Glioblastoma with BRAFV600E mutation and numerous metastatic foci: a case report

Introduction

Glioblastoma (GB), the most malignant astrocytic tumour, constitutes one of the biggest challenges in the oncology field. Its infiltrative nature and location in the brain often forbid complete surgical resection and even with continuous developments in diagnostic and therapeutic approaches, median survival rate of patients still does not exceed one year [18]. Intriguingly, GB metastases are rarely detected and if so, tumours are mostly located within the neuroaxis [5]. Extra-central nervous system (CNS) metastases (only up to 2% of cases) can be usually found in lungs, regional lymph nodes or bones [3,15,17]. Moreover, it was indicated that such tumours are characterized with different vasculature than primary focus [23]. Such rare occurrence of distant metastases may be caused by the fact that the brain is immunologically and anatomically separated by the blood-brain barrier – semipermeable membrane impeding access to the brain. Nevertheless, despite the fact that the exact mechanism of extracerebral metastasis has been poorly understood [22], this is mostly detected in patients with a longer survival rate [1] or tending to be associated with disruption of the cellular integrity during surgical procedures [12]. Currently, standard GB treatment involves the maximal feasible surgical resection followed by 60 Gy radiotherapy with concomitant and adjuvant temozolomide-based chemotherapy [26]. In case of glioblastoma patients with no effective therapeutic options available, off-label use of targeted drugs tends to be exceptionally employed, especially since particular targetable molecular alterations are detected in this tumour type. Indeed, BRAFV600E, mutation that occurs most commonly in melanomas [2], is detected in pleomorphic xanthoastrocytomas [11] and in low percentage of glioblastomas [9]. Therefore, mutation-specific inhibitors (such as vemurafenib or dabrafenib) can be possibly effective not only in melanoma brain metastases [16], but also glioblastoma tumours with this mutation [6]. Still, administration of targeted therapeutics requires the precise molecular diagnostics of the tumour.
In this paper, a case of glioblastoma patient diagnosed with primary tumour, recurrence and extra-CNS metastases is presented. The aim of the conducted analyses was to define molecular background of this tumour, corresponding with the acquisition of metastasis-prone features, and to molecularly compare the three tumour foci (primary, recurrent and metastatic).

Case presentation

Clinical summary

A 51-year-old non-smoking Caucasian male was admitted to the hospital due to headaches, memory impairment, dropping right mouth corner, psychomotor retardation and confusion. Computed tomo­graphy (CT) and magnetic resonance imaging (MRI) revealed a tumour in the right temporal lobe (Fig. 1A,B). The tumour was macroscopically completely resected.
Histopathological analysis shown highly cellular neoplasm with marked cytological atypia, high mitotic activity, focal geographic and pseudopalisading necrosis and extensive microvascular proliferations highlighted by the reticulin stain (Fig. 1C,D). Morphology, immunohistochemical and histochemical stains were consistent with the diagnosis of glioblastoma [GFAP/+/; Ki-67/+/ up to 20% of cells; p53/+/ (Fig. 1E)]. Further sequencing analysis of IDH1 gene indicated isocitrate dehydrogenase (IDH)-wild type glioblastoma, according to the latest WHO classification [14]. The patient was qualified to adjuvant radiotherapy (60 Gy/30 fractions) and temozolomide treatment (150 mg/m2/day). After 16 months, radiological follow-up revealed recurrent tumour at the base of the right temporal lobe (Fig. 2A) and possible new focus in the left parietal lobe (Fig. 2B). Histochemical and immunohistochemical analyses indicated glioblastoma with TP53 accumulation (Fig. 2C,D). The patient was under strict observation, however, after three months he was admitted to the hospital once again complaining of nonproductive cough, general weakness, night sweats and shivers. Initial diagnosis suggested pulmonary embolism, however, angio-CT revealed diffuse foci in parenchyma of both lungs (Fig. 3A). Histopathological analysis of bronchoscopically collected material confirmed glioblastoma (Fig. 3B,C). Additional tomograms revealed numerous metastatic foci located e.g. in iliacus muscle (Fig. 3D), thoracic vertebrae and soft tissues of lower limbs (data not shown). Material from primary and recurrent tumour samples as well as lung metastatic focus was then delivered to the laboratory in order to isolate DNA and conduct precise molecular analyses, with the emphasis on possible targets of experimental therapy. Unfortunately, the patient died 3.5 months following metastases detection, 90 weeks after the initial diagnosis.

