Context.—

The diagnosis of classic Hodgkin lymphoma (CHL) traditionally requires surgical tissue biopsy because of the paucity of diagnostic Hodgkin and Reed-Sternberg cells. Diagnosis can be challenging in small core needle and cytologic biopsies, which are increasingly used because of reduced costs and minimal invasiveness. Flow cytometric (FC) identification of Hodgkin and Reed-Sternberg cells is possible, but FC test efficacy is not well studied outside of validation settings, especially in small specimens.

Objective.—

To assess the testing efficacy of FC performed on small biopsy and cytology specimens for the diagnosis of CHL.

Design.—

We reviewed 131 patients with CHL and 459 patients without CHL during a 3-year period who underwent a small biopsy procedure, including core biopsy and/or cytology evaluation, with concurrent routine clinical FC testing for CHL, assessing performance of FC in small specimens.

Results.—

Evaluating testing efficacy, sensitivity was 95.4% and specificity was 98.2%, whereas positive and negative predictive values were 92.2% and 99.0%, respectively. Although there were more false-positive results than compared with published validation studies, expert review identified distinct diagnostic pitfalls; awareness of these may improve testing efficacy.

Conclusions.—

Although FC diagnosis of CHL was historically considered unfeasible, our findings in a real-world clinical setting suggest that FC adds diagnostic value to small biopsy evaluation, reducing time to treatment, costs, and invasive excisional procedures.

The diagnosis of classic Hodgkin lymphoma (CHL) requires morphologic and immunophenotypic identification of neoplastic Hodgkin and Reed-Sternberg cells (collectively referred to as HRS cells) intermixed with a background of reactive lymphocytes, histiocytes, plasma cells, and eosinophils.13  The diagnosis of CHL may pose challenges specifically in limited specimens, such as core needle biopsies (CNBs) and fine-needle aspiration (FNA) biopsies, because the neoplastic HRS cell population often represents less than 1% and frequently less than 0.01% of the total number of cells in a variable pool of background inflammatory cells.1,2,4  Use of immunohistochemical staining can help better characterize both HRS cells and the background reactive lymphocytes. HRS cells are usually positive for CD30, CD15, MUM1, PAX5 (dim/weak), CD40, and CD95; they are usually negative for CD45 and CD20 but can show weak positive expression of these markers, and they are weak to negative for CD79a.1,2  Most of the background lymphocytes are of T-cell lineage with a high CD4 to CD8 ratio. Rosetting of the HRS cells by CD4 positive T cells is a known and diagnostically useful phenomenon.1,2 

Evaluation of lymphadenopathy and diagnosis of lymphoma increasingly rely upon small biopsy procedures, such as CNB and FNAs, rather than excisional or incisional biopsies.5,6  Core needle biopsies and FNAs are minimally invasive, cost-effective, and can still produce material for ancillary tests, including flow cytometry (FC), cytogenetics, and molecular genetic testing.512  However, FNA specimens have higher rates of nondiagnostic specimens and may not allow for complete subclassification of lymphoproliferative disorders due to loss of tissue architecture, particularly in deep-seated lesions.1315  Evaluation of CHL in particular may pose challenges in small biopsy specimens because of low numbers of diagnostic neoplastic HRS cells.2,12  In addition to low numbers of diagnostic cells, the evaluation of small samples can be confounded by extensive fibrosis, particularly in the nodular sclerosis subtype of CHL.1,2,8,12,15  Therefore, the use of specific and sensitive ancillary techniques applied to small biopsies can increase the diagnostic value of these specimens.710 

Flow cytometry studies have been used extensively as part of the workup of most lymphoproliferative disorders, including B- and T-cell lymphomas. The relatively rapid analytic time and minimal requirements for diagnostic samples make FC an attractive initial diagnostic modality, especially when combined with cytology specimens that often have scant cellularity.10,1618  Flow cytometry immunophenotyping evaluates antigen expression and light scatter characteristics of single cells in liquid phase, using antibody-linked, laser-activated fluorescent tags. Newer digital acquisition, multilaser flow cytometers allow for the rapid detection of millions of individual cells with simultaneous evaluation of more than 10 antigens in a single analysis.17 

