by either VAD or combination of prednisolone and thalidomide. Response criteria were defined as (a) remission, <5% plasma cells in the bone marrow with disappearance of M protein; (b) residual, >50% reduction in the M protein levels in serum with >5% plasma cells in bone marrow; and (c) refractory, no response to the initial therapy.
Correlation between the manual and computerized image analysis in evaluating the angiogenesis parameters were done by Pearson’s correlation coefficient (pcc). Clinical and laboratory parameters related to prognosis of MM were correlated with the MVD using Spearman and Pearson’s correlation coefficient. Significance of association between the bone marrow findings and response to chemotherapy with the microvessel density and total vascular area was evaluated using the tests of proportion. P value <0.05 was considered as statistically significant. The statistical analysis was carried out with the SPSS Software Version 15.
Demographic, clinical, hematological, biochemical, immunological, and histopathological details of 50 cases
Age range (years)
S. creatinine (>2 mg/dl)
Mean age (years)
S. calcium (>12 mg/dl)
Median age (years)
Peripheral blood findings
Hb (<10 g/dl)
Normal platelet count
Platelet count (<1,000,000/µl)
Physical examination findings
Plasma cells percentage
Soft tissue/bony swelling
Plasma cell morphology
Stage I 03
Mature (low grade)
Immature (intermediate grade)
Stage III 3
Plasmablastic (high grade)
Electrophoresis for M component
Pattern of infiltration
“M” component in serum
“M” component in urine
Immunofixation results (n = )
Response to chemotherapy
Grade of angiogenesis parameters and correlation between them in pre-treatment biopsies
Grades of angiogenesis parameters
Correlation between angiogenesis parameters (MVD-A, MVD-B, TVA)
MVD-A vs MVD-B
MVD-A vs TVA
MVD-B vs TVA
Pearson correlation coefficient
Significance (p value)a
Factors associated significantly with MVD and TVA
Prognostic factors vs high grade MVD-A
Plasma cell percentage >50%
Immature plasma cell morphology
Diffuse pattern of infiltration
Prognostic factors vs TVA
Plasma cell percentage >50%
Diffuse pattern of infiltration
When the MVD was correlated with the extent of infiltration, it was noticed that most of the patients with a higher grade of angiogenesis had a diffuse pattern of plasma cell infiltration in bone marrow biopsy (P value < 0.001). It was also found that complete responders had a lower grade of angiogenesis than those who had residual disease (P value < 0.05; Table 3).
Total vascular area was also correlated with other factor like microvessels density. Total vascular area was higher, i.e., TVA >3% in patients with high tumor load as compared to cases with low tumor burden (P < 0.05). Diffuse pattern of infiltration was also more frequently encountered in cases with highest total TVA≥3% (Table 3).
Immunohistochemistry has provided an objective method for assessing degree of neovascularization. It has been shown to be a significant and independent prognostic indicator of various malignancies including myeloma. MM was the first hematological malignancy in which a significant correlation of angiogenesis with prognosis and survival could be identified . Vacca et al.  demonstrated for the first time that BM-MVD was significantly increased in MM compared to MGUS and moreover active versus non-active myeloma. They hypothesized that progression from MGUS to myeloma is accompanied by an increase in BM-MVD.
There are only a few studies on angiogenesis in multiple myeloma. In all these studies angiogenesis was measured as microvessel density in the “hot spots”. These measurements were done mostly based on light microscopic analysis of bone marrow biopsies. There are only two studies by Bhatti et al.  and Rajkumar et al.  in literature that used computerized image analyzer to study the various angiogenesis parameters in MM. Bhatti et al.  performed both MVD as well as TVA using CIA and compared them with the various prognostic factors of MM.
We also used light microscopy as well CIA and found that all angiogenesis parameters were significantly higher in myeloma cases as compared to controls (P value < 0.0001). These results were in accordance with the results of previous studies done by Rajkumar et al.  and Bhatti et al. . MVD by light microscopy as well as CIA correlated with each other, hence any of the two can be used for MVD analysis, the only difference being that CIA provides easy visualization of microvessel and helps in more accurate counting by avoiding overlapping. Since CIA is costly method, hence light microscopy can be used where computerised image analyser is not available.
Vacca et al.  postulated that higher plasma cell labeling index and plasma cell percentage are associated with active myeloma as compared to non-active myeloma. While Rajkumar et al., Munshi et al. , and Singhal et al.  found no correlation between plasma cell infiltration and MVD. Due to these conflicting results, we investigated this relationship and demonstrated a significant correlation between high tumor burden (plasma cells >50%) and grade III MVD. In our study, there were five patients with plasma cells <50% but had a grade III MVD. When these patients were followed-up, it was found that all of them had a residual or refractory disease after treatment. These findings indicate that angiogenesis is a better factor for predicting the outcome of the patient after treatment as compared to the plasma cell number.
We also demonstrated that patients with mature plasma cells morphology had a grade I MVD, while most of the patients with plasmablastic morphology had higher angiogenesis, i.e., grade III MVD. Carter et al.  had predicted a shorter median survival for plasmablastic myeloma as compared to mature and immature myeloma. Similarly, diffuse pattern of infiltration was also associated with grade III MVD and there was not even a single case of diffuse pattern of infiltration with grade I MVD or TVA.
Only two studies have calculated the total vascular areas in BM biopsies of MM. Vacca et al.  used planimetric method while Bhatti et al.  used CIA. We also studied the total vascular area using CIA and found that grade III MVD was significantly associated with high tumor burden (plasma cell >50%) and diffuse pattern of infiltration had grade III MVD (P < 0.05).
Our study demonstrated that angiogenesis was higher in patients with residual disease as compared to complete responders. This indicates that angiogenesis can a good indicator of response and hence should be done in all newly diagnosed cases of MM as this could predict the outcome of the patient.
Since angiogenesis is known to play a very important role in progression of myeloma from non-active stage to active stage, it can be a fruitful target for treatment of multiple myeloma. Experimental evidences have been provided that angiogenesis is not negatively affected by drug resistance due to genetic stability of endothelial cells in contrast to tumor cells. On this basis various strategies for anti-angiogenic treatment have been developed which include interference with angiogenic stimulators (e.g., vascular endothelial growth factor (VEGF), HGF, and bFGF), angiogenic factor receptors (e.g., VEGF-receptor signaling), extracellular matrix interactions, inhibiting oncogenes controlling the angiogenic response, and proteolysis .
It can be concluded from our study that angiogenesis was increased in patients with MM as compared to controls. Manual (MVD-A) and computerized microvessel density (MVD-B) correlated with each other as well as the total vascular area occupied by the microvessels. Secondly, angiogenesis is higher in patients with residual disease as compared to those with complete response after treatment, hence when performed in newly diagnosed cases of MM degree of angiogenesis can predict the outcome. Thirdly, patients with increased plasma cell burden, immature plasma cell morphology, and diffuse pattern of infiltration had a higher microvessel density. Since the abovementioned factors are known independent prognostic factors and angiogenesis correlate with them, angiogenesis can be included as one of the prognostic factor for multiple myeloma. As stated above, it can even be a better factor; however, a more extensive study is required to prove it. Finally, angiogenesis can be a target for various anti-angiogenic therapies thereby opening gateways towards new treatment approach which could probably be less harmful and more effective thereby improving patient survival from this devastating disease.