Biallelic MGMT loss in a case of IDH-wild-type adult glioblastoma: a case for concurrent epigenomic and molecular karyotype testing

To the Editor, Molecular genetic investigation has become established practice for many solid tumours, including low and high grade gliomas.1WHO Classification of Tumours Editorial BoardCentral Nervous System Tumours. WHO Classification or Tumours Series.5th ed., Vol. 6. International Agency for Research on Cancer, Lyon2021https://tumourclassification.iarc.who.int/chapters/45Google Scholar Both acquired somatic DNA sequence and copy number variants are gaining increasing importance to further refine the histological diagnosis by identifying the underlying genetic features that have important diagnostic, prognostic and therapeutic implications. This includes identifying epigenetic changes such as altered methylation of the gene MGMT [O(6)-methylguanine-DNA methyltransferase] in glioblastoma, with its status well documented in the literature as an independent favourable prognostic marker and indicator of increased responsiveness to alkylating chemotherapy agents (e.g. temozolomide).2Hegi M.E. Diserens A.C. Gorlia T. et al.MGMT gene silencing and benefit from temozolomide in glioblastoma.N Engl J Med. 2005; 352: 997-1003Crossref PubMed Scopus (5432) Google Scholar,3Wick W. Platten M. Meisner C. et al.NOA-08 Study Group of Neuro-oncology Working Group (NOA) of German Cancer Society. Temozolomide chemotherapy alone versus radiotherapy alone for malignant astrocytoma in the elderly: the NOA-08 randomised, phase 3 trial.Lancet Oncol. 2012; 13: 707-715Abstract Full Text Full Text PDF PubMed Scopus (835) Google Scholar This has led to MGMT promoter methylation status driving treatment decision making in cases of newly diagnosed glioblastoma and those aged 60 years and over.5Stupp R. Hegi M.E. Mason W.P. et al.European organisation for research and treatment of cancer brain tumour and radiation oncology groups; national cancer institute of Canada clinical trials group. Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial.Lancet Oncol. 2009; 10: 459-466Abstract Full Text Full Text PDF PubMed Scopus (5677) Google Scholar Treatment standard for glioblastoma is currently surgical excision, followed by radiotherapy and alkylating chemotherapy. In older patients, the presence of MGMT promoter methylation has resulted in monotherapy with alkylating chemotherapy (temozolomide), rather than dual chemotherapy and radiotherapy, or radiotherapy alone in unmethylated tumours. MGMT encodes a DNA damage repair enzyme which reverses mutagenic alkylation of the O6-guanine in DNA.4Brandes A.A. Franceschi E. Tosoni A. et al.Temozolomide concomitant and adjuvant to radiotherapy in elderly patients with glioblastoma: correlation with MGMT promoter methylation status.Cancer. 2009; 115: 3512-3518Crossref PubMed Scopus (184) Google Scholar Temozolomide, an alkylator, induces DNA crosslinking and causes single- and double-strand DNA breaks, resulting in cell cycle arrest, apoptosis and tumour cell death. Hypermethylation of its 5′ CpG island promoter region results in MGMT gene silencing. Paradoxically, a MGMT hypermethylated tumour can accumulate cancer promoting mutations over time but also has enhanced sensitivity to alkylating agents as MGMT based DNA damage repair is inhibited. Therefore, epigenetic silencing of MGMT increases the effectiveness of temozolomide.5Stupp R. Hegi M.E. Mason W.P. et al.European organisation for research and treatment of cancer brain tumour and radiation oncology groups; national cancer institute of Canada clinical trials group. Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial.Lancet Oncol. 2009; 10: 459-466Abstract Full Text Full Text PDF PubMed Scopus (5677) Google Scholar MGMT promoter methylation is the main pathway by which MGMT gene expression is reduced in glioblastoma. The mainstay of determining MGMT methylation status is by standard bisulfite pyrosequencing, which has been described as the gold standard for correlating methylation with overall patient survival and progression free survival in published major clinical trials.6Philteos J. Karmur B.S. Mansouri A. MGMT testing in glioblastomas: pitfalls and opportunities.Am J Clin Oncol. 