Historically, despite the widespread use of FC, certain hematolymphoid neoplasms, including CHL, were deemed unsuitable for FC because of the inability to detect the diagnostic HRS cells. This could be attributed to a number of factors, including rarity of diagnostic cells, size of diagnostic cells, rosetting of HRS cells by reactive T cells, and effects of sample preparation.18,19  More recently, several studies have described FC methods to identify HRS cells with high sensitivity and specificity.1,18,20,21  Detection of HRS cells can be performed in a single tube, with as few as 6 antibodies.20,22  However, validation studies typically have used a mixture of small biopsy specimens and surgical biopsy specimens; there are no systematic studies of the efficacy of FC testing specifically evaluating CNB and cytology specimens. Therefore, we report here a 3-year experience using FC in clinical CNB and cytology specimens for the diagnosis of CHL.

Sample Selection

The study was approved by the Institutional Review Board. Pathology archives were searched from 2015 to 2018 for all histologically confirmed diagnoses of CHL. In addition, archives were queried for all FC cases evaluated with a CHL assay within the same time frame. For the purposes of calculating test efficacy, tissue biopsy was considered the gold standard for diagnosis of CHL; samples with a final tissue diagnosis of CHL were considered positive, and samples with non-CHL final tissue diagnoses were considered negative. We defined a “small biopsy” as any CNB or cytology specimen, including FNA and cytology specimens derived from needle rinses after core biopsy.

FC Specimen Preparation

Flow cytometry was performed on all samples as part of routine clinical practice at a major tertiary cancer center. As part of this routine clinical evaluation, all new tissue biopsies (other than bone marrow specimens) submitted for lymphoma evaluation were assessed using separate FC assays to evaluate for B- and T-cell lymphoproliferative disorders, and an FC assay evaluating for CHL. Tissue biopsy specimens were prepared in the standard manner used for routine clinical FC; tissue was finely minced in RPMI 1640 growth medium using scalpel blades, then filtered and resuspended in RPMI. Needle aspirates and biopsy needle rinses were filtered and suspended in RPMI. Following resuspension, cells were stained with a 9-antibody panel designed to assess for HRS cells, consisting of fluorescent tagged antibodies against CD64-FITC (Beckman Coulter, Brea, California), CD30-PE (Beckman Coulter), CD20-PC7 (Beckman Coulter), CD15-APC (BD Biosciences, San Jose, California), CD40 PerCP eFluor 710 (Thermo Fisher eBiosciences, Waltham, Massachusetts), CD45-APC-H7 (BD Biosciences), CD95 PAC BLUE (Thermo Fisher Life Technologies, Waltham, Massachusetts), CD5-BV510 (BioLegend, San Diego, California), and CD71 APC-A700 (Beckman Coulter), and incubated at room temperature. Following staining and incubation, cells were lysed, fixed, washed, and resuspended. Up to 500 000 cells were acquired on a fluorescence-activated cell sorting Canto 10-color flow cytometer (BD Biosciences).

CHL Analysis by FC

Results were analyzed with custom Woodlist software (a generous gift from Brent Wood, MD, Childrens Hospital of Los Angeles, Los Angeles, California). All assays were initially assessed by a senior technologist and a single pathologist. Abnormal CD30+ populations were identified by visual assessment of populations with antigen expression fitting previously reported phenotypes of CHL. Abnormal CD30+ populations compatible with Hodgkin cell phenotype were detected by the following characteristics: (1) expression of CD30, CD40, and CD95; (2) increased forward and side scatter indicative of large size; (3) absence of bright CD20 expression; (4) absence of CD64 expression; and (5) forming a discrete cluster of events. Some CD20 expression was accepted in the abnormal populations, if expression was not as bright as background B cells. CD15 was not required to define an abnormal population.20  A sample was considered diagnostic if it met 1 of the following conditions: (1) abnormal population was detected by FC; or (2) at least 100 000 viable cells were acquired for analysis. Samples were considered nondiagnostic if both of the following conditions were met: (1) fewer than 100 000 viable cells were acquired for analysis; and (2) no abnormal population was detected. The FC detection of a suspicious (10–20 abnormal cells) or abnormal (>20 abnormal cells) population was considered positive.

