case_images: PMC4549912_01
case_images: Clinical history, therapeutic interventions and TP53 sequencing results. a-d Immunohistochemistry slides showing Burkitt lymphoma medium-sized cells (Giemsa stain) expressing CD20 (b) and CD10 (c) (x400); c-MYC positivity in the inset (c). Peritoneal fluid collected at the moment of initial diagnosis (d), showing monomorphic BL cells with frequent mitotic figures. e FISH analysis of cells from peritoneal fluid using the Whole Chromosome Painting (WCP) probes of chromosomes 8 and 22, respectively pseudo-colored in red and green. The results showed the occurrence of the recurrent t(8;22)(q24;q11) translocation. f-i Clinical course of the patient depicted over a 4 months period with time points of CT scans performed at initial diagnosis (f), after a first chemotherapy cycle (g), and at disease progression (h), and different therapeutic interventions (i). j, k
  TP53 deep targeted sequencing study of cells from the bulky mass and peritoneal fluid, showing the presence of R282W mutation in the peritoneal (ascitic) fluid cells but not in the bulky mass. l Sanger sequencing analysis confirming the presence of a heterozygous R282W mutation in the cells from peritoneal fluid, and lack of mutation in the bulky mass.
case_images: 40164_2015_19_Fig1_HTML.jpg
case_images: PMC4549912_01_40164_2015_19_Fig1_HTML.jpg
case_images: Fig1
case_images: Surgical biopsy of the bulky mass (left ovary), cytology of the malignant cells from ascitic fluid, and immunophenotype (CD20+, CD19+, CD10+, BCL6+, CD38+, c-MYC) led to the diagnosis of BL (Fig. 1a-d). Fluorescence in situ hybridization (FISH) on malignant cells from both bulky mass and ascitic fluid showed a t(8;22)(q24;q11) translocation involving the MYC oncogene and the lambda light chain locus (IGL) (Fig. 1e). Initial treatment consisted in 5 days of debulking cyclophosphamide (200 mg/m2/die) followed by 1 CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone) cycle, which was complicated by severe tumor lysis syndrome, and bowel perforation requiring surgical intervention (Fig. 1f-i). A computed tomography (CT) scan performed after the first cycle showed marked reduction of the bulky lesions, with persistence of the abdominal effusion (Fig. 1g). After recovering from surgery, she received two additional Rituximab-CHOP-14 cycles but a CT scan performed right after showed marked disease progression (Fig. 1h). TP53 wild type, whereas lymphoma cells from the abdominal effusion harbored an heterozygous R282W mutation (Fig. 1j, k), which resulted in a 844C>T aminoacidic change, known to negatively affect p53 function and being associated to shorter survival in different cancer models (IARC database http://p53.iarc.fr/TP53GeneVariations.aspx). These findings were confirmed by Sanger sequencing (Fig. 1l). CT scan despite multiple repeated paracenteses (Fig. 1g), indicating a similar chemoresistant behavior also in vivo. Cell viability assays were performed by using WST-1 reagent (Roche).
case_images: Functional ex vivo studies showing doxorubicin resistance and sensitivity to DDR inhibition in TP53 mutant cells from peritoneal effusion. a WST-1 viability assay of primary ascitic fluid BL cells and KM-H2 cells treated with DMSO and doxorubicin 500 nM for 24 h. The percentage of viable cells after treatment in each cell line was normalized to its own untreated (DMSO) control. b Western blot analysis of BL TP53 mutant primary cells and TP53 wild type HL-derived KM-H2 cells showing p21 induction in KM-H2 cells after doxorubicin (doxo) treatment (500 nM for 6 and 24 h). c Western blot analysis of BL TP53 mutant primary cells and TP53 wild type HL-derived KM-H2 cells showing relative overexpression of pCHK1 S345 and pH2AX S139 in primary BL TP53 mutant BL cells, compared to TP53 wild type KM-H2 cells. d-i Immunocytochemistry for p-CHK1 S345, p-H2AX S139, in cultured primary cells from peritoneal fluid (d, e), the bulky mass (f, g), and KM-H2 cells (g, h) confirming western blot findings (x400). j WST-1 viability assay of primary BL cells from ascitic fluid and KM-H2 cells treated with DMSO and PF-0477736 250 nM for 24 h. The percentage of viable cells after treatment in each cell line was normalized to its own untreated (DMSO) control. k Western blot assay of TP53 mutant primary BL cells from ascitic fluid and TP53 wild type HL-derived KM-H2 cells showing pH2AX S139 induction in primary BL cells after PF-0477736 treatment (250 nM for 24 h).
case_images: 40164_2015_19_Fig2_HTML.jpg
case_images: PMC4549912_01_40164_2015_19_Fig2_HTML.jpg
case_images: Fig2
case_images: TP53 mutant primary BL cells from the ascitic fluid with either DMSO 0.01 % or doxorubicin 500 nM (Fig. 2a). The TP53 wild type Hodgkin lymphoma cell line KM-H2 was used as a control. Consistent with these data, doxorubicin induced p21 expression in KM-H2 cells but not in primary BL cells (Fig. 2b). Since we recently reported constitutive DDR activation and high efficacy of CHK inhibitors in TP53 mutant aggressive B-cell lymphomas (DLBCL and BL), we evaluated the expression levels of genomic instability and DDR activation markers in peritoneal fluid cells and in the bulky mass by western blotting (Fig. 2c) and immunohistochemistry (Fig. 2d-i) confirming that peritoneal fluid cells demonstrated constitutive gammaH2AX (H2AX S139) and p-CHK1 S345 expression (Fig. 2c-e). TP53 WT bulky mass (Fig. 2f, g), suggesting that the acquisition of genomic instability and of a DDR+ phenotype was an intrinsic feature of this neoplasm that preceded the development of the TP53 mutation. The TP53 WT KM-H2 cells, used as negative control of DDR activation, were negative for both p-CHK1 and gammaH2AX (Fig. 2h, i). Next, in order to assess whether the TP53 mutant subclone was sensitive to DDR inhibition, we treated primary ascitic fluid BL cells (DDR+) and KM-H2 cells (DDR-) with the CHK inhibitor PF-0477736, finding that peritoneal fluid cells were exquisitely sensitive to CHK inhibition whereas KM-H2 cells were resistant (Fig. 2j). Following CHK inhibition, gammaH2AX levels increased in primary peritoneal fluid cells, indicating that in these cells the blockade of DDR leads to accumulation of endogenous DNA damage (Fig. 2k).