Cytological examination of peripheral blood cell block to diagnose small cell variant ALK‐positive anaplastic large cell lymphoma

In small cell variant ALK-positive anaplastic large cell lymphoma (SC-ALCL), large hallmark cells are few and the preponderance are small- to medium-sized tumour cells. The cell block method is advantageous in SC-ALCL with small numbers of CD30-positive hallmark cells, in order to evaluate cell morphology and marker expression simultaneously. Accurate diagnosis of ALK-positive anaplastic large cell lymphoma (ALK+ ALCL) requires detection of CD30-positive hallmark cells. In small cell variant ALCL (SC-ALCL), large hallmark cells are few with the preponderance being small- to medium-sized tumour cells. The cell block method is advantageous in SC-ALCL with small numbers of CD30-positive hallmark cells in order to evaluate cell morphology and marker expression simultaneously.


| INTRODUC TI ON
Anaplastic lymphoma kinase-positive anaplastic large cell lymphoma (ALK+ ALCL) is characterised by large lymphocyte-like cells with abundant cytoplasm and variable-shaped nuclei, often resembling horseshoes or kidneys.These cells have a specific genetic translocation involving the ALK gene and are positive for ALK and CD30 by immunohistochemistry. 1 In children, ALK+ ALCL is the most common subtype of T-cell lymphoma, accounting for 10%-30% of lymphomas.ALK+ ALCL exhibits a wide range of morphological patterns, with the common variant comprising 60% of cases, followed by lymphohistiocytic (10%), small-cell (5%-10%), and Hodgkin-like (3%) variants. 2 small cell variant ALCL (SC-ALCL), large hallmark cells are few with the preponderance being small-to medium-sized tumour cells.
Tumour cells in peripheral blood indicate the leukaemic phase, which is associated with poor prognosis.Among the morphological variants, patients with SC-ALCL often show the leukaemic phase, thus accurate and prompt diagnosis is essential for effective treatment. 3curate diagnosis requires detection of CD30-positive hallmark cells.5][6] Flow cytometry is also commonly used to detect CD30-positive hallmark cells, but it can be challenging in SC-ALCL due to the small number of hallmark cells.This method carries a greater risk of false negative results.
We report a case of SC-ALCL in the leukaemic phase wherein immunocytochemistry using a cell block (CB) of peripheral blood proved useful for diagnosis.The CB method is advantageous in cases with small numbers of CD30-positive hallmark cells to evaluate cell morphology and marker expression simultaneously.

| C A S E REP ORT
A 10-year-old boy was admitted to our hospital with symptoms including a 1 week history of fever, swelling in the posterior region of his left ear, dyspnoea, and weight loss.Contrast-enhanced computed tomography scan revealed left cervical lymph node swelling, and fluorodeoxyglucose positron emission tomography (FDG-PET) showed (18)F-FDG uptake in the left cervical and subclavicular regions (Figure 1A,B).The differential blood count showed a high presence of tumour cells in a peripheral blood smear.Since neither needle biopsy of the lymph nodes nor bone marrow biopsy provided sufficient tumour cells for analysis, cytological diagnosis using peripheral blood was attempted.
Blood obtained from a peripheral vein was centrifuged to separate its components.Samples of the buffy coat fraction that contains most of the white blood cells and platelets was smeared onto two glass slides and stained separately with Papanicolaou and Giemsa stains.Cytology was performed on the smear preparations.In addition, CB specimens were prepared from the peripheral blood sample using the centrifuge tube method (Figure 1C).The CB was sliced into 3 μm thick sections using a microtome and underwent haematoxylin and eosin (HE) staining.Immunocytochemical staining was also conducted on the CB sections, using the same conditions as for tissue specimens analysed by the BenchMark ULTRA system.

| DISCUSS ION
The leukaemic phase of ALK+ ALCL is uncommon.However, since it can take a highly aggressive clinical course, prompt and accurate diagnosis is critical for effective treatment.We encountered a paediatric case of SC-ALCL where the disease had progressed to a leukaemic phase.In this case we could not obtain sufficient biopsy tissue, so we had to rely solely on blood samples for a definitive diagnosis.immunocytochemistry is essential for a definitive diagnosis of ALCL to differentiate it from the other types of lymphomas. 1,2 the present case, we prepared CB specimens of peripheral blood.The process of creating them allowed us to perform immunocytochemical staining, which ultimately proved essential in making a definitive diagnosis.By CB examination, we were able to diagnose SC-ALCL at an early stage and promptly initiate the treatment protocol most beneficial to the patient.This approach allowed us to use peripheral blood as a valuable specimen at an early stage of the disease, which is especially important in cases of aggressive lymphomas like ALCL.

