Flow cytometry is helpful in differentiating between B-cell lymphoma (BCL) and reactive lymphocytic proliferation (RLP) in FNA biopsies. However; the presence of inconclusive surface immunoglobulin light chains (sIg LC) poses a problem. We investigated the usefulness of additional tests; namely Bcl-2 expression and expression of cytoplasmic Ig LC (cIg LC), mainly on samples with inconclusive sIg LC. Both tests were performed on 232 FNA samples from lymph nodes. Bcl-2 alone was determined qualitatively and quantitatively on 315 samples. The quantitative test was correctly positive in 76% of cases and falsely negative in 24%. The correctly positive results of the qualitative test were 11% points lower. cIg LC correctly identified 65% of BCL with dual positive sIg LC; 36% of BCL with difficult to interpret sIg LC and only 7% of BCL with negative sIg LC. The best results in differentiating between BCL and RLP were obtained when all three tests were used together. In samples with inconclusive sIg LC and additional monoclonal or polyclonal populations the κ:λ ratios did not differentiate between RLP and BCL. We propose that in case of inconclusive sIg LC Bcl-2 test is used first. The addition of cIg LC test is sensible only in cases with dual positive and difficult to interpret sIg LC.
Keywords: B-cell lymphoma; flow cytometry; inconclusive surface immunoglobulin light chains; Bcl-2; cytoplasmic immunoglobulin light chains; fine needle aspiration
Flow cytometric immunophenotyping (FCI) has made a great contribution towards higher accuracy in lymphoma recognition from fine needle aspiration (FNA) biopsies over the past years. Several papers in the literature have reported from 80–99% correct diagnoses in differentiating between lymphoma and reactive lymphocytic proliferation (RLP) [[
The aim of our study was to test the diagnostic value of two additional tests, using FCI in differentiating between BCL and RLP in FNA samples from lymph nodes, most of which had inconclusive sIg LC: firstly, the qualitative and quantitative Bcl-2 tests were used; Secondly the clonality determination was conducted by cIg LC ratio alone, and in combination with the Bcl-2 test.
We determined Bcl-2 expression in 315 FNA samples from lymph nodes of 282 patients. There were 143 males and 139 females. The age of patients ranged from four to 92 years. Final diagnoses were RLP in 159 cases and BCL in 156 cases. There were 118 primary and 38 secondary lymphomas. Among BCL there were 68 follicular lymphomas (FL), 42 diffuse large B-cell lymphomas (DLBCL), 37 marginal zone lymphomas (MZL), four chronic lymphatic leukaemia (CLL), three Burkitt lymphomas (BL), one mantel cell lymphoma (MCL) and one unclassified lymphoma. Histological diagnoses were available for 187 patients.
FCI test defined 19 samples with monotypic sIg LC expression, 16 were polytypic and 280 samples had inconclusive sIg LC (208 negative, 54 dual positive and 18 difficult to interpret). The percentage of cells with negative sIg LC ranged from 17% to 100% (median = 60%), percentage of cells with dual positive sIg LC ranged from 7% to 100% (median 32%). The monotypic group contained only BCL, the polytypic group contained 15 RLP and one BCL. Among samples with inconclusive sIg LC the dual positive sIg LC group contained the highest number of BCL (49/54; 91%), followed by the "difficult to interpret group" (13/18; 72%). The negative sIg LC group contained only 36% BCL (74/208).
When we determined Bcl-2 expression qualitatively 32% of samples (102/315) showed overexpression. The test was correctly positive in 101/156 (65%) and falsely negative in 55/156 (35%) samples of BCL. The 55 false negative samples originated from 20/42 DLBC, 16/37 MZL, 14/68 FL, 3/3 BL and 2/4 CLL. There was one false positive Bcl-2 test. It was a sample from a patient with cytological diagnosis of CLL, who experienced complete regression of enlarged lymph nodes. Except for the cytologically diagnosed CLL with spontaneous regression, none of the samples from RLP showed Bcl-2 overexpression.
