Keywords Hodgkin lymphoma - primary diagnosis - bone marrow biopsy
Introduction
Hodgkin lymphoma (HL) is a malignant lymphoid neoplasm usually involving the lymph
nodes. HL infiltrating bone marrow (BM) is relatively rare with an incidence ranging
from 4% to 18%.[1 ]
[2 ]
[3 ]
[4 ] As the bone marrow lacks lymphatics, bone marrow infiltration indicates vascular
dissemination (stage IV disease). BM examination is usually done for clinical staging
and BM involvement upgrades it to stage IV with a poorer prognosis.[5 ]
[6 ]
[7 ] Extranodal HL is very rare and primary HL of marrow has an incidence of 0.25%.[7 ] To the best of our knowledge, the present study is the largest study on HL, initially
diagnosed on marrow including 36 cases, and highlights the role of BM examination
in diagnosing HL based on its clinicopathological, histological, and immunohistochemical
features.
Materials and Methods
This was a retrospective study done in a tertiary care center that included 36 cases
of HL diagnosed initially on the BM from January 2012 to December 2020. The study
was designed to emphasize the characteristic clinicopathological picture, i.e., pyrexia
of unknown origin with cytopenias (primary outcome) in unsuspected cases of HL and
create awareness on the requirement of lymph node biopsies with ancillary testing
(secondary outcome) in such cases. All known cases (n = 157) of HL, where BM examination was done for staging were excluded from the study.
Clinical data, peripheral blood, and BM findings (as mentioned in the supplementary
file) collected were retrospectively reviewed and analyzed. Under aseptic conditions,
aspiration and biopsy were done from the posterior superior iliac spines. Six to 10
smears, each of peripheral smears, aspirates, and imprints were prepared, air dried,
and stained. Smears were stained with Giemsa. BM biopsies done were decalcified, processed,
and sections were stained with hematoxylin and eosin stain, and reticulin. Special
stains such as acid-fast bacilli (AFB) were used wherever necessary. Based on the
presence of large mononuclear or binucleate Reed–Sternberg (RS)-like cells in a polymorphous
inflammatory background, HL was suspected and immunohistochemistry (IHC) was done
on a fully automated immunostainer (X matrix Elite; BioGenex). The markers in the
IHC panel included LCA/CD45, CD3, CD20, CD15, CD30, CD68, ALK, EMA, and PAX5. IHC
was done on the marrow in 29 cases. In seven cases that had subsequent lymph node
biopsy, the morphology of lymph node with IHC results was reviewed.
Statistical Analysis
For statistical analysis, Microsoft Excel 2019 was used and frequencies for each variable
were calculated.
Ethics
The study was approved by the NIMS Institutional Ethics Committee with approval number
of EC/NIMS/2705/2021, PBAC No. 1349/2021, dated 15.3.2021. The procedures followed
were in accordance with the ethical standards of the responsible committee on human
experimentation (institutional) and with the Helsinki declaration of 1964, as reviewed
in 2013. Waiver of the informed consent form was obtained from the Ethics Committee
due to the retrospective nature of the study.
Results
The study included 36 cases of HL diagnosed initially on the bone marrow in 9 years
duration. In view of high-grade fever with associated bi/pancytopenias, all 36 cases
were suspected as tuberculosis/lymphoma clinically and none of these cases had a prior
diagnosis of HL.
There was a wide age distribution with a mean of 40 years (±6.7, 95% confidence interval
[CI]). There was a male predominance with a male to female ratio of 3:1, of which
27 were males and 9 were females. Three cases were positive for HIV with very low
CD4 counts (24, 57, and 39 cells/mm3 ) and were on HAART therapy. Five cases were previously diagnosed as cases of tuberculosis
and were on anti-tuberculous therapy (ATT). One case each was diagnosed with autoimmune
hemolytic anemia, poliomyelitis, ileal perforation, digital gangrene, and mitral valve
regurgitation. The clinical presentation of the 36 cases is depicted in [Table 1 ]. Among “B” symptoms, fever (34/36) (94.4%) was the most common symptom, followed
by loss of weight (24/36) (66.7%). As HL was not suspected before diagnosis on marrow,
the clinical staging was not done. PET CT was done only in 14 patients due to financial
constraints, of which 9 cases showed central lymphadenopathy (mediastinal, retroperitoneal,
intra-abdominal), 2 cases had both central and peripheral nodes, 2 cases had space-occupying
lesions, one each in liver and spleen and one case had no other masses or nodes anywhere
else in the body. In cases (n = 22) where PET CT was not done, 15 cases had peripheral lymphadenopathy. Altogether,
lymphadenopathy (peripheral or central or both) was seen in (26/36) 72.2% cases. Other
findings included hepatomegaly (19/36) (52.7%) and splenomegaly (18/36) (50%). In
view of the presence of bi/pancytopenias, bone marrow aspiration and biopsy was performed
in all cases and this was followed or accompanied by biopsy of the lymph node in only
seven cases with peripheral lymphadenopathy. In patients who had only mediastinal
or retroperitoneal lymphadenopathy, biopsies could not be obtained due to difficulty
in accessing biopsy site.
