Open Access

Therapeutic implications of intratumor heterogeneity for TP53 mutational status in Burkitt lymphoma

  • Enrico Derenzini1Email author,
  • Ilaria Iacobucci1,
  • Claudio Agostinelli2,
  • Enrica Imbrogno1,
  • Clelia Tiziana Storlazzi3,
  • Alberto L`Abbate3,
  • Beatrice Casadei1,
  • Anna Ferrari1,
  • Andrea Ghelli Luserna Di Rora`1,
  • Giovanni Martinelli1,
  • Stefano Pileri2 and
  • Pier Luigi Zinzani1
Experimental Hematology & Oncology20154:24

https://doi.org/10.1186/s40164-015-0019-9

Received: 3 July 2015

Accepted: 4 August 2015

Published: 27 August 2015

Abstract

Therapeutic implications of intra-tumor heterogeneity are still undefined. In this study we report a genetic and functional analysis aimed at defining the mechanisms of chemoresistance in a 43-year old woman affected by stage IVB Burkitt lymphoma with bulky abdominal masses and peritoneal effusion. The patient, despite a transient initial response to chemotherapy with reduction of the bulky masses, rapidly progressed and died of her disease. Targeted TP53 sequencing found that the bulky mass was wild-type whereas peritoneal fluid cells harbored a R282W mutation. Functional studies on TP53 mutant cells demonstrated an impaired p53-mediated response, resistance to ex vivo doxorubicin administration, overexpression of DNA damage response (DDR) activation markers and high sensitivity to pharmacologic DDR inhibition. These findings suggest that intra-tumor heterogeneity for TP53 mutational status may occur in MYC-driven cancers, and that DDR inhibitors could be effective in targeting hidden TP53 mutant clones in tumors characterized by genomic instability and prone to intra-tumor heterogeneity.

Keywords

Burkitt lymphoma Intra-tumor heterogeneity Genomic instability CHK1 γ-H2AX MYC

Background

Genomic instability, defined as the tendency to acquire DNA damage determining accumulation of genomic alterations over time, is a hallmark of cancer conferring evolutionary advantages, and resulting in resistance to chemotherapy and increased metastatic potential [1]. A common mechanism determining genomic instability in tumors is oncogene-induced replication stress, leading to DNA damage accumulation during the S phase of the cell cycle [2]. Our group and others recently reported that MYC-driven cancers such as Burkitt lymphoma (BL) and Diffuse large B-cell lymphoma (DLBCL) overexpress active components of the DNA damage response pathway (DDR) such as checkpoint kinases (CHK1/2), in order to cope with the high levels of replication stress deriving from MYC overexpression, and are sensitive to pharmacologic DDR inhibition [35]. BL is characterized by a high level of MYC expression due to the occurrence of chromosomal translocations which are hallmarks of the disease, and G1/S checkpoint dysfunction with frequent TP53 mutations (30 % of cases) [6, 7]. TP53 mutations drive chemoresistance in many different cancers including aggressive B-cell lymphomas [8, 9], and cooperate with MYC by preventing its intrinsic proapoptotic effects and by further increasing genomic instability [10]. Intra-tumor heterogeneity, intended as the occurrence of genomic diversities within the same tumor over space and time, is intimately related to genomic instability, and has been recently unraveled by next generation sequencing (NGS) studies [11]. Nevertheless, its clinical significance and therapeutic implications in aggressive B-cell lymphomas are yet to be elucidated.

In the current study we report a genetic and functional analysis aimed at defining the mechanisms of chemoresistance in a 43-year old woman affected by stage IVB Burkitt lymphoma with bulky abdominal masses and peritoneal effusion. The patient, despite a transient initial response to chemotherapy with reduction of the bulky masses, rapidly progressed and died of her disease. Targeted TP53 sequencing found that the bulky mass was wild-type (WT) whereas peritoneal fluid cells harbored a R282W mutation, depicting a paradigmatic example of intra-tumor heterogeneity for the TP53 mutational status at disease onset in BL. Functional studies on the TP53 mutant clone confirmed an impaired p53-mediated response and resistance to ex vivo doxorubicin administration. Finally, we demonstrated that these cells were characterized by overexpression of markers of genomic instability and DDR pathway activation, and were sensitive to pharmacologic inhibition of CHK kinases.