2019; 42: 117-122Crossref PubMed Scopus (11) Google Scholar However, there are alternate pathways through which reduction of MGMT gene expression can occur. Herein, we describe a rare case of glioblastoma with biallelic loss of MGMT, and describe the divergent results obtained by traditional pyrosequencing and cytogenomic methods. Single nucleotide polymorphism (SNP) chromosome microarray (CMA) is an important tool to assess chromosomal copy number abnormality in both low grade and high grade gliomas. In glioblastoma, hallmark cytogenetic features involve co-occurring chromosome 7 gain/chromosome 10/10q loss (including PTEN), CDKN2A/2B biallelic loss, and EGFR amplification (without chromosome 7 gain). Loss or copy neutral loss of heterozygosity (cnLOH) of chromosome 17p (with TP53 loss-of-function somatic variants) is also important.6Philteos J. Karmur B.S. Mansouri A. MGMT testing in glioblastomas: pitfalls and opportunities.Am J Clin Oncol. 2019; 42: 117-122Crossref PubMed Scopus (11) Google Scholar Of interest, MGMT, located within chromosome 10q, can also be lost but single copy loss is not usually considered prognostic.7Richard S. Tachon G. Milin S. Wager M. Karayan-Tapon L. Dual MGMT inactivation by promoter hypermethylation and loss of the long arm of chromosome 10 in glioblastoma.Cancer Med. 2020; 9: 6344-6353Crossref PubMed Scopus (3) Google Scholar The recent World Health Organization (WHO) Classification of Tumours of the Central Nervous System1WHO Classification of Tumours Editorial BoardCentral Nervous System Tumours. WHO Classification or Tumours Series.5th ed., Vol. 6. International Agency for Research on Cancer, Lyon2021https://tumourclassification.iarc.who.int/chapters/45Google Scholar now integrates the recommendations of the previously published cIMPACT-NOW guidelines, whereby molecular events must be integrated with demographic factors (patient age, tumour location) and histopathological findings to determine tumour diagnosis and grade.1WHO Classification of Tumours Editorial BoardCentral Nervous System Tumours. WHO Classification or Tumours Series.5th ed., Vol. 6. International Agency for Research on Cancer, Lyon2021https://tumourclassification.iarc.who.int/chapters/45Google Scholar,8Louis D.N. Perry A. Wesseling P. et al.The 2021 WHO Classification of Tumours of the Central Nervous System: a summary.Neuro Oncol. 2021; 23: 1231-1251Crossref PubMed Scopus (2279) Google Scholar Therefore, understanding the molecular information gained by utilising molecular testing is imperative to arriving at an ‘integrated diagnosis’, as encouraged by the WHO. We present a case of a 70-year-old male with new irregularly enhancing lesions of the high left paramedian and parietal lobe with distant non-contiguous lesions in the left parietal occipital junction, left corpus callosum splenium and the left major forceps. Histology and immunohistochemical findings showed an IDH-wild-type glioblastoma. Fresh tissue was sent for chromosome microarray testing at time of specimen receipt, as per protocol in our laboratory for all gliomas (a diagnosis of glioblastoma usually results in cancellation of array testing, but this was not cancelled in time). Subsequent MGMT promoter methylation testing was performed on formalin fixed, paraffin embedded tissue (FFPE) after histological diagnosis. Molecular karyotyping by SNP-CMA: DNA was obtained using the Wizard Genomic DNA purification kit (Promega, USA) with modification involving overnight 56°C digestion with protease (Sigma, USA), followed by 37°C RNAse treatment. The Infinium 850K CytoSNP Beadchip v1.2 microarray (Illumina, USA) was analysed using NxClinical v6.2 (BioDiscovery, USA) and genome reference GRCh37, according to the manufacturer's instructions. Copy number abnormality (CNA) loss or gain was called when >50 kb and probe Log R values were below –0.2 and above 0.1, respectively. cnLOH was called when >5 Mb. Tumour load (%) was estimated from the B-allele frequency (BAF).9Conlin L.K. Theil B.D. Bonnemann C.G. Mechanisms of mosaicism, chimerism and uniparental disomy identified by single nucleotide polymorphism array analysis.Hum Mol Genet. 2010; 19: 1263-1275Crossref PubMed Scopus (330) Google Scholar Chromothripsis was defined as alternating CNAs in a chromosome arising from ≥10 breakpoints. Pyrosequencing of MGMT promoter region: DNA was extracted from FFPE tissue using the Qiagen QIAmp DNA FFPE Tissue Kit as per manufacturer's protocol (Qiagen, Germany), after adequacy of tumour cellularity was assessed by a pathologist. Sodium bisulfite modification of the tumour DNA was then performed. Quantitative methylation measurement was undertaken with the Qiagen Therascreen MGMT Pyro Kit, on the Qiagen PyroMark Q24 system. Data from four CpG island sites in exon 1 of the MGMT promoter region were analysed using PyroMark Q24 Software. Both external methylated and unmethylated controls were run simultaneously with expected results. Results of cytogenetics