Cytological analysis of peripheral blood
To the best of our knowledge, only one case report has been published describing the diagnosis of SC-ALCL aided by CB prepared from peripheral blood, but that case also included a cutaneous nodule biopsy. 7Ours is the first report of a case to be diagnosed using only peripheral blood.Immunostaining with a CB of peripheral blood enabled confirmation of ALK-and CD30-positivity in large cells, which are difficult to detect with flow cytometry.In the diagnosis of the leukaemic phase of SC-ALCL, CB of peripheral blood is a valuable diagnostic tool.
The major variants of ALCL are the common type, small cell, lymphohistiocytic, and Hodgkin-like. 1,2All variants are positive for CD30 by immunohistochemical staining, but the positivity rate for ALK differs by variant.Small cell variants have been reported to be positive for ALK in all cases. 4ALCL is characterised by different immunostaining patterns of ALK depending on the type of partner gene fusion with the ALK gene. 1,8Therefore, the location of ALK expression by immunostaining should be carefully evaluated.When the partner gene of ALK is nucleophosmin 1 (NPM1), ALK is positive in both the nucleus (especially in the nucleolus) and the cytoplasm.
Currently, only NPM-ALK is known to exhibit nuclear positivity in ALK immunohistochemistry. 3,8 The ALK-positive signal was positive in both the nucleus and the cytoplasm in the present case, indicating that the partner gene was NPM1.Subsequently, genetic mutation analysis by real-time polymerase chain reaction using peripheral blood revealed an ALK-NPM1 fusion gene.
Papanicolaou and Giemsa staining revealed a mixture of two cell types.One type was numerous small cells with lobulated nuclei, This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.© 2023 The Authors.Cytopathology published by John Wiley & Sons Ltd.
(arrows, Figure2A,B) and the other was a few large cells with vacuoles and basophilic cytoplasm (arrowheads, Figure2A,B).The large cells had a distinctive feature called "hallmark cells" characterised by horseshoe or kidney-shaped nuclei.HE staining of the CB specimens revealed slightly hyperchromatic nuclei.The cytological findings were almost similar to those observed in the cytology smear as shown (Figure2C).Immunocytochemical staining of the CB specimens showed positive for CD30 (Figure2D).ALK was strongly positive in the cytoplasm and nucleus, especially in the nucleolus (Figure2E).Positive staining for CD30 and ALK was prominent in the large cells, but sparse in the small cells.CD3, CD8, TIA-1, Granzyme B, and Perforin were positive in the small, atypical cells, while CD4, CD5, and B cell markers were negative.Fluorescence in situ hybridisation showed numerous ALK split signals in the atypical cells as shown in Figure 2F.Similar ALK split signals were observed in the cytology smear specimens (Figure 2G).The final diagnosis based on cytological sampling was SC-ALCL in the leukaemic phase.The patient successfully underwent allogeneic bone marrow transplantation while in first complete remission, 2 months after starting alectinib.
from the patient revealed the presence of both small and large atypical cells with characteristic nuclear shapes, including lobulated, horseshoe-, and kidney-shaped, which are typical characteristics of ALCL.Differential diagnoses for the present case of SC-ALCL in a child include several types of lymphomas, such as Hodgkin, diffuse large B-cell, Burkitt, precursor B-cell lymphoblastic, precursor T-cell lymphoblastic, and peripheral T-cell lymphoma.Among these, Hodgkin and peripheral T-cell lymphoma are particularly important to consider in patients with the two-cell pattern of large and small atypical cells.In cytological samples, the presence of hallmark cells, which are large with characteristic nuclear shapes, is a useful finding for diagnosing ALCL.However, F I G U R E 1 (A) Contrast-enhanced computed tomography: Swollen left cervical lymph nodes.(B) Fluorodeoxyglucose (FDG) positron emission tomography image findings.FDG uptake visible in the left neck to subclavian region.(C) Cell block preparation method using peripheral blood.