Case presentation

The patient was hospitalized in August 2011 in critical conditions with two bulky abdominal masses originating from both ovaries, a massive abdominal effusion and small bowel obstruction. 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). Detailed description of Immunohistochemistry and FISH studies is available in Additional file 1. The principal comorbidity was a severe bipolar disorder and anorexia nervosa that was still active at the time of disease onset, so that the patient was severely underweight (body mass index <17 kg/m2) and deemed initially unfit for intensive chemotherapy. 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). At this point the patient underwent therapy intensification according to the B-NHL-2002 regimen [12] but was unresponsive and ultimately died of rapidly progressing disease. Since recent studies confirmed that TP53 mutations occur in about 30 % of BL cases [6, 7], in order to investigate the mechanisms underlying resistance to standard and intensive chemotherapy in this patient, we performed TP53 targeted DNA sequencing of the tumor tissue available from the initial biopsy (left ovary), of tumor cells initially collected from the ascitic fluid, and of matched normal saliva using the 454 GS Junior platform (Roche diagnostics) (Additional file 1). The patient gave informed consent for the use of surplus tissue in research, and the protocol was approved by the Institutional Review Board (Study n 12/2009/U/Tess, protocol 148/2009). We found that the tumor tissue from the initial bulky mass was entirely 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 ) [13, 14]. These findings were confirmed by Sanger sequencing (Fig. 1l). Notably, both samples were chemonaive being collected before the start of chemotherapy. In order to define the impact of the R282W mutation on response to therapy in this specific case, we treated cultured 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. According to the TP53 status, primary mutant BL cells were resistant to doxorubicin, whereas KM-H2 cells were sensitive. Consistent with these data, doxorubicin induced p21 expression in KM-H2 cells but not in primary BL cells (Fig. 2b). Interestingly, as shown in Fig. 1, while the TP53 WT bulky masses rapidly responded to chemotherapy, the malignant TP53 mutant ascites was still present at the time of second 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). Detailed description of western blot protocols, antibodies and reagents is available in Additional file 1.
Fig. 1

Clinical history, therapeutic interventions and TP53 sequencing results. ad Immunohistochemistry slides showing Burkitt lymphoma medium-sized cells (Giemsa stain) expressing CD20 (b) and CD10 (c) (×400); 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. fi 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.

Fig. 2

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. di 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 (×400). 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).

Since we recently reported constitutive DDR activation and high efficacy of CHK inhibitors in TP53 mutant aggressive B-cell lymphomas (DLBCL and BL) [5], we evaluated the expression levels of genomic instability and DDR activation markers [5, 15] 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 γH2AX (H2AX S139) and p-CHK1 S345 expression (Fig. 2c–e). Notably, although to a lesser extent, we observed positivity for these markers also in the 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 [5], were negative for both p-CHK1 and γH2AX (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, γH2AX 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). These findings are consistent with a model in which constitutive activation of CHK kinases cooperates with MYC and is crucial to prevent untolerable levels of genomic instability deriving from MYC-induced replication stress and G1/S checkpoint dysfunction.

Conclusions

These observations could have broad implications in clinical practice, suggesting that multiple tumor samples from different regions should be evaluated before tailoring therapies based on genome sequencing results. Although no definitive conclusions can be drawn from single case studies, this report strongly corroborates previous findings from our group and others showing efficacy of CHK inhibitors in MYC-driven and TP53 mutant lymphoma models, suggesting that: (1) the occurrence of clonal heterogeneity at disease onset for mutations driving chemoresistance, such as those in TP53, should be taken into account in aggressive MYC-driven lymphomas; (2) CHK inhibitors could be effective in targeting hidden TP53 mutant clones in tumors characterized by genomic instability and prone to intra-tumor heterogeneity. In conclusion, these data indicate that multiregion sequencing will be a crucial step for the development of precision therapy in aggressive B-cell lymphomas and confirm that inhibition of CHK kinases could be a suitable therapeutic strategy for MYC-driven tumors, which should be evaluated in future clinical trials.

Consent

Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.

Abbreviations

DDR: 

DNA damage response

BL: 

Burkitt lymphoma

DLBCL: 

diffuse large B cell lymphoma

CHK: 

checkpoint kinase

NGS: 

next generation sequencing

WT: 

wild type

FISH: 

fluorescence in situ hybridization

IGL: 

immunoglobulin lambda light chain locus

DMSO: 

dimethyl sulfoxide

Declarations

Authors’ contributions

ED and II designed the study and wrote the manuscript; EI performed experiments; CA performed immunohistochemical studies and revised the manuscript; CTS and AL performed FISH studies and critically revised the manuscript; AGLDR performed experiments; BC collected clinical data and revised the manuscript; AF and II performed TP53 sequencing studies; GM, SP and PLZ revised the manuscript critically, providing important intellectual contribution. All authors read and approved the final manuscript.

Acknowledgements

This study was partially funded by BolognAIL ONLUS and AIRC (CTS), European LeukemiaNet, AIL, AIRC (GM), Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, Ateneo RFO Grants, Programma di Ricerca Regione—Università 2010–2012, NGS-PTL project, Grant agreement number 306242, funded by the EC Seventh Framework Programme theme FP7-HEALTH-2012-INNOVATION-1.

