PMC10154527_01
The bone marrow biopsy of the first disease recurrence showed the tumor cells composed of small lymphoid cells, plasmacytoid lymphocytes and plasma cells infiltrated the bone marrow (HEX400). Flow cytometric analysis of bone marrow specimen shows: CD20+, CD19+, CD5-, CD10-, Kappa+, Lambda-, CD200-, CD43-, CD38+, CD23p+, CD79B+, FMC7p+ and CD103-.
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F1
The bone marrow flow cytometry results were as follows: CD19+, CD20+, CD79B+, CD38+, CD138-, CD5-, CD10-, CD103-, Kappa+, Lambda-, CD200-, CD43-, FMC7p+, and CD23p+ (Figure 1).
Pathological features of inguinal lymph node biopsy specimen before BTK inhibitor treatment. The structure of lymph node was destroyed and replaced by infiltration of small lymphocytes, amyloid material (black arrow) was present among tumor cells (HEX200) (A). The inserted lower right picture was the magnification of tumor cells and amyloid material (HEX400). Immunohistochemical staining demonstrated the tumor cells were positive for CD20 (B), CD38 (C), IgM (D) and was negative for P53 (E) and showed a low proliferation rate by Ki-67 staining (F) (Envision X200).
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F2
Immunohistochemical (IHC) analysis of the left inguinal lymph node biopsy showed that the tumor cells were positive for CD20, CD19, CD38, CD138, and IgM and negative for CD5, CD10, kappa, lambda and p53, which was consistent with the diagnosis of WM, with Ki-67 = 15% (Figure 2).
Treatment after disease recurrence and the time and type of obtained specimens over the disease course (A). The red arrow shows chest wall mass on chest CT scan (B).
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F3
His hemoglobin level returned to 126 g/L, and his IgM level dropped to 26.4 g/L, with shrinkage of the spleen and retroperitoneal lymph nodes after treatment, thus achieving partial remission in December 2020 (Figure 3). CT showed a new mass in the left chest wall (8.5 x 3.1 cm) (Figure 3). Chest wall tissue biopsy showed that the tumor cells were medium to large in size with round or oval nuclei, and small nucleoli could be seen in some tumor cells.
Pathological features of high grade transfomation in the biopsy from the chest wall after BTK inhibitor treatment. The tumor cells were medium to large in size with round or oval nuclei and small nucleoli could be seen in some tumor cells (HEX400) (A). Immunohistochemical staining demonstrated the tumor cells was positive for CD20 (B) but negative for CD38 (C), diffusely and strongly positive for P53 (D, inserted upper right picture was high power view, X400) and showed a much higher proliferation rate by Ki-67 staining (E) (Envision X200). FISH test showed P53 gene lost (white arrow, the red signal was P53 gene, the green signal was CEP17) (F).
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F4
Fluorescence in situ hybridization test showed that the P53 gene was lost (Figure 4).
Mutated genes and gene mutation types of inguinal lymph node (Tissue 1), transformed chest wall tissue (Tissue 2), peripheral blood, and ascites (A). Relationships of mutant genes in different specimens (B). Variant Allele Frequency of mutated genes (C).
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F5
In addition to these mutations, we detected genetic mutations (BTK  T316A , PLCG2  D334G,R337W ) associated with resistance to BTK inhibitors in ctDNA derived from ascites (Figure 5).