In quantitative determination of Bcl-2 expression the median value of Bcl-2 index was 1.0 in RLP (range 0.5–4.7) while the median value in BCL was 2.19 (range 0.6–13.0). The highest value of Bcl-2 index among RLP was in the sample from the patient with cytological diagnosis of CLL with spontaneous regression of enlarged lymph nodes. After we excluded this sample, the highest value was 1.5.
Since area under curve (AUC) for Bcl-2 index values was 0.89 (95% confidence interval: 0.85–0.93, p < 0.001) we considered Bcl-2 test as excellent for differentiating between RLP and BCL. We obtained the best combination of sensitivity and specificity at Bcl-2 index value of 1.5 for differentiating between RLP and BCL (sensitivity 75%, specificity 99%). The positive quantitative Bcl-2 test (Bcl-2 index › 1.5) had a high positive prediction value (99%) and somewhat lower negative predictive value (80%). Bcl-2 indexes were statistically significantly higher (p < 0.001) in BCL compared to RLP. The same was true for the following specific lymphoma types: FL, DLBCL and MZL. CLL, BL and MCL also showed higher Bcl-2 indexes compared to those in RLP. However, they were represented in too few numbers for conclusive statistical analysis (Figure 1).
Quantitative Bcl-2 test was correctly positive in 118/156 (76%) cases, falsely negative in 38/156 (24%) cases and falsely positive in one case, the same one as explained for the falsely positive qualitative Bcl-2 test. Among the false negative cases, there were 13/42 DLBCL, 11/68 FL, 13/37 MZL and 1/4 CLL. Except for the cytologically diagnosed CLL with spontaneous regression, none of the RLP cases showed Bcl-2 overexpression. Therefore, quantitative Bcl-2 test correctly identified 88% of all cases, among them 76% of BCL.
Among samples with inconclusive sIg LC the Bcl-2 index was most frequently positive in the dual positive sIg LC group (84%; 41/49 BCL), followed by the "difficult to interpret" sIg LC group (77%; 10/13 BCL) and the negative sIg LC group (70%; 52/74 BCL). In the group with monotypic sIg LC, Bcl-2 test was positive in 79% (15/19 BCL). In the group with polytypic sIg LC one case showed positive Bcl-2 test (1/16).
Results of the qualitative and quantitative Bcl-2 tests were the same in all cases of RLP and in the case of CLL with spontaneous regression of lymph nodes. The two tests were not compatible in 20 cases (16%) of BCL (Figure 2).
In 18 samples only the quantitative test was positive while in two samples only the qualitative test was positive. The latter two samples contained many reactive B-cells in addition to the neoplastic ones. In one of these two samples, the quantitative Bcl-2 test was negative because there were too few B-cells with Bcl-2 overexpression, while in the other sample the quantitative test was negative because there were too few T cells (Figure 3a,b). In 18 samples the qualitative test was negative because the difference in median expression of Bcl-2 between B-cells and T cells was too low. Among these 18 samples there were 8 DLBCL, 4 FL, 3 MCL and 3 BL.
Sensitivity, specificity, PPV and NPV for each type of Bcl-2 test as well as for the combination of both types are shown in Table 1.
We determined expression of cIg LC and Bcl-2 in 232 FNA samples from lymph nodes of 211 patients. There were 106 males and 105 females. The age of patients ranged from 14 to 92 years. Final diagnoses were RLP in 102 cases and BCL in 130 cases, among them 100 primary and 30 secondary lymphomas. Histology was available for 153 patients.
In this study group, five samples had monotypic sIg LC, seven were polytypic and 220 had inconclusive sIg LC. The percentage of cells with negative and dual positive sIg LC was the same as in the study group where only Bcl-2 testing was performed. Among 130 BCL, five samples expressed monotypic sIg LC, one expressed polytypic sLC and 124 sIg LC were inconclusive (70 negative, 43 dual positive and 11 difficult to interpret). Among 102 RLP, there were no samples with monotypic sIg LC, six expressed polytypic sIg LC and 96 expressed inconclusive sIg LC (89 negative, four dual positive and three difficult to interpret).