Compliance with ethical guidelines

Competing interests The authors declare that they have no competing interests.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Department of Experimental, Diagnostic and Specialty Medicine, DIMES, Institute of Hematology and Medical Oncology L.A. Seragnoli, University of Bologna
(2)
Hematopathology Unit, Department of Experimental, Diagnostic and Specialty Medicine, DIMES, University of Bologna
(3)
Department of Biology, University of Bari “Aldo Moro”

References

  1. Negrini S, Gorgoulis VG, Halazonetis TD. Genomic instability—an evolving hallmark of cancer. Nat Rev Mol Cell Biol. 2010;11:220–8.View ArticlePubMedGoogle Scholar
  2. Halazonetis TD, Gorgoulis VG, Bartek J. An oncogene-induced DNA damage model for cancer development. Science. 2008;319:1352–5.View ArticlePubMedGoogle Scholar
  3. Ferrao PT, Bukczynska EP, Johnstone RW, McArthur GA. Efficacy of CHK inhibitors as single agents in MYC-driven lymphoma cells. Oncogene. 2012;31:1661–72.View ArticlePubMedGoogle Scholar
  4. Höglund A, Nilsson LM, Muralidharan SV, Hasvold LA, Merta P, Rudelius M, et al. Therapeutic implications for the induced levels of Chk1 in Myc-expressing cancer cells. Clin Cancer Res. 2011;17:7067–79.View ArticlePubMedGoogle Scholar
  5. Derenzini E, Agostinelli C, Imbrogno E, Iacobucci I, Casadei B, Brighenti E, et al. Constitutive activation of the DNA damage response pathway as a novel therapeutic target in diffuse large B-cell lymphoma. Oncotarget. 2015;6:6553–69.PubMed CentralPubMedGoogle Scholar
  6. Love C, Sun Z, Jima D, Li G, Zhang J, Miles R, et al. The genetic landscape of mutations in Burkitt lymphoma. Nat Genet. 2012;44:1321–5.PubMed CentralView ArticlePubMedGoogle Scholar
  7. Schmitz R, Young RM, Ceribelli M, Jhavar S, Xiao W, Zhang M, et al. Burkitt lymphoma pathogenesis and therapeutic targets from structural and functional genomics. Nature. 2012;490:116–20.PubMed CentralView ArticlePubMedGoogle Scholar
  8. Kaneko H, Sugita K, Kiyokawa N, Iizuka K, Takada K, Saito M, et al. Lack of CD54 expression and mutation of p53 gene relate to the prognosis of childhood Burkitt’s lymphoma. Leuk Lymphoma. 1996;21:449–55.View ArticlePubMedGoogle Scholar
  9. Xu-Monette ZY, Wu L, Visco C, Tai YC, Tzankov A, Liu WM, et al. Mutational profile and prognostic significance of TP53 in diffuse large B-cell lymphoma patients treated with R-CHOP: report from an International DLBCL Rituximab-CHOP Consortium Program Study. Blood. 2012;120:3986–96.PubMed CentralView ArticlePubMedGoogle Scholar
  10. Rowh MA, DeMicco A, Horowitz JE, Yin B, Yang-Iott KS, Fusello AM, et al. Tp53 deletion in B lineage cells predisposes mice to lymphomas with oncogenic translocations. Oncogene. 2011;30:4757–64.View ArticlePubMedGoogle Scholar
  11. Gerlinger M, Rowan AJ, Horswell S, Larkin J, Endesfelder D, Gronroos E, et al. Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. N Engl J Med. 2012;366:883–92.View ArticlePubMedGoogle Scholar
  12. Intermesoli T, Rambaldi A, Rossi G, Delaini F, Romani C, Pogliani EM, et al. High cure rates in Burkitt lymphoma and leukemia: a Northern Italy Leukemia Group study of the German short intensive rituximab-chemotherapy program. Haematologica. 2013;98:1718–25.PubMed CentralView ArticlePubMedGoogle Scholar
  13. Xu J, Wang J, Hu Y, Qian J, Xu B, Chen H, et al. Unequal prognostic potentials of p53 gain-of-function mutations in human cancers associate with drug-metabolizing activity. Cell Death Dis. 2014;5:e1108.PubMed CentralView ArticlePubMedGoogle Scholar
  14. Joerger AC, Ang HC, Fersht AR. Structural basis for understanding oncogenic p53 mutations and designing rescue drugs. Proc Natl Acad Sci USA. 2006;103:15056–61.PubMed CentralView ArticlePubMedGoogle Scholar
  15. Mah LJ, El-Osta A, Karagiannis TC. gammaH2AX: a sensitive molecular marker of DNA damage and repair. Leukemia. 2010;24:679–86.View ArticlePubMedGoogle Scholar

Copyright

© Derenzini et al. 2015

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