Jean-François Lesesve1, Maxime Cravat2, Laura Boulangé2, Marie-Thérèse Rubio3 and Marcelo De Carvalho2.
Services d’Hématologie Biologique1 et Clinique3, d’Immunologie2, CHRU Brabois, Nancy, France.
JF Lesesve. Service d’Hématologie Biologique, CHRU Nancy Brabois, 54511 Vandœuvre. Tel: 0623120423. Email: email@example.com
Published: March 1, 2022
Received: November 9, 2021
Accepted: February 11, 2022
Mediterr J Hematol Infect Dis 2022, 14(1): e2022024 DOI 10.4084/MJHID.2022.024
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To the editor
infusion of T cells with a chimeric antigenic receptor (CAR-T cells)
represents a recent therapeutic innovation in managing hemopathies. As
an indication for large B-cell lymphoma, CAR-T cells are used for
patients in relapse after several conventional treatment lines. Nowadays, clinical trials propose this therapeutic approach as second-line therapy (ZUMA-7 clinical trial) and even as first-line therapy in high-risk patients (ZUMA-12 trial).
In practice, the collection of patients' T cells is performed by
leukapheresis: these T cells will then be genetically modified using a
viral vector to make them express a chimeric receptor able to recognize
a surface antigen expressed by tumor cells. After being transduced, the CAR-T cells are reinjected by intravenous infusion.
The morphologic and immunophenotypic description of CAR-T cells
observed in residual cells of CAR-T cells infusion bag has just been
described by Galli et al. in this journal. Here we
confirm their findings in the peripheral blood (PB) and report about
our experience of CAR-T cells automated and microscopic detection and
A 54-year-old woman was diagnosed in October
2017 with stage III large cell B-cell lymphoma. She was first treated
with chemotherapy until complete remission but relapsed in December
2018, and the second line of chemotherapy was undertaken to allow the
achievement of a new complete remission. Then, an autologous transplant
was performed in April 2019 to intensify the treatment. Unfortunately,
a second relapse occurred in May 2020, and a CAR-T-cells therapy
project was validated in July. The anti-CD19 CAR-T cells (Axicabtagene
Ciloleucel, Yescarta, Kite Gilead) were infused in September after
usual conditioning by cyclophosphamide and fludarabine. Blood counts
(XN 9000 automated counter, Sysmex, Japan) after CAR-T cells infusion
revealed expected pancytopenia due to the depletion conditioning
regimen. However, the follow-up of the white differential fluorescence
(WDF) channel from the automated counter during the first four days was
On the fifth day, dots were observed in the area corresponding to high fluorescence cells, flagged as "atypical lymphocytes" (Figure 1).
Seven days after infusion, the automated counter detected unusual cells
in another area (white dots) where abnormal lymphocytes and blasts are
usually observed, leading to rejection of the automated leucocytes
differential analysis. The WDF channel becomes without peculiarity
again from the tenth day, and no more dots were observed in
the window above the normal lymphocytes. In the blood smear, several
unusually large cells were observed (day 5 post-infusion of CAR-T cells
in Figure 2).
||Figure 1. WDF channel (Sysmex-XN counter). Dots corresponding to lymphocytes are pink-colored (days 5 to 11).
||Figure 2. Peripheral blood atypical lymphocytes observed after CAR-T cells infusion (day 5; May-Grunwald-Giemsa stain; x1000).
Their sizes were about 15 to 20 μm.
These cells were composed of atypical lymphocytes, some with a
nucleole, others with immature blastic-like appearance (corresponding
to undifferentiated cells at J7), and often with a lobulated nucleus.
The cytoplasm of these cells was basophil and sometimes with peripheral
strengthening. Very frequently, small vacuoles and small azure
granulations were observed, both at the limit of visibility with an x50
microscopic average objective. All these lymphoid cells were observed
in the first few days when no monocytes and only a few neutrophils were
observed, stressing their potential relationship with CAR-T. As
mentioned by Galli et al., a possible relapse of the malignant disorder
might have been suspected in the absence of context knowledge.
From day seven post-infusion, immuno-phenotyping of whole blood by flow
cytometry demonstrated the existence of a strong CD45 lymphocyte
population, composed of CD3+ T lymphocytes, with a CD4+ (56%) and CD8+
(36%) distribution, a CD3-CD16/56+ NK component (8%). There was a
complete lack of CD19 expression by lymphoid cells (Figure 3). Repeated flow cytometry analysis confirms initial data, and a full decrease of CAR-T cells was confirmed within 60 days (Table 1 and Figure 4).
||Figure 3. Lymphocytes immunophenotypic assessment at day 5.
||Table 1. Immuno-phenotypic follow-up (day 5 to 91).
||Figure 4. CAR-T cells repartition (day 21) and CAR-T cells among total T cells (follow-up, days 10 to 60).
seemed likely to predict the potential presence of atypical lymphoid
cells by observing the WDF channel of the Sysmex XN counter. We
confirmed in the PB the heterogeneous cytomorphology described by Galli
et al. in the leftovers, including few large granular lymphocytes but
notable medium-sized basophilic lymphocytes, large promonocytic-like
cells, and giant blast-like cells. Large basophilic
cells with convoluted nuclei were very easily detected under the
microscope during the expansion of CAR-T (focused on Figure 2).
of patient PB after CAR-T cells show a mixture of CD4+ and CD8+ T
cells, sometimes NK cells. These cells correspond, among others, to the
reinjected cells, observed a few days after infusion and linked to the
well-documented expansion peak of CAR-T cells generally between 5 to 10
days post-infusion. Our case report allowed us to document that (i) day
5 is the day of detectable CAR-T in whole blood, (ii) day 7 is the peak
of expansion, (iii) the total time exposure to CAR-T lasted more than
21 days, (iv) CD8+ cells decrease with time, and (v) that B-cell
aplasia persisted for more than 90 days. Pharmacokinetics revealed that
the circulation of CAR-T cells in peripheral blood was linked to a
better response to the treatment.
Furthermore, according to the
ZUMA-2 study of CAR-T cells Tecartus (Brexucabtagene Autoleucel, Kite
Gilead), the number of anti-CD19 CAR-T cells in the blood was
positively correlated with an objective response (complete or partial
response). Concerning prolonged B-cell aplasia, an
indirect marker of anti-CD19 CAR-T cells potency, it highlights that
immune monitoring using flow cytometry is mandatory. CAR-T cells
currently promise hopeful options for curing malignant diseases
(nowadays mainly in onco-hematology field) and represent a new
challenge for assessment in hematology laboratories from initial to
long-term patient follow-up.
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