In 5% of all cases both sIg and cIg LC were monotypic or polytypic. 20% of samples had inconclusive sIg LC and conclusive cIg LC. 75% of samples had inconclusive both sIg and cIg LC. Expression of cIg LC in groups with specific expression of sIg LC is shown in Table 2. With the use of cIg LC test we identified 7% (8/70) of BCL within the group of samples with negative sIg LC, 65% (28/43) of BCL within the group of dual positive sIg LC and 36% (4/11) of BCL within the group of sIg LC which were difficult to interpret. There was one BCL among samples with inconclusive sIg LC and polytypic cIg LC.
In Table 3 we present sensitivity, specificity, PPV and NPV of Bcl-2 test (qualitative and quantitative); of the cIg LC test; and of the combination of both tests together. For all three groups of inconclusive sIg LC sensitivity and specificity were highest when we performed both tests together. In the group with negative sIg LC, the sensitivity of Bcl-2 test was only 1.5 percentage points lower than the sensitivity for the combination of Bcl-2 and cIg LC tests together, while specificity stayed the same. In the other two groups of samples with inconclusive sIg LC there was nine percentage points difference in sensitivity. Determination of cIg LC is the least sensitive test for differentiation between RLP and BCL in samples with inconclusive sIg LC (Table 3, Figure 4).
In the both above mentioned study groups we detected additional monotypic or polytypic B-cell populations among those with inconclusive sIg LC. The Bcl-2 test group contained 180/280 (64%) such cases, 160 with additional polytypic sIg LC populations and 20 with additional monotypic sIg LC. Samples with additional polytypic populations originated from 112 RLP and 48 BCL. Samples with additional monotypic populations originated from six BCL and one RLP. The cIg LC study group contained 129/220 samples with inconclusive sIg LC and additional monotypic (18/129) or polytypic (111/129) sIg LC populations. Samples with additional polytypic populations originated from 68 RLP and 43 BCL. Samples with additional monotypic populations originated from 17 BCL and one RLP.
We tried to differentiate between BCL and RLP on the basis of ratios between sIg LC of these additional populations by the use of ROC curve. However, the AUC for κ/λ fractions was too small to enable such a differentiation. The differentiation by κ:λ ratios of additional monotypic/polytypic populations was unsuccessful in both study groups.
The results of our study demonstrated that qualitative Bcl-2 test correctly identified 82% of cases as BCL or RLP, while the quantitative Bcl-2 test correctly identified 88% of all cases when we used Bcl-2 index of 1.5 as the cut-off value. In both tests types, there was one, the same, falsely positive case. Only 21% of specimens with inconclusive sIg LC were correctly identified as BCL or RLP when clonality was determined by the cIg LC test. The best results were obtained when all three tests were applied together. Depending on the specific subgroup of samples with inconclusive sIg LC, the sensitivity ranged from 78% to 91% and the specificity from 99% to 100%.