Statistical Evaluation

Statistical testing was performed using Prism (GraphPad, San Diego, California). Mann-Whitney U test was used for comparisons between 2 continuous variables, and Fisher exact test was used for comparisons between categoric variables. Receiver-operator curve statistics were also assessed. Findings were considered statistically significant for P values of <.05.

Clinicopathologic features are summarized in Table 1. From 2015 through 2018, a total of 131 patients with CHL underwent a small biopsy assessment with concurrent FC; 125 had CNB performed, and 109 had cytologic material evaluated by FNA or needle rinse. The median age in the cohort was 37 years (range, 11–82 years). There were 77 male patients and 54 female patients. Sample types submitted for FC included tissue CNBs, FNA material, and biopsy needle rinse material. A total of 87 patients eventually received a diagnosis of CHL nodular sclerosis subtype; 8 were diagnosed with mixed cellularity subtype, and the remainder could not be further subclassified because of the small biopsy nature of the specimen. A total of 42 biopsies were from cervical sites, 23 from the mediastinum, 20 from supraclavicular sites, 11 from the retroperitoneum, 9 from axillary nodes, 7 from inguinal nodes, 5 from soft tissue sites, and 4 from pelvic sites, with the remainder coming from other assorted sites. For a negative control, we reviewed FC diagnoses from 459 small biopsy specimens that were confirmed negative for CHL by tissue biopsy and were evaluated for CHL by FC through routine clinical testing.

Table 1

Clinicopathologic Features of Study Cohort

Clinicopathologic Features of Study Cohort
Clinicopathologic Features of Study Cohort

We reviewed the FC diagnoses for all 131 patients with FC from small biopsies. Based on the preestablished criteria described above, 87 small biopsy specimens (66.41%) were diagnostic. However, 100 000 or more cells were acquired in only 58 of 131 specimens (44.28%). Flow cytometry samples specifically from FNAs represented 35 specimens, of which 15 (42.86%) had at least 100 000 cells acquired; nonetheless, 24 (68.57%) were considered diagnostic specimens. In the 459 negative control specimens, 397 (86.49%) were considered diagnostic; there were 213 FNAs, of which 186 (87.32%) were diagnostic.

Among the 87 confirmed CHL small biopsy specimens considered diagnostic, FC was deemed to be positive in 83, leading to a sensitivity of 95.40%. For an analysis of the 24 FNA specimens with confirmed CHL, FC was positive in 22, resulting in a sensitivity of 91.67%. Among the negative control specimens, 390 small biopsy–derived specimens were considered to be negative by FC, thus leading to a specificity of 98.20%; 185 FNA-derived specimens were considered negative by FC, resulting in a specificity of 99.46%. Among all small biopsy FC samples, there were 7 false-positive (FP) and 4 false-negative (FN) results, whereas FNA samples yielded 1 FP and 2 FNs. Positive predictive values (PPV) and negative predictive values (NPV) of small biopsies were 92.22% and 98.98%, respectively. For FNA, the PPV was 95.65% and the NPV was 98.93%. Receiver-operator curve statistics demonstrated an area under the curve of 0.9682. These results are summarized in Table 2.

Table 2

Contingency Tables and Testing Performance

Contingency Tables and Testing Performance
Contingency Tables and Testing Performance

Immunophenotypes of HRS by FC testing typically matched those reported in prior validation studies (Figure 1). In the 83 specimens reported as positive for CHL, CD30 was expressed in every case (by definition). CD15 was expressed in 69 of 83 cases (83.1%), CD20 was partially expressed in 12 (14.5%), CD40 was brightly expressed in 83 (100%), CD64 was weakly expressed in 2 (2.4%), and CD95 was expressed in 83 (100%).

Figure 1

Flow cytometric (FC) immunophenotype of Hodgkin/Reed-Sternberg (HRS) cells. A through E, HRS cells (red) show high forward and side scatter, and positive CD30 expression without significant CD64 expression. They also show bright expression of CD95 and CD40. CD40 is notably brighter than background B cells (blue). The HRS cells show variable expression of CD15 and dim CD20 compared with background B cells. They also show variable expression of CD5 and CD45 due to T-cell rosettes. F and G, Evaluation of background T cells by FC shows increased expression of CD5 and CD7 by CD4+ T cells (red), as well as increased CD25+ T cells.