and pyrosequencing: SNP-CMA showed a near-tetraploid (4n) tumour and complex molecular karyotype at an estimated 70–80% tumour load (Fig. 1A). The more significant abnormalities included tetrasomy 7 (including EGFR), CDKN2A/CDKN2B biallelic loss due to overlapping deletions within chromosome 9p, 17p cnLOH (including TP53) as part of chromosome 17 complex structural changes, and chromosome 10 chromothripsis. The latter resulted in MGMT biallelic loss due to complete loss of bands 10q26.2 to 10q26.3 (Fig.1B,C). PTEN was retained. MGMT promoter methylation status by pyrosequencing reported the tumour to be unmethylated (Fig. 1D). As per the SNP array result, the tumour cells no longer harboured the MGMT gene or its promoter region due to biallelic loss. Therefore, the MGMT promoter region assessed by pyrosequencing reflected the background proportion of germline (normal) cells with an intact MGMT promoter region, within the sample submitted for pyrosequencing. Interrogation of publicly available cancer genomic databases: Genomic datasets pertaining to glioblastoma (Columbia10Zhao J. Chen A.X. Gartrell R.D. et al.Immune and genomic correlates of response to anti-PD-1 immunotherapy in glioblastoma.Nat Med. 2019; 25: 462-469Crossref PubMed Scopus (429) Google Scholar and TCGA11Brennan C.W. Verhaak R.G. McKenna A. et al.Research Network: The somatic genomic landscape of glioblastoma.Cell. 2013; 155: 462-477Abstract Full Text Full Text PDF PubMed Scopus (3162) Google Scholar) were identified via cBioPortal for Cancer Genomics (https://www.cbioportal.org/).12Cerami E. Gao J. Dogrusoz U. et al.The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data.Cancer Discov. 2012; 2: 401-404Crossref PubMed Scopus (9801) Google Scholar Six hundred IDH-wild-type glioblastoma samples were identified. Data including single nucleotide variants (IDH1/2 status), copy number variants and methylation data were collected and analysed.10Zhao J. Chen A.X. Gartrell R.D. et al.Immune and genomic correlates of response to anti-PD-1 immunotherapy in glioblastoma.Nat Med. 2019; 25: 462-469Crossref PubMed Scopus (429) Google Scholar, 11Brennan C.W. Verhaak R.G. McKenna A. et al.Research Network: The somatic genomic landscape of glioblastoma.Cell. 2013; 155: 462-477Abstract Full Text Full Text PDF PubMed Scopus (3162) Google Scholar, 12Cerami E. Gao J. Dogrusoz U. et al.The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data.Cancer Discov. 2012; 2: 401-404Crossref PubMed Scopus (9801) Google Scholar Three of 600 cases (0.5%) demonstrated biallelic (homozygous) deletion of MGMT. MGMT promoter status for all three cases was unmethylated, much like our case. In neuropathology, as with other areas which require molecular features for histological diagnosis, triage of tissue, molecular testing techniques and integration of molecular findings are important in providing standard of care for patients. Clinicians and scientists within the neuropathology sphere are now required to be versed in a range of techniques available in order to elicit clinically relevant and personalised pathological information from tumours. SNP-CMA is an important diagnostic tool in many cancers, including glioma. It has the advantage that it can detect CNA (loss, gain, amplification) and cnLOH; the latter frequently harbour activating/inactivating gene mutations. It also allows assessment of tumour ploidy, tumour load (purity) and genomic content containing cancer-related genes within identified chromosomal aberrations relevant for the tumour type under investigation. However, it cannot detect balanced structural rearrangements or DNA sequence variants and is limited in detection of CNAs when <20 kb in size or present in <20% subclonal fraction/tumour load. SNP-CMA performed in most clinical diagnostic laboratories cannot detect DNA methylation, however methylation microarrays are available in the research setting. In our case, taken together with the pyrosequencing results, we theorise that this tumour would behave like a methylated tumour due to biallelic loss of MGMT repair genes. Examination of publicly available cancer genomic databases revealed three cases with the same histological and molecular phenomena; however, a review of the literature failed to find other reports describing biallelic loss of chromosome bands containing MGMT, or even 10q in glioblastoma. Therefore, to our knowledge, this is the first describing such a case. This case illustrates the advantages and limitations of both techniques, the outcome of which have implications for clinical management and prognosis. This tumour