Although we were primarily interested in differentiating between BCL and RLP with inconclusive sIg LC, we also included in our study 57 cases with conclusive sIg LC for which cytomorphology was not concordant with FCI outcome (35 cases in the group where only Bcl-2 was determined and 12 cases where Bcl-2 and cIg LC were determined). These cases also served as a control group. The results showed that the percentage of correctly positive Bcl-2 test determined quantitatively was almost the same in both groups: 76% in the group with inconclusive sIg LC and 79% in the group with monoclonal sIg LC. Among the 16 cases with polytypic sIg LC there was one case of BCL (DLBCL), however, it did not show Bcl-2 overexpression. Lymphomas with polytypic sIg LC have been observed previously. Laane et al., for example, reported 9/222 BCL with polytypic sIg LC [[
Our results demonstrated a very high PPV of the Bcl-2 test. The only false positive case was the sample with morphologic characteristics of CLL and positive expression of CD5 and CD23. There was no histologic confirmation of the diagnosis since enlarged lymph nodes regressed spontaneously. However, the patient returned recently with enlarged lymph nodes, FNA was performed again and FCI demonstrated the same immunophenotype as ten years ago. The patient is still under diagnostic investigation. However, we believe that our diagnosis was correct and that the case was in reality not a false positive one regarding the Bcl-2 overexpression. There are reports in the literature of spontaneous regression of histologically confirmed lymphomas [[
We have found very few reports on Bcl-2 expression in BCL and RLP determined by FCI and none where these two tests would be investigated in samples with predominantly inconclusive sIg LC. Cornfield et al. investigated Bcl-2 expression on material from surgical biopsies and were concerned solely on the ability to distinguish between follicular hyperplasia and FL [[
Additional differences among the above mentioned papers and our report are also in the use of qualitative or quantitative Bcl-2 determination and in the manner in which Bcl-2 expression was detected. Tarafder et al. [[
In the report of Cook et al. [[
Despite similarities between our study and the one reported by Laane et al. [[
The Bcl-2 test has a very high PPV and somewhat lower NPV which varies in different BCL types and is the main drawback of the test. The major weakness of our study is that we could not calculate reliably the percentage of Bcl-2 negative individual BCL types because some of them were represented in very few numbers. For example, only four CLL were included in the study because they were mostly monotypic with a characteristic immunophenotype and additional FCI analyses were not necessary. FL, DLBC and MZL, on the other hand, were represented in moderately high numbers and in these three groups of BCL Bcl-2 was overexpressed in 84%, 69% and 65% respectively. These results differ only by one to two percentage points from the results reported by Lai et al. [[
In our study group where we determined cIg LC in addition to the Bcl-2 expression 79% of samples with inconclusive sIg LC also expressed inconclusive cIg LC. Only 18% of samples with inconclusive sIg LC had monotypic cIg LC and they all originated from BCL. Three percent of samples had inconclusive sIg LC and polytypic cIg LC, however, one of them was a DLBCL. The Bcl-2 was not overexpressed in this case.
The reasons for inconclusive Ig LC have not been definitely explained. Some speculate that dual positive expression of LC is the result of unspecific binding of free immunoglobulins from serum and from tissues to the Fc receptors on lymphatic cells [[
We have found few articles which report on the cIg LC [[
Whenever we fail to differentiate between BCL and RLP by using morphology and FC, including the analysis of BCL-2 and cIg LC, it is advisable to use molecular techniques. In our previous paper we have already reported that we were able to demonstrate monoclonality in 77% of BCL by detecting immunoglobulin heavy chain rearrangement using PCR [[
We included FNA samples of lymph nodes obtained between the years 2007 and 2013 for which Bcl-2 and cIg LC were determined in addition to routinely performed FCI antibody panels. Most of these samples had inconclusive sIg LC, however, all were not difficult cases for interpretation since many had characteristic morphology and/or immunophenotype. In addition, we included some cases with conclusive sIg LC because their cytomorphology was not concordant with FCI outcome. From the hospital information system we obtained clinical data, cytological and histological diagnoses for all patients included in the study. The study was approved (February 18th, 2014) by The National Medical Ethics Committee of the Republic of Slovenia (109/02/14) and was performed in compliance with the Helsinki declaration.
Histological diagnosis was considered final and was based on microscopic examination and on the results of immunohistochemistry. For patients without histological diagnosis we determined final diagnosis on the basis of cytological diagnosis and data on clinical course of the disease. Cytological diagnoses of RLP or BCL were based on morphological criteria of lymphoid cells combined with the results of FCI analysis, namely the expression of CD45, CD20, CD19, CD3, FMC7, CD10, CD5, CD23, sIg LC, cIg LC and Bcl-2. The average follow-up period was 4.8 years.