Figure 1

Flow cytometric (FC) immunophenotype of Hodgkin/Reed-Sternberg (HRS) cells. A through E, HRS cells (red) show high forward and side scatter, and positive CD30 expression without significant CD64 expression. They also show bright expression of CD95 and CD40. CD40 is notably brighter than background B cells (blue). The HRS cells show variable expression of CD15 and dim CD20 compared with background B cells. They also show variable expression of CD5 and CD45 due to T-cell rosettes. F and G, Evaluation of background T cells by FC shows increased expression of CD5 and CD7 by CD4+ T cells (red), as well as increased CD25+ T cells.

Close modal

Seven cases initially considered as FP and 4 FN cases based on the original diagnosis were reevaluated by 3 authors (A.C., O.L., and M.R.) in a blinded fashion. The FP and FN cases were mixed with 18 mixed true-positive and true-negative cases to avoid recognition and bias. A total of 6 of 7 FP cases and 3 of 4 FN cases in which the reevaluation by at least 2 of 3 pathologists was concordant with the final tissue diagnosis were reclassified. When considering only cases from samples considered diagnostic, this reevaluation resulted in an improved sensitivity and specificity of 98.85% and 99.75%, respectively, and positive and negative predictive values of 98.85% and 99.75%, respectively.

False-positive cases showed distinct differences from true-positive cases (Figure 2). Most notably, FP cases showed dimmer CD40 expression in HRS cells than in true positive cases. Mean fluorescence intensity measurements demonstrated that the median CD40 mean fluorescence intensity in FP cases was 6128 (interquartile range [IQR], 1029–10 938), compared with a median CD40 mean fluorescence intensity of 24 030 (IQR, 10 827–43 686) in a random sampling of 25 true-positive cases, a statistically significant difference (Mann-Whitney test, P = .003). In addition, only 1 of the 7 FP cases demonstrated increased CD7 and/or CD45 expression in background T-cell evaluation, a so-called Hodgkin sign (Figure 1, F), whereas 21 of the 25 randomly selected true-positive cases demonstrated this finding, a statistically significant difference (Fisher exact test, P = .001; Figure 2, C and F).23,24  All FN results were due to the low number of events suggestive of HRS cells, leading to conservative reporting. A total of 3 of the 4 FN cases demonstrated nodular sclerosis subtype of CHL on morphologic review, and 1 was diagnosed as mixed cellularity subtype. Of note, 3 of the 4 FN results showed increased CD7 and/or CD45 expression in the background T cells.

Figure 2

Comparison of example true-positive and false-positive cases. A, Example true-positive case demonstrates very bright CD40 in Hodgkin/Reed-Sternberg (HRS) cells (red) compared with background B cells (blue). B, It also shows CD15 expression and dim CD20 expression. C, Background T cells show increased CD7 expression. D, An example false-positive case, diagnosed as “suspicious for abnormal CD30 positive population,” demonstrates dim CD40 in HRS cells, similar to or weaker than background B cells. E, In addition, HRS cells show bright CD20. F, Background T cells do not show increases in CD7 compared with the true-positive case.

Figure 2

Comparison of example true-positive and false-positive cases. A, Example true-positive case demonstrates very bright CD40 in Hodgkin/Reed-Sternberg (HRS) cells (red) compared with background B cells (blue). B, It also shows CD15 expression and dim CD20 expression. C, Background T cells show increased CD7 expression. D, An example false-positive case, diagnosed as “suspicious for abnormal CD30 positive population,” demonstrates dim CD40 in HRS cells, similar to or weaker than background B cells. E, In addition, HRS cells show bright CD20. F, Background T cells do not show increases in CD7 compared with the true-positive case.

