Introduction:
Patients with underlying hematological diseases have an elevated risk to develop SARS-CoV-2
infection with significant morbidity and mortality. Waldenström’s macroglobulinemia
(WM) is an indolent low-grade lymphoma accounting for 1% to 2% of lymphoproliferative
disorders
1
. MW is characterized by the infiltration of bone marrow (BM) by clonal lymphoplasmacytic
cells that produce monoclonal immunoglobulin M (IgM)
2
. We report here a case of severe and irreversible bone marrow aplasia related to
SARS-CoV-2 infection in a 61-years old woman with WM.
Case report:
Smoldering WM diagnosis was initially made in March 2015 based on an IgM monoclonal
component at 25 g/L, associated with 50% BM infiltration by lymphoplasmacytic cells.
At the time of diagnosis, no tumoral syndrome had been identified on thoraco-abdominal
scan and initial international prognostic scoring system for MW
3
(IPSS MW) was low. BM karyotype revealed in 8/20 metaphases a t(6;8)(q27;p12). Without
treatment indication according to the Mayo Clinic mSMART consensus
4
, a clinical and biological monitoring was proposed in first intention. In February
2017, the molecular biology analysis revealed a MYD88L265P mutation. The patient was
monitored for almost 3 years. In October 2018, she developed pancytopenia (hemoglobin
8.5 g/dL, platelets count 81x109/L, neutrophils count 0.81x109/L) whereas the IgM
component remained stable around 30 g/L. A treatment by Bendamustine 90 mg/m2 and
Rituximab was then initiated. In April 2019, after 6 cycles, a very good partial response
(VGPR) was obtained with normalization of blood counts and decrease monoclonal component
at 0.9 g/L. In December 2019, the patient was still in VGPR with a normal hematologic
count, no clinical tumoral syndrome, no constitutional symptom and an IgM component
at 1.36 g/L.
Three months later, during the SARS-CoV-2 epidemic, the patient was admitted in the
emergency department because of a fast deterioration of the general status with fever
(39.1°C), dyspnea, high respiratory rate (> 30 breaths/min) associated with an oxygen
saturation of 89% in ambient air. At admission, a severe pancytopenia was discovered
(hemoglobin 4 g/dL, platelets count 4x109/L, neutrophils count 0.01x109/L) associated
with a lymphocytosis at 12x109/L. The patient also presented a major biological inflammatory
syndrome (C-Reactive Protein: 298 mg/L, serum ferritin: 3965 μg/L and fibrinogen:
7.96 g/L) and increased plasma concentrations of Interleukin 6 (IL-6: 110 pg/mL) and
Interferon gamma induced Protein 10 (IP-10: 1609 pg/mL). Besides, an endothelial injury
was objective by an important elevation of the Circulating Endothelial Cells (261
elements/mL, normal rate < 10) in the peripheral blood, consistent with a severe form
of COVID-19, as described in previous study
5
.
This deep pancytopenia was not explained by any drugs or toxic exposure leading to
an exhaustive microbiological screening of putative responsible bacterial (repeated
blood cultures), viral (Cytomegalovirus, Epstein-Barr Virus, Enterovirus, Parvorirus
B19, Adenovirus, Dengue, Hepatitis B, C, E) and parasitological (Plasmodium, Leishmania)
organisms. All of these investigations were negative.
Histologically, BM biopsy showed a dense and diffuse interstitial infiltrate predominantly
composed of relatively monotonous small lymphocytes and plasmacytoïd lymphocytes (Figure
1A
) admixed with plasma cells and few large transformed cells. The neoplastic lymphocytes
and plasmacytoïd lymphocytes expressed the B-cell associated antigen CD20 (Figure
1B) and the Bcl2 protein, whereas neoplastic plasma cells expressed CD138 (Figure
1C) and a monotypic cytoplasmic kappa light chain. Several CD138-positive plasma cells,
referred to as Mott cells, contained multiple round cytoplasmic hyaline inclusions.
The Ki-67 proliferation index was high, >50% (Figure 1D). CD3 and CD68 immunostaining
highlighted an associated reactive T-cells and histiocytic infiltrate (Figure 1E and
1F). Reticulin staining showed a loose network of reticulin fibers with many intersections
corresponding to early-stage myelofibrosis. In parallel, the Real Time-Polymerase
Chain Reaction (RT-PCR) for SARS-CoV-2, Mycobacterium, Histoplasma and Leishmania
were all negative in the BM.
