Severe and Prolonged Myelosuppression during Concomitant Temozolomide and Radiotherapy Treatment in a Patient with Glioblastoma Multiforme

Aims: We describe the case of a patient with glioblastoma (GBM) who developed severe and prolonged myelosuppression during concomitant daily temozolomide (TMZ) and radiotherapy (RT) Case Study treatment. Analysis of polymorphisms in genes correlated with TMZ-induced myelotoxicity was also performed. Presentation of the Case: A 67–year-old man with diagnosis of GBM undergoing concomitant RT-TMZ treatment developed severe and prolonged pancytopenia that led to discontinuation of TMZ and required frequent platelet and red cells transfusions. Analysis of single nucleotide polymorphisms (SNPs) in the genes NAD(P)H dehydrogenase, quinone 1 (NQO1) and glutathione S-transferase pi 1 (GSTP1) was carried out. Both SNPs were found to be wild-type. Discussion: TMZ is an oral alkylating agent used for the treatment of glioblastoma. TMZ is usually considered well tolerated and safe, with nausea and mild myelosuppression being the most common side effects. However, severe haematologic adverse events have been also reported. Recently, there has been growing interest in gene polymorphisms that might be associated with an increased risk of hematologic toxicity. Conclusion: Myelosuppression is a side effect that can occur relatively early during concomitant TMZ treatment and can negatively impact on patient’s quality of life. Further studies are warranted to find out a correlation between genetic factors and the occurrence of severe hematologic toxicity.


INTRODUCTION
Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor in adults. The standard treatment for newly diagnosed glioblastoma consists of maximum safe surgical resection followed by radiotherapy (RT) with concomitant and adjuvant chemotherapy with Temozolomide (TMZ) [1]. The use of TMZ, an oral alkylating agent, in association with RT can improve overall survival of patients with GBM [1][2]. TMZ is relatively safe and generally welltolerated.
We describe the case of a patient with GBM who developed severe and prolonged myelosuppression during concomitant daily TMZ and radiotherapy treatment. Analysis of polymorphisms in genes correlated with TMZinduced myelotoxicity was also performed.

PRESENTATION OF THE CASE
A 67-year-old man with a history of arterial hypertension was admitted to the emergency department with paresthesias of the left upper extremity.Magnetic resonance imaging of the brain showed an enhancing solid mass (20 mm) in the right parietal lobe and diffuse signal hyperintensity on T2-weighted images in the right hemisphere suggestive of gliomatosis cerebri (Fig. 1). The patient subsequently underwent a right temporal craniotomy and subtotal excision of the mass. The histopathological findings of the tumor were consistent with the diagnosis of GBM. Molecular analysis revealed methylation of the O 6 -methylguanine-DNA-methyltransferase (MGMT) gene promoter in the tumor sample.
The patient was put on antiepileptic medication with levetiracetam and referred to us for adjuvant treatment. He began treatment with RT and concomitant daily TMZ at a dose of 140 mg/die (75 mg/m 2 ). The planned RT dose was of 59.4 Gy to be given in 33 fractions of 1.8 Gy. Prior to treatment the patient had a white blood cell (WBC) and platelet (PLT) count of 8.8 x 10 3 /uL and 197 x 10 3 /uL, respectively. He did not receive co-Trimoxazole prophylaxis. During treatment, complete blood count (CBC) was carried out regularly once a week. He tolerated the combined therapy quite well, being mild fatigue the main side effect. After 21 of 33 planned fractions of radiation therapy and 30 of 47 days of TMZ, a platelet count of 11 x 10 3 /uL was found. TMZ and RT were immediately stopped. One week later, when the platelet count dropped to 8 x 10 3 /uL and multiple petechiae appeared on the trunk and arms, he was hospitalized in the medical oncology department. During hospitalization he became pancytopenic and required repeated platelet and red cell transfusions. He was treated with growth factors,

b) a)
represented but without dysplasia. Several plasma cells with reactive features were observed in the marrow interstitium. Cytogenetic analysis showed a normal karyotype. Analysis of single nucleotide polymorphisms (SNPs) in the genes NAD(P)H dehydrogenase, quinone 1 (NQO1; rs1800566) and glutathione Stransferase pi 1 (GSTP1; rs1695) was carried out as previously described by Sylvester et al. [16]. Both SNPs were found to be wild-type (Table 2). No further treatment was administered until 20 months from GBM diagnosis when, following disease progression, the patient underwent a second course of RT (37.5 Gy in 15 fractions of 2.5 Gy) without TMZ. MRI of the brain performed one month after the end of RT showed further tumor progression and the patient died two years after the initial diagnosis of GBM. The values of the last CBC were: WBC: 3.6 x 10 3 /uL; PLT: 130 x 10 3 /uL; Hb: 9.3 g/dl.

DISCUSSION
TMZ is an oral alkylating agent with activity against brain tumors. TMZ is usually considered well tolerated and has a good toxicity profile. The cytotoxic effect of TMZ is caused by the generation of O 6 -methylguanine DNA adducts; when the enzymes of the mismatch repair system attempt to repair this alteration, the mechanism of apoptosis is activated. Myelosuppression is the most common haematologic adverse event of TMZ but it is a non-cumulative and reversible effect, and bone marrow recovery usually occurs within 28 days [17]. However, severe myelosuppression has been also reported.  [18] reviewed all the TMZ-associated haematologic adverse events reported in the FDA Med-Watch database between 1997 and 2008, and they identified 228 cases of pancytopenia. Death occurred in 56 patients because of pancytopenia or its complications. The median duration of TMZ treatment was less than 3 weeks. Most of the cases were diagnosed within a week from the end of combined RT-TMZ treatment and, in 59 patients, during concomitant TMZ treatment, as in our case.
The association between genetic alterations in genes involved in DNA repair and drug metabolism and the risk of severe myelosuppression has been addressed in several studies [12][13][14][15]. Armstrong et al. [13] observed that the presence of the A allele of NQO1 and of the G allele of GSTP1 decreased the risk of toxicity. Reduced levels of NQO1 could lead to reduced activation of TMZ, while the relationship between GSTP1 and myelotoxicity is not explained. Moreover, the presence of the G allele of MGMT in peripheral blood cells resulted in a significant increase in risk of toxicity, as a result of lower DNA repair and increased DNA damage. In our case, it was possible to perform only analysis of NQO1 and GSTP1 polymorphisms but not of MGMT. Both SNPs were found to be wild-type and no conclusions can be drawn from these findings. Data from a greater number of patients could help in treatment decision. However, currently genetic analysis is not routinely performed at our institution.

CONCLUSION
In conclusion, our case shows that severe and prolonged myelosuppression is a side effect that can occur relatively early during concomitant TMZ treatment and can negatively impact on patient's quality of life. Physicians should be aware of this potential long-term complication and should carefully evaluate hematopoietic function during TMZ treatment in order to eventually detect early bone marrow failure. Further studies are warranted to find out a correlation between genetic factors and the occurrence of severe hematologic toxicity.

CONSENT
Written consent for publication of case details was obtained from the patient's next of kin.

ETHICAL APPROVAL
Not applicable.