The preparation of cell suspension from FNA lymph node sample, cell counting, the sample preparations for FCI (including preparation for determination of cIg LC and Bcl-2), acquisition of cells with flow cytometer and measurement result analysis have already been described in our previous paper [[
The Ig light chains (Ig LC) were considered conclusive when B-cell populations were present only within κ or/and λ positive area on FCI histogram. The Ig LC were considered inconclusive when B-cells were in the negative or dual positive area of the histogram or when we could not definitively determine the ratio between Ig LC. The later situation presented in cases with more than two populations of cells expressing κ or λ LC and in cases with few CD19 positive cells. This group of inconclusive Ig LC was named "difficult to interpret". Among samples with inconclusive sIg LC there were many cases with additional monotypic or polytypic B-cell populations. In such samples we investigated the possibility to differentiate between BCL and RLP on the basis of the ratio between sIg LC of these additional populations.
We determined the expression of Bcl-2 qualitatively and quantitatively. Qualitative method represented visual determination of FCI results. The method was already explained in our previous paper. We determined Bcl-2 quantitatively by calculating the Bcl-2 index, which is the ratio between mean fluorescence intensity (MFI) of B-cells and MFI of T-cells T within the same sample.
For statistical analysis of results we used the programme Statistical Package for Social Sciences (IBM Corporation, New York, NY, USA). We used the curve of receiver operating characteristics (ROC) for analyzing the following results: 1. For determining cut-off point values for Bcl-2 index, which enables most accurate differentiation between RLP and BCL; 2. For determining if it is possible to differentiate between RLP and BCL on the basis of sIg LC ratio in samples with inconclusive sIg LC and expressing additional polytypic or monotypic lymphoid cell populations; 3. By comparing the sIg LC and cIg LC tests we investigated whether additional analysis of cIg LC increases recognition between RLP and BCL.
ROC curve demonstrates the ratio between fractions of true positive and false positive diagnoses at various values of Ig LC ratios or various values of Bcl-2 index. To produce an ROC curve we set the number of samples with true positive diagnosis (sensitivity-y coordinate) and the number of samples with false positive diagnosis (1-specificity –x coordinate) for each value of the Ig LC ratio or Bcl-2 index. Then we selected the cut-off value of the Ig LC ratio or Bcl-2 index at which we obtained the highest sensitivity at maximum specificity for differentiating between RLP and BCL. We constructed separate ROC curves for all RLP and BCL with predominant expression of κ sIg LC and separate curves for RLP and BCL which predominantly expressed λ sIg LCs.
By calculating the area under the ROC curve we determined the probability of a certain test to differentiate between RLP and BCL. The larger the AUC the more successful is the test. Values of AUC range from 0 to 1. The usefulness of the test is divided into five categories: unsatisfactory (AUC = 0.5), poor (0.5 < AUC <0.7), acceptable (0.7 ≤ AUC < 0.8), excellent (0.8 ≤ AUC < 0.9), and outstanding (AUC ≥ 0.9) [[
On the basis of our results we propose the following algorithm for the use of Bcl-2 and cIg LC in samples with inconclusive sIg LC. The Bcl-2 test should be used first. In the case that sIg LC are negative, the addition of cIg LC is not recommended since the sensitivity of Bcl-2 and cIg LC tests together is only 1.5 percentage points higher compared to the use of Bcl-2 test alone. The use of both tests together is advisable only in the groups of dual positive and difficult to interpret sIg LC where the sensitivity is nine percentage points higher compared to the sensitivity of the Bcl-2 test alone.
Graph: Figure 1 Quartile diagram of Bcl-2 indexes in RLP and various BCL types. The Bcl-2 index of 1.5 is shown with a dotted line. RLP—reactive lymphocytic proliferation. FL—follicular lymphoma. DLBC—diffuse large B-cell lymphoma. MZL—mantle zone lymphoma. CLL—chronic lymphatic leukaemia. BL—Burkitt lymphoma. MCL—mantel cell lymphoma.
Graph: Figure 2 Results of the qualitative and quantitative Bcl-2 tests (Bcl-2 index of 1.5). RLP—reactive lymphocytic proliferation. BCL—B-cell lymphoma.