Close modal

We also reviewed the clinical histories of all patients who had subsequent excisional biopsy on a site that was previously assessed by FC and cytologic evaluation. We identified 23 patients with CHL who underwent an excisional biopsy after FC and cytologic evaluation of the same site. A total of 17 of those patients underwent excisional procedures because of nondiagnostic results in FC, cytology, and/or CNB review. A total of 6 patients were initially assessed by FC and FNA alone, without tissue biopsy. These 6 patients had positive FC testing with immunophenotype consistent with CHL and cytologic evaluation that was compatible with CHL. All 6 of these patients also had FC studies that were negative for other abnormal B- or T-cell populations, and the clinical and radiologic impression was suggestive of CHL. Only 1 patient with CHL had a positive FC test with negative morphology on both tissue and cytologic assessment, which was thought to be a sampling issue. Follow-up assessment confirmed a diagnosis of CHL in all patients who underwent surgical excisional procedures.

Advances in ancillary studies have allowed an increased use of small biopsy specimens in the diagnosis of lymphoproliferative disorders. Fine-needle aspiration/CNB have become the primary diagnostic procedures for patients with lymphadenopathy in many centers despite the recommendations from major societies or organizations, such as the European Society of Medical Oncologists and the National Comprehensive Cancer Network.25,26  The National Comprehensive Cancer Network clinical practice guidelines in oncology recommend the use of excisional biopsies at the time of initial diagnosis. Frederiksen et al8  performed a review of the literature evaluating how often these small biopsies could provide an actionable diagnosis in cases of lymphoma. They reported an actionable rate of 74% in a review of 42 studies; however, the extent to which advanced ancillary techniques was used in each study is not clear.

The diagnosis of CHL can be challenging in small biopsies because of the paucity of the neoplastic cells. Flow cytometry has not been traditionally used as an ancillary test in the diagnosis of CHL because isolating Reed-Sternberg cells from the background inflammatory cells had been difficult. More recently, Fromm and Wood22  have reported the use of a 6-color flow panel, including CD3, CD20, CD30, CD40, CD64, and CD95, to diagnose CHL in lymph node biopsy samples.27  The HRS cells show increased forward and side scatter compared with normal lymphocytes, are positive for CD30, CD40, and CD95, while variably positive for CD45 (in part due to bound T cells), mostly negative for CD20, negative for CD64, and may be positive for CD3 (due to bound T cells). They also show an immunophenotype distinct from immunoblasts, which can be seen in increased numbers in reactive conditions and may express CD30.20 

Flow cytometry studies can be helpful to distinguish from other entities that demonstrate the presence of interspersed large cells, such as anaplastic large cell lymphoma, nodular lymphocyte–predominant Hodgkin lymphoma, and T-cell–rich large B-cell lymphoma. For instance, CD30+ anaplastic large cell lymphomas do not show expression of CD40, and they can show CD5, CD45, CD71, and CD95 expression with absence of CD15, CD20, and CD64. As a T-cell lineage malignancy, anaplastic large cell lymphoma can also show other T-cell marker expression, which can be identified in FC assessment of T cells. CD30 expression in nodular lymphocyte–predominant Hodgkin lymphoma and T-cell–rich large B-cell lymphoma is infrequent.4,18,28,29 

Other B-cell lymphomas can occasionally show CD30 expression, including diffuse large B-cell lymphoma, primary mediastinal large B-cell lymphoma, and B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and CHL (so-called grey zone lymphoma).2,4  In this study the neoplastic populations in these cases were usually detected through assessment of B cells rather than the CHL assay. In a single case these B cells were only detected in the CHL assay but showed CD40 and CD95 too dim for HRS cells and strong CD20 expression. In this case the reporting pathologist noted that the abnormal CD30+ cells were unlikely to represent CHL; we did not consider this a FP.

In addition to events suggestive of HRS, CHL shows alternations in background lymphoid cells. These can include an increase of the CD4/CD8 ratio among T cells, a decrease of B cells, increased expression of CD5, CD7, and CD45 on CD4+ T cells (Figure 1, F through H), and an increase in CD25+ Tregs cells (Figure 1, H) compared with reactive lymphoid hyperplasia.21,30  This so-called Hodgkin sign can also aid in FC assessment for CHL.