Molecular analyses

All analyses concerning tumour material were approved by the Bioethical Committee of the Medical University of Lodz (Approval No. RNN/234/17/KE). QIAamp DNA Mini Kit (Qiagen) was used for DNA isolation from formalin-fixed and paraffin-embedded tissues prepared for primary tumour, recurrence and lung metastasis. Isolation was preceded by the deparaffinization step with xylene (Sigma). DNA was analysed using a wide range of molecular techniques, as described previously [24,25], what enabled to compare molecular profiles of these three tumour foci. BRAF gene sequencing was conducted with the following primer pair: 5’-AACTCTTCATAATGCTTGCTCTGAT-3’ and 5’GTAACTCAGCAGCATCTCAGGG-3’. MLPA analysis using P105 Glioma-2 and P175 Tumor Gain probemixes (MRC Holland) revealed various alterations in the copy number of glioblastoma-associated genes, e.g. EGFR amplification or CDKN2A and PTEN deletion (Fig. 4). Unequivocal identification of the pulmonary mass origin was, however, impossible based on MLPA results only. Therefore, next generation sequencing (NGS) analysis using AmpliSeq Cancer Hotspot Panel v2 (Life Technologies) was performed. This revealed non-hotspot mutations (homozygous in FLT3 and heterozygous in IDH1 (synonymous) and APC; Table I) in all analysed samples. Other hotspot mutations were detected in EGFR (R776C) and PTPN11 (E69K) in the recurrent tumour and in EGFR (W731* – nonsense mutation leading to the formation of stop codon) and TP53 (M246I) in lung metastasis. Despite the fact that TP53 mutation was detected neither in primary nor in recurrent tumour in sequencing analyses, IHC analysis for TP53 turned out to be clearly positive. It is consistent with previous report indicating that in case of gliomas, not only TP53 mutations, but also disturbed TP53 pathway (e.g. by MDM2 or MDM4 amplification as well as promoter methylation of CDKN2A or TP53) may result in abnormal TP53 expression, hence positive IHC staining [28].
Interestingly, a hotspot mutation in BRAF gene (V600E) was detected in the primary tumour (20.7%) and lung metastasis (16.2%), which clearly confirmed the origin of metastatic focus. This codon was additionally re-analysed using Sanger sequencing in an attempt to verify the status of BRAF gene in the recurrent tumour with the latter method. For this purpose, DNA isolated from SK-MEL-28 cell line (ATCC), characterized by endogenous homozygous BRAFV600E mutation, and from BJ fibroblasts (control wild-type cells; ATCC) was used to obtain dilution series with various percentage of mutated template, in order to verify the detection threshold of Sanger sequencing. Results indicated that in all analysed samples, the percentage of BRAF-mutated template was below or near Sanger detection threshold, estimated to be 10-20% of mutated template [7]. Therefore, only NGS results were considered. To be consistent with the latest WHO classification, IDH1 status was also evaluated. Both, NGS and additional Sanger sequencing analysis revealed that all analysed tumour foci were IDH-wild type. Importantly, all the remaining mutations detected by means of NGS in both primary and recurrent tumours or recurrent and metastatic foci were novel (non-hotspot).