Graph: Figure 3 Positive qualitative Bcl-2 test in two samples with negative quantitative Bcl-2 test. Results of Bcl-2 measurement in B-cell and in T-cells, where we observe two populations of B-cells with different expression of Bcl-2 and a population of T-cells. (a) a small B-cell population with Bcl-2 overexpression which was not detected by the quantitative test. (b) Positive qualitative Bcl-2 test with few T-cells which resulted into false negative quantitative Bcl-2 test. Red circles show B-cells with Bcl-2 overexpression.
Graph: Figure 4 Performance of Bcl-2 and cIg LC tests for diagnosing BCL in cases with inconslusive sIg LC. Samples with conclusive and inconclusive sIg LC (blue columns, left axis), % BCL (red dots, right axis), % monotipic cIg LC (yellow line, right axis), % positive Bcl-2 test (green dots, right axis).
Table 1 Sensitivity, specificity, PPV and NPV of the qualitative and quantitative Bcl-2 test for differentiation between RLP and BCL.
Test Bcl-2 Sensitivity [%] Specificity [%] PPV [%] NPV [%] Qualitative 65 99 99 74 Quantitative 75 99 99 80 Both tests together 76 99 99 81
Table 2 Expression of cIg LC in groups with specific expression of sIg LC in FNA of lymph nodes.
sIg LC cIg LC Negative N (L) Double Positive N (L) Difficult to Interpret N (L) Monotypic N (L) Polytypic N (L) Total N (L) Negative 145 (61) 7 (5) 1 (1) - - 153 (69) Double positive - 8 (7) - - - 8 (7) Difficult to interpret 1 (0) 3 (3) 9 (6) - - 13 (9) Monotypic 8 (8) 28 (28) 4 (4) 5 (5) - 45 (45) Polytypic 5 (1) 1 (0) - - 7 (1) 13 (2) Total 159 (70) 47 (43) 14 (11) 5 (5) 7 (1) 232 (130)
Table 3 Sensitivity, specificity, PPV and NPV of the Bcl-2 test (qualitative and quantitative), of the cIg LC test and of the two tests together in groups with inconclusive sIg LC.
Groups with Inconclusive sIg LC Test Sensitivity [%] Specificity [%] PPV [%] NPV [%] Bcl-2 76.92 98.94 98.04 86.11 cIg LC 5.19 80.00 88.89 2.67 Bcl-2 and/or cIg LC 78.46 98.94 98.08 86.92 Bcl-2 86.05 100.00 100.00 40.00 cIg LC 65.22 100.00 100.00 5.88 Bcl-2 and/or cIg LC 95.35 100.00 100.00 66.67 Bcl-2 81.82 100.00 100.00 60.00 cIg LC 28.57 - 100.00 - Bcl-2 and/or cIg LC 90.91 100.00 100.00 75.00
A.B.; conceptualization, investigation, formal analysis, resources, visualization, writing- original draft preparation; Z.P.M.; data curation, writing-original draft preparation, language correction; S.B.; formal analysis; M.C.; writing-critical review and editing; V.K.P.; supervision, conceptualization, data curation, visualization, writing-original draft preparation. All authors read and approved the final manuscript.
This research received no external funding.
The authors declare no conflict of interest.
BCL B-cell lymphoma RLP reactive lymphocytic proliferation FNA fine needle aspiration sIg LC surface immunoglobulin light chains cIg LC cytoplasmic immunoglobulin light chains FCI flow cytometric immunophenotyping RLP reactive lymphocytic proliferation FL follicular lymphoma DLBCL diffuse large B-cell lymphoma MZL marginal zone lymphoma CLL chronic lymphatic leukaemia BL Burkitt lymphoma MCL mantel cell lymphoma AUC area under curve
By Andreja Brozic; Ziva Pohar Marinsek; Simon Bucek; Maja Cemazar and Veronika Kloboves Prevodnik
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