Our results demonstrate that small biopsy and FNA material can be used successfully for CHL evaluation by FC, with very high specificity and relatively high sensitivity. Sensitivity and specificity are comparable to those in previously reported validation studies.20,22  However, there were a greater number of FN and FP cases than published validation studies, particularly FP cases. This is most likely reflective of “real-world” clinical testing analysis in this study, which typically offers less control of specimens and more confounding variables than a validation environment. Of note, 5 of the 7 FP cases were from patients with a clinical history of CHL, and the knowledge of this likely led the initial reviewers to overinterpret CD30+ events seen in the FC assay.

Review of FP and FN cases revealed potential pitfalls in FC evaluation of CHL. All the FN cases demonstrated the presence of very few HRS cells. Because the HRS cells are unevenly and often sparsely distributed throughout affected tissue, this confirms that adequate sampling plays a critical role in successful diagnosis by FC. False-positive cases also shared similar findings because all FP cases demonstrated dim CD40 expression, which was mistakenly interpreted as positive. True HRS cells express CD40 at a level brighter than background B cells, including immunoblasts. This finding highlights the importance of the inclusion of CD20 antibody in the panel of antibodies used to analyze potential HRS cells, as well as knowledge of the differences in phenotypes between HRS cells and immunoblasts, which can also be CD30+.20  Increased CD7 and CD45 expression on background CD4+ T cells might be helpful in challenging cases because this feature was noted in most FN cases, whereas background T cells in most of the FP cases did not show this finding. However, in clinical practice we do not routinely report these T-cell findings because they have not been validated as a diagnostic tool.23,24 

Our data emphasize the need for adequate sampling of the lesion to obtain sufficient material for analysis, because approximately one-third of all small biopsy samples in cases diagnosed as CHL will be nondiagnostic by FC because of the low number of cells collected. The fibrosis associated with nodular sclerosis CHL might be partly responsible for the low number of cells collected in some cases. Multiple dedicated FNA biopsies or submission of additional tissue cores is recommended to obtain enough material for FC studies.

Flow cytometry testing is also not only useful for immunophenotypic confirmation of HRS cells. The use of cell sorting techniques by FC may improve the ability to perform ancillary molecular and cytogenetic testing of HRS in CHL. The cells could be sorted for more accurate analysis of PD-L1/PD-L2 amplification status, which has been associated with inferior progression-free survival.31  The ability to concentrate HRS cells might improve the sensitivity of cytogenetic and molecular analysis of HRS cells. Cytogenetic testing in CHL is typically challenging because of the low number of HRS cells available for analysis.32  Furthermore, increased sample purity through FC cell sorting has allowed for in-depth study of the genomics of CHL.33,34 

When considering our findings regarding subsequent excisions, our data suggest that out of 87 patients with diagnostic FC specimens, 6 patients might have avoided a subsequent surgical excisional biopsy. Although tissue biopsy is and should remain the gold standard for CHL diagnosis, if a patient's clinical and radiologic contexts are compatible with CHL, cytology and FC assessments are consistent with CHL with adequate cellularity, and there is no FC or cytologic evidence of alternative B- or T-cell lymphoproliferative disorder, it seems likely that CNB would suffice for diagnosis in these patients, which could spare the patient an invasive and costly surgical excision. Of course, decisions regarding subsequent tissue sampling would require full assessment of the clinical context, including the patient's condition and anatomic site of lesion. In our cohort, these situations are uncommon, so further evaluation would be required to make any recommendations regarding clinical management.

The high diagnostic specificity and high PPV and NPV of FC studies described here suggest that the combination of small biopsies and FC can provide rapid and early diagnostic information. They have the potential for faster turnaround time in urgent clinical situations, and FC can aid when immunohistochemical workup is limited for technical reasons. In addition, there are situations in which cytology and FC alone can provide important guidance for further clinical assessment. In summary, FC evaluation provides additional diagnostic value to small biopsies for the diagnosis of CHL, with the potential to reduce time to treatment, costs, and invasive excisional procedures.

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Author notes

Roshal and Lin contributed equally as senior authors.

This study was funded by the Center for Hematologic Malignancies at Memorial Sloan Kettering Cancer Center and in part through the National Institutes of Health/National Cancer Institute Cancer Center Support Grant P30 CA008748.

The authors have no relevant financial interest in the products or companies described in this article.