Discussion

Glioblastoma, despite its highly aggressive nature, is rarely associated with de­tectable metastases. None­theless, with expected progress in treatment of patients and more precise diagnostic imaging methods in common practice, there will be probably more cases with detected GB metastases. Nevertheless, so far, extra-CNS glioblastoma foci still constitute an interesting research material.
Glioblastoma metastases outside the CNS frequently tend to occur late in the disease course, with patients survival rates reaching approximately two years [1,22]. Therefore, the long survival of the described patient (22.5 months) might have been associated with detection of various metastatic foci. In this case it is not clear whether the tumour location in the frontal lobe close to the frontal corner of the lateral ventricle might have facilitated tumour spread via cerebrospinal fluid. However, it may be suggested by the presence of a metastatic focus in the parietal lobe of the opposite hemisphere. Moreover, in case of the analysed patient, extra-CNS metastases, detected e.g. in lungs or kidneys, might have been caused by hematogenous spread via sphenoparietal or superior petrosal sinus, etc. [12,21]. Nevertheless, when considering all the factors that may have an impact on the pattern of recurrence, including its location (local re-growth vs. new focus), surgical resection range, possible ventricular entry, TMZ administration, long progression-free survival or GB recurrence spreading, as in the case of this patient, it still remains difficult for interpretation [10]. Although the profound molecular analysis was not necessary to confirm the same origin of the metastases and primary tumour in the reported case, such molecular comparison not only indicates possible therapeutic options but also enables better understanding of glioblastomas biology.
Molecular analyses of patient’s tumour samples revealed a hotspot mutation in the BRAF gene (BRAFV600E), which is frequently detected in melanoma [2] and constitutes a perfect target for specific small molecule inhibitors, such as vemurafenib or dabrafenib. Interestingly, various alterations in BRAF gene are found in gliomas, with BRAFV600E mutation present in up to 50% of pleomorphic xanthoastrocytoma [11] and in 2-5% of glioblastomas [9] (with more than half of cases of the epithelioid glioblastoma type). There are several reports of anticancer efficacy of BRAFV600E-targeted inhibitors in patients diagnosed with melanoma metastases to the brain, indicating that these drugs may penetrate CNS tumours [16]. There are also single, successful reports of vemurafenib administration in BRAFV600E-positive anaplastic pleomorphic xanthoastrocytoma [13] and glioblastoma [6,20]. It is not clear whether this mutation may be associated with metastatic potential. Therefore, it may be suggested to put emphasis on analyses of molecular profiles of patients with detected glioblastoma metastases.
So far, there has never been such a large group of GB patients with detected metastases to enable correlation of some molecular markers with metastasis occurrence. There was one report presenting analysis of several genetic alterations in six GB patients, suggesting that TP53 mutation may have an impact on metastases occurrence [19]. Beyond any doubt one case may not constitute a basis to confirm that BRAF mutation may be associated with longer survival rate and metastasis development. This is, however, in line with literature data indicating that glioblastoma patients harbouring BRAFV600E mutation tend to be characterized by prolonged survival, even up to 4 years following diagnosis [27], as well as by younger age [8].
In case of the analysed patient, BRAFV600E inhibitor administration as an adjuvant therapy might have possibly prolong survival. Moreover, hotspot mutations detected only in the recurrent tumour (EGFR R776C and PTPN11 E69K) might have a significant clinical impact. The former hotspot is reported to be associated with reduced sensitivity to particular EGFR TKIs, while the latter constitutes a possible target for SHP2 inhibitors, which are currently under extensive development. Undoubtedly, as early detection of molecular targets as possible is crucial in cancer management. Analyses of circulating tumour DNA, tumour microRNAs or circulating tumour cells may constitute an interesting option in detection of new, targetable molecular alterations, especially those conferring drug resistance [4].

Acknowledgments

This study was supported by the National Centre for Research and Development, EU Smart Growth Operational Programme grant No. POIR.01.02.00-00-0035/15 (NGS and Sanger sequencing analyses) and the National Science Centre Grant No. 2016/21/D/NZ3/02616 (tumour material handling and MLPA analyses).

Disclosure

The authors report no conflict of interest.

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