PMC5572605_01
Tosillar tissue: H&E sections showing large to medium sized lymphoid cells with round to oval vesicular nuclei (a). The malignant cells are positive for CD20 immunostain (b). Immunohistochemistry (40x): Retrospectively performed on original diagnostic tonsillar tissue. Negative for TDT (c) and positive for MYC immunostains (d).
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PMC5572605_02
PMC5572605_03
Tosillar tissue: H&E sections showing large to medium sized lymphoid cells with round to oval vesicular nuclei (a). The malignant cells are positive for CD20 immunostain (b). Immunohistochemistry (40x): Retrospectively performed on original diagnostic tonsillar tissue. Negative for TDT (c) and positive for MYC immunostains (d).
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CSF cytospin preparation smear (50x) shows infiltration with many malignant lymphoid cells, some of which are larger with irregular nuclear contours, less clumped nuclear chromatin, and prominent nucleoli with few mitotic figures (black arrow) (Wright stain, x500).
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BM aspirate smear shows numerous abnormal medium to large-sized lymphoid cells. The cells showed slightly irregular nuclear contours, dispersed nuclear chromatin, and basophilic cytoplasm (Wright stain, 1,000x) (a). BMB biopsy (H&E 50x): interstitial infiltration with malignant lymphoid cells (b).
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Restaging BM showed infiltration by abnormal medium to large-sized lymphoid cells (~40%) with slightly irregular nuclear contours, dispersed nuclear chromatin, few nucleoli, and basophilic cytoplasm (Figure 3(a)). BM biopsy was hypocellular (20-30%) with interstitial infiltration by many malignant lymphoid cells (Figure 3(b)). By immunohistochemistry (IHC), the lymphoid cells were positive for PAX-5, BCL-2 (more than 50%), cMYC, and
Immunohistochemistry performed on bone marrow biopsy (first relapse). The abnormal lymphoid cells are positive for PAX-5, BCL-2, cMYC, and TDT (a-d). KI-67 shows high proliferative index (e). The neoplastic cells are negative for CD20 (f). PAX-5 50x (g), cMYC 40x, and Tdt 40x (i).
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Tdt immunostains (Figures 4(a)-4(d)) with high mitotic index reflected by strong KI-67 positivity (Figure 4(e)). The neoplastic cells were negative for CD20 (Figure 4(f)), CD5, BCL6, CD23, MUM-1, and Cyclin D1.
Flow cytometry on bone marrow aspirate: a population of monotypic B-cells, expressing CD45, CD10, CD38, and surface kappa light chain restriction. The monotypic B-cells are negative for CD 5 with downregulation of pan B markers (partial CD79 (dim), loss of CD19 and CD20).
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Flow cytometry (FCM) of the BM aspirate (Figure 5) revealed a population of kappa-restricted monotypic B-cells (~15%), expressing CD45, CD10, and CD38 (bright) and showed surface kappa light chain restriction. Moreover, the malignant population showed partial dim expression of Tdt (Figure 5(g)).
CEP8/MYC/IGH probes on tonsils (a) and bone marrow biopsy (b). The FISH analysis was performed using probes from Vysis; MYC-Orange spectrum, CEP8-Aqua spectrum, and IGH-Green spectrum. White arrows indicate the fusion signal a result of MYC/IGH translocation.
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Conventional cytogenetics studies revealed a complex karyotype: (48,XY,+X,t(8;14)(q24;q32),+12,13,der(14)t(1;14)(q21;p11.2),+der(?)t(1;?)(q21;?)[8]/50,idem,+der(?)t(1;?),+mar[2]/46, XY[22]) and subsequent FISH analysis confirmed the MYC/IGH, t(8;14) rearrangement in 41% of cells analyzed (Figure 6(b)). Screening of the original diagnostic tonsillar tissue for double hit gene rearrangement IGH/BCL2, MYC/IGH by FISH analysis was also performed at this stage and revealed positivity for MYC/IGH (Figure 6(a)) and negativity for BCL-2 gene rearrangement.
PMC5572605_04
PMC5572605_05
Peripheral smear at time of second relapse: Neoplastic cells show more pronounced nuclear irregularities with variable cytoplasmic vacuolation (a). Wright stain, 1,000x magnification. Flow cytometry on peripheral blood showed malignant cells with CD5 acquisition (b).
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Unfortunately, the patient relapsed again within few weeks where circulating malignant cells ~10% were detected in peripheral smear (Figure 7(a)), for which flow cytometry was performed and revealed a population of monotypic B-cells ~10% expressing CD45, CD10, CD20, and CD38, with kappa light chain restriction, loss of CD19, and acquisition of CD5 expression (Figure 7(b)).