Table 1
Clinical presentation in cases with bone marrow involvement by Hodgkin Lymphoma
Clinical presentation
No. of cases (%)
(n = 36)
Fever
34 (94.4%)
Cough
11 (30.5%)
Shortness of breath
8 (22.2%)
Abdominal pain
2 (5.5%)
Abdominal distension
5 (13.9%)
Vomiting
2 (5.5%)
Diarrhea
3 (8.3%)
Malena
1 (2.7%)
Loss of weight
24 (66.7%)
Loss of appetite
10 (27.7%)
Urinary tract infection
1 (2.7%)
Generalized weakness
11 (30.5%)
Jaundice
5 (13.9%)
Hepatomegaly
19 (52.7%)
Space-occupying lesion liver
1 (2.7%)
Splenomegaly
18 (50%)
Space-occupying lesion spleen
1 (2.7%)
Lymphadenopathy
Peripheral
15 (41.6%)
Central
9 (25%)
Both
2 (5.5%)
Altered sensorium
5 (13.9%)
Pedal edema
1 (2.7%)
Pruritus
1 (2.7%)
Pallor
8 (22.22%)
The hematological profile of all cases is depicted in [Table 2 ]. Cytopenia/s was seen in all cases with a mean number of cytopenias of 2.4 ± 0.295%
CI. All cases had anemia, of which 15/36 cases (41.6%) had normocytic normochromic
anemia. Leucopenia was seen in 21/36 cases (58.3%), of which two cases showed atypical
lymphocytes on peripheral smear examination. Thrombocytopenia was seen in 26/36 cases
(72.2%) and 15/36 cases (41.6%) had pancytopenia.
Table 2
Hematological profile in cases with bone marrow involvement by Hodgkin lymphoma
Parameter
Hematological profile
No. of cases (%) (n = 36)
Hemoglobin
Mild anemia (10–11 g/dL)
18 (50%)
Moderate anemia (7–9 g/dL)
15 (41.6%)
Severe anemia (< 7 g/dl.)
3 (8.4%)
Red blood cells
Normocytic normochromic
15 (41.6%)
Anisopoikilocytosis
19 (52.7%)
Microcytes
5 (13.9%)
Macrocytes
6 (16.7%)
Tear drop cells
2 (5.5%)
Polychromatophils
5 (13.9%)
Nucleated RBCs
2 (5.5%)
Total leucocyte count
Normal WBC count
10 (27.7%)
Leucopenia
21 (58.3%)
Differential leucocyte count
Normal differential
24 (66.7%)
Relative neutrophilia
1 (2.7%)
Relative lymphocytosis
4 (11.11%)
Eosinophilia
6 (16.7%)
Monocytosis
1 (2.7%)
Shift to left
4 (11.11%)
Atypical lymphocytes
2 (15.5%)
Platelet count
Normal counts
10 (27.7%)
Thrombocytopenia
26 (72.2%)
Others
Leucoerythroblastic picture
1 (2.7%)
Pancytopenia
15 (41.6%)
Abbreviations: RBC, red blood cell; WBC, white blood cell.
Bone marrow aspirate was particulate in 24/36 cases (66.7%), out of which smears were
normocellular in 12/36 cases (50%), hypercellular in 8/36 cases (33.3%), and hypocellular
in 4/36 cases (16.6%). Only one case showed suspicious mononuclear RS cells on BM
aspirates and the rest of the cases were diagnosed only on trephine biopsy. Trephine
imprints showed variable cellularity in 13/36 (36.1%) cases.
Marrow cellularity on trephine biopsy varied from 50% to 90% with a mean of 68% ± 4.8,
95% CI. Diffuse involvement was seen in 24/36 cases (66.7%) and focal nodular aggregates
were seen in 12/36 cases (33.3%). Marrow fibrosis was seen in 16/36 cases (44.4%)
that included 12 cases with aparticulate aspirates and 4 cases with hypocellular aspirates.
