Therapeutic implications of intratumor heterogeneity for TP53 mutational status in Burkitt lymphoma
© Derenzini et al. 2015
Received: 3 July 2015
Accepted: 4 August 2015
Published: 27 August 2015
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.
KeywordsBurkitt lymphoma Intra-tumor heterogeneity Genomic instability CHK1 γ-H2AX MYC
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 . 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 . 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 [3–5]. 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 . 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 . 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.
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 [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 , 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.
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.
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.
DNA damage response
diffuse large B cell lymphoma
next generation sequencing
fluorescence in situ hybridization
immunoglobulin lambda light chain locus
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.
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.
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