Figure 1
Histological and immunohistochemical features of bone marrow biopsy A. Hematoxylin
and eosin staining showing a dense proliferation of neoplastic lymphocytes, plasmacytoïd
lymphocytes and plasma cells including Mott cells (arrowhead). B. Diffuse CD20 expression
by neoplastic lymphocytes and plasmacytoid lymphocytes. C. CD138 immunohistochemical
staining highlighting scattered plasma cells. D. Ki-67 staining reveals a high proliferation
index. E-F. CD3 (E) and CD68 (F) immunostaining demonstrating respectively an associated
reactive T-cells and histiocytic infiltrate. All the pictures were taken at 400X magnification
using the Hamamatsu’s virtual slide scanner Nanozoomer 2.0-HT with the NDP.view2 viewing
software (ver. 2.6.17).
Since the patient has been confined at home for 3 weeks with her son and daughter
who were both symptomatic and positive for SARS-CoV-2 (RT-PCR) in nasopharyngeal swab,
a chest CT-scan was performed showing typical images of COVID-19 intermediate to severe
stage. Due to the familial virus exposure, the blood, BM and CT-scan results and despite
repeated negative SARS-CoV-2 RT-PCR tests in different samples (oropharynx, blood,
BM and urine); we considered BM aplasia accelerated by SARS-CoV-2 infection as likely
and performed further investigations. First, the presence of anti-Spike SARS-CoV-2
IgG antibodies was detected in both serum and BM samples by a commercial ELISA test
(Euroimmun, Luebeck, Germany). Then, a SARS-CoV-2-specific virus neutralization test
was performed and the presence of high neutralizing antibody titers (1/160 in BM and
1/80 in serum) confirmed that the patient had been previously exposed to SARS-CoV-2.
Moreover, performing immunofluorescence using a known antisera obtained from an infected
patient, we were able to detect for the first time to our knowledge, the presence
of infected cells by SARS-CoV-2 in the BM (Figure 2
). These virological investigations brought the direct and indirect proof of SARS-CoV-2
infection in this patient’s BM.
Figure 2
Immunofluorescence detection of SARS-CoV-2 infected cells in bone marrow. Detection
of SARS CoV-2 (green) in bone-marrow lymphoid cells stained with known antisera from
infected patient and using a 400X magnification. The cell nucleus was stained by Hoechst
33342 (blue). Images were acquired using a Leica DMi8.
A therapeutic association of oral of Ibrutinib (140 mg three times a day) and 300mg/d
IV Anakinra
6
was initiated for 10 days within 48 hours of admission leading to a rapid and significant
decrease in both fever and blood inflammation, with a good clinical tolerance but
without hematopoietic reconstitution. One month later, the patient was still in deep
pancytopenia and developed a fatal invasive pulmonary fungal infection despite appropriate
antifungal treatment.
Discussion:
This unexpected hematologic complication of SARS-CoV-2 infection in our WM patient
is consistent with another recent reports of pancytopenia associated with SARS-CoV-2
infection in immunocompromised patients with hematological diseases
7
,
8
. Nevertheless, we noted significant differences between these reports, notably on
the methods used to detect SARS-CoV-2 in BM. Issa and colleagues showed for the first
time the persistence of SARS-CoV-2 nucleic acids in blood and BM at least 45 days
in a patient with a medical history of mantle-cell lymphoma. In contrast, in this
report, we highlighted the presence of infected cells in BM by labelling of lymphoplasmacytic
cells by an anti-SARS-CoV-2 serum.
Moreover, as Hersby et al., we described nonspecific reactive T lymphocytes in the
BM biopsy. Other hematological cell morphological changes such as pronounced granulocytic
reaction with immaturity, dysmorphism, apoptotic-degenerative morphology and circulating
atypical reactive lymphocytes have been largely described in the subsequent phases
of COVID-199-11 and particularly in the early phase of symptom aggravation.
Conclusion:
To our knowledge, we report here the first evidence of SARS-CoV-2 infected cells and
neutralizing antibodies in BM samples of a patient suffering from MW despite negative
RT-PCR results. This case confirms that patients with compromised immunity or underlying
hematological malignancies have an elevated risk of severe and/or atypical forms of
SARS-CoV-2 infection and highlights the importance of BM investigations in case of
severe and persistent pancytopenia, even if repeated SARS-CoV-2 RT-PCR are negative.
Uncited reference
9., 10., 11..