Reticulin condensation was variable from grade II to grade IV. Cases with diffuse
involvement of marrow on biopsy showed acellular smears on imprint cytology with increased
reticulin condensation of grade IV. Cases with focal nodular aggregates on biopsy
showed predominantly variable cellularity on imprint cytology with a variable reticulin
grading of grade II to grade III in involved marrow ([Fig. 1 ]). Marrow necrosis was seen in 3/36 cases (8.4%). Granulomas were seen in 8/36 cases
(22.2%), of which 1 case was HIV-positive. Stains were done for AFB and fungus and
were negative ([Fig. 2 ]).
Fig. 1 Patterns of marrow involvement in HL. (A, B ). Diffuse pattern. (A ) Diffuse involvement on trephine biopsy (×100, H&E), (B ) Increased reticulin condensation of grade IV (×400, reticulin); (C, D ). Focal pattern. (C ) Focal nodular aggregates (above) with uninvolved marrow (below) on trephine biopsy
(×100, H&E), (D ) Variable reticulin grading of grade III in involved marrow (above) and grade II
in normal marrow (below) (×400, reticulin). H&E, hematoxylin and eosin; HL, Hodgkin
lymphoma.
Fig. 2 Granulomas in marrow involved by HL. (A ) Aggregates of epithelioid macrophages (X400, H&E), (B ) Negative for AFB (×100, Ziehl–Neelsen stain), (C ) Negative for fungus (×100, Grocott methenamine silver). AFB, acid-fast bacilli;
H&E, hematoxylin and eosin; HL, Hodgkin lymphoma.
The main diagnostic criterion of HL on marrow was the presence of large mononuclear
or binucleate RS-like cells in a polymorphous inflammatory background that was seen
in all 36 cases. Classical binucleated RS cells, as well as mononuclear Hodgkin cells,
were seen in 26/36 cases (72.3%), while mononuclear Hodgkin cells alone were seen
in 10/36 cases (27.7%). The background of the polymorphous population of inflammatory
cells comprised lymphocytes, plasma cells, eosinophils, neutrophils, and histiocytes
([Fig. 3 ]). IHC with CD30 and CD15 were done on trephine sections only in 29 cases. The IHC
results on marrow were CD30+ /CD15+ in (6/29) (20.7%) cases, CD30+ /CD15− in (7/29) (24.1%) cases, and CD30− /CD15+ in (6/29) (20.7%) cases, and CD30− /CD15− in 10/29 cases (34.4%).
Fig. 3 Reed–Sternberg cells. (A ) Binucleated (arrowhead) and mononucleated (arrow) RS cells. (B ) Polymorphous background of eosinophils, plasma cells, and histiocytes (×400, H&E),
(C ) IHC with CD30 showing Golgi area positivity (×1000), (D ) IHC with CD15 showing Golgi area positivity (×1000). H&E, hematoxylin and eosin;
IHC, immunohistochemistry.
In cases (n = 10) with negative IHC results (CD15− , CD30− , PAX5+ , CD20+ , ALK− , EMA− , CD3− , CD45− ) done on marrow, two cases had subsequent lymph node biopsy with positive IHC results
(CD15+ , CD30+ , PAX5+ , CD20+ , ALK− , EMA− , CD3− , CD45− ) and were of mixed cellularity subtype. Of the remaining eight cases, three patients
died and five got discharged against medical advice and were lost to follow-up.
In seven cases, IHC was not done on marrow because five cases had subsequent lymph
node biopsies with positive IHC results, while two cases got discharged against medical
advice and lost to follow-up. All seven cases with lymph node biopsy showed mixed
cellularity HL, with IHC positivity for both CD15 and CD30. In total, there were 26/36
confirmed cases (72.2%), of which only 1 case had no mass or lymph nodes anywhere
else in the body even after extensive radiological workup and was finally diagnosed
as primary HL of marrow.
Treatment strategies: Marrow diagnosis based on morphology alone indicates suspicion
and needs IHC confirmation to start the treatment. All the cases of HL initially diagnosed
on marrow with confirmed IHC results were treated as per the NCCN (National Comprehensive
Cancer Network) recommendations with ABVD (adriamycin, bleomycin, vinblastine, and
dacarbazine) regimen. In cases where IHC is inconclusive, a tissue diagnosis with
positive IHC results is mandatory to start the treatment. If the nodes are not accessible,
even an invasive procedure is attempted to retrieve the representative biopsy sample.
In our study, only 26 (72.2%) cases had a confirmed diagnosis of HL and were started
on treatment. The remaining cases (10/36) (27.8%) that were diagnosed only based on
marrow morphology but had inconclusive IHC results, remained suspicious of HL. A confirmed
diagnosis could not be made in these cases as three patients died of sepsis within
a few days of diagnosis and the rest (n = 7) got discharged against medical advice and lost to further follow-up.
Discussion
BM studies are usually done as a part of the staging workup for HL because its involvement
is considered as stage IV disease with poorer prognosis and reduced survival.[1 ]
[5 ]
[6 ]
[8 ] When compared with non-Hodgkin lymphomas, HL involving marrow is relatively rare
with an incidence of 3 to 18%.[1 ]
[9 ]
[10 ]
[11 ] HL invariably arises in the lymph nodes, and primary extranodal HL is very rare.
Isolated bone and bone marrow Hodgkin lymphoma is extremely rare, seen in less than
0.25% of cases, and very few case reports are reported in the literature.[12 ]
[13 ]
[14 ]
[15 ]
[16 ] It is important to confirm whether extranodal involvement represents a primary manifestation
or dissemination of systemic disease with a poor prognosis.[7 ]
Our study group comprised 36 cases that were first diagnosed on the marrow. They account
for 18.7% of all HL cases (n = 193) that were diagnosed and underwent BM examination during the study period.
The high number could be due to the longer duration of the study and only one case
was finally diagnosed as primary HL of marrow, which accounted for 0.5% (1/193) of
all the HL diagnosed in the study period. The rest of the cases (17/36) (18.2%) were
HL with secondary involvement of marrow but diagnosed initially on the marrow.
When clinicopathological features associated with HL (“B” symptoms) were studied,
we noted that 94.4% of the cases presented with fever followed by loss of weight (66.7%).
Lymphadenopathy was seen in 55.5% of cases. The hematological profile revealed that
58.3% of cases had leucopenia, 72.2% of cases had thrombocytopenia, and 41.6% of cases
had pancytopenia. Many studies revealed that the presence of “B” symptoms and cytopenias,
especially leucopenia and thrombocytopenia, were frequently associated with BM involvement
by HL.[17 ]
[18 ]
[19 ]
[20 ]
BM aspirates are comparatively of lesser diagnostic value in diagnosing HL, as the
involved marrow is difficult to aspirate due to marrow fibrosis.[2 ]
[21 ] As the aspirates were aparticulate or hypocellular, there is no/limited role of
flow cytometry in the diagnosis of HL involving marrow. In our study, only one case
showed suspicious RS cells on aspirate. Variable cellularity on imprint smears is
possibly due to marrow fibrosis and this should raise the suspicion of HL involving
BM.[4 ]
[5 ]
[22 ] We observed variably cellular imprint smears in 36.1% of cases. HL involving the
marrow can be diffuse or focal, and an adequate length of BM needs to be sampled especially
in cases with focal involvement.[5 ] Diffuse involvement of BM was seen in 66.7% of our cases and focal nodular aggregates
were seen in 33.3% of cases, similar to other studies.[17 ]
Diagnosis of marrow involvement by HL is challenging, especially when there is no
prior diagnosis on a lymph node or other site in the body. Knowledge of histomorphology
in correlation with clinicopathological features is essential to diagnose HL on the
marrow.[23 ]
[24 ] As per the recommendations drawn at the Ann Arbor conference (1971), criteria to
establish a marrow diagnosis of HL differ according to whether or not a tissue diagnosis
of HL is already established.[25 ] To diagnose HL initially on marrow without a tissue diagnosis, the presence of CD30-
and/or CD15-positive “classic” binucleated RS cells in a polymorphous inflammatory
background of lymphocytes, plasma cells, eosinophils, and neutrophils is required.
An exception to this is that, in the nodular sclerosis subtype, the presence of “variant
RS cells (lacunar cells)“ in an appropriate cellular background is also sufficient
to make the diagnosis.[25 ]
As per these diagnostic criteria, finding a classic binucleate RS cell is particularly
challenging and wherever suspicious, we used IHC to confirm the diagnosis. In cases
where tissue diagnosis is already made and bone marrow biopsy is done for staging,
the criteria are less stringent, which include the presence of mononuclear Hodgkin
cells in an appropriate cellular background. The presence of only a few large atypical
cells (or) diffuse fibrosis with appropriate inflammatory cells is suggestive but
not diagnostic of HL. In such suspicious cases, serial sections may reveal more diagnostic
material and/or IHC is needed to confirm the diagnosis.[25 ] Although CD30 is a specific marker for RS cells of HL, studies reveal that the sensitivity
and specificity of CD15 in detecting cases of Hodgkin disease is 80% and 80.6%, respectively.[26 ] CD15 expression in HL differs between studies.[18 ]
[19 ]
In this study, immunophenotype of the RS cells on marrow CD30+ /CD15+ in 20.7% of cases, CD30+ /CD15− in 24.1% of cases, and CD30− /CD15+ in 20.7% of cases. Cases, where only CD30 was positive, showed positivity for PAX5
and negativity for CD3, CD45, ALK, and EMA, ruling out the possibility of anaplastic
large cell lymphoma. CD68 was done to differentiate histiocytes from mononuclear RS
cells, especially in cases where additional findings such as granulomas were seen.
Out of 10 cases with negative IHC results on marrow, 2 cases had subsequent lymph
node biopsy and were diagnosed as HL. IHC on trephine sections often gives good results
but occasionally, acid decalcification interferes with antigen retrieval due to the
destruction of the antigenic epitopes by fixation and decalcification process used
for BM biopsy specimens.[27 ]
[28 ] This could be the possible explanation for the negative IHC results on marrow but
positive on lymph nodes. So far, this is the largest study of HL diagnosed on marrow,
where 72.2% (26/36) of cases had IHC confirmation, of which 19 cases (52.7%) were
confirmed on trephine sections and the rest on subsequent lymph node biopsies.
HL is a unique neoplasm that exhibits geographical variations in its histological
subtypes as well as in the incidence of BM infiltration.[3 ] Among the subtypes of HL, lymphocyte-depleted HL has a higher frequency (54%) of
involving the marrow, followed by mixed cellularity HL (20%).[3 ] Nodular sclerosis HL rarely involves the marrow with an incidence of 5%.[3 ] We observed that the seven cases in our study that had subsequent lymph node biopsies
were diagnosed as mixed cellularity HL. Similar studies observed that BM involvement
was more common in the lymphocyte-depleted subtype, followed by mixed cellularity.[17 ]
[18 ]
[29 ]
HL is one of the most common non-AIDS defining tumors that can occur in HIV-positive
cases.[5 ] Primary HL of the marrow is relatively rare but can be seen in HIV-positive individuals,
and the secondary involvement of marrow by HL is relatively more common in HIV-positive
patients.[30 ] In our study, three cases were HIV-positive and all had lymphadenopathy. The term
“isolated bone marrow HIV-associated Hodgkin lymphoma (IBM-HIV-HL)” refers to cases
with BM involvement by HL in the absence of lymphadenopathy, even after extensive
clinical and imaging evaluation.[30 ]
[31 ] Because all three cases had lymphadenopathy, the term “IBM-HIV-HL” does not apply
to these cases. In all three cases, CD15- and CD30-positive RS cells were seen on
trephine sections. All three cases presented with pancytopenia and this could be secondary
to marrow infiltration along with extensive stromal changes, such as fibrosis and
necrosis were seen in HIV-positive patients.[5 ]
In our study, granulomatous inflammation was found in eight cases (22.2%), of which
one case was HIV-positive. Studies revealed that ∼9% of HL cases are accompanied by
non-necrotizing epithelioid granulomas.[32 ]
[33 ]
[34 ] The presence of epithelioid granulomas in cases with HL may reflect a host response
to the tumor with favorable prognostic implications.[34 ]
[35 ]
Limitations of the Study
IHC confirmation of HL on marrow was not possible in all suspected cases as the results
were altered by the decalcification process.
In cases with associated lymphadenopathy, subsequent lymph node biopsies were not
available for all the cases as few of the cases had inaccessible central lymphadenopathy,
few patients succumbed to death early and few got discharged against medical advice.
Future Research Directions
Future Research Directions
The current decalcification and fixation processes that are used for BM biopsy specimens
interfere with antigen retrieval due to the destruction of the antigenic epitopes.
Future research should be aimed at developing an efficient decalcification process
that does not affect the overall results.
Conclusion
Initial diagnosis of HL on marrow without a tissue diagnosis is very difficult and
has stringent diagnostic criteria. So far, this would be the largest study in the
literature describing 36 clinically unsuspected cases of HL that were diagnosed initially
on the marrow. In cases presenting with fever and cytopenias where BM examination
is done, the presence of RS cells in a polymorphous cell background should always
raise the suspicion of HL. Bone marrow biopsy is preferable over aspiration in such
cases due to associated secondary marrow fibrosis, and IHC plays a major role in the
confirmation of diagnosis. The study concludes that the knowledge of clinical and
histological features of HL can aid in the diagnosis of these lesions on the marrow
in clinically unsuspected cases. This study also shows that the presence of “B” symptoms
or cytopenias is associated with the highest risk of BM involvement by HL. When these
risk factors are coexistent, the likelihood of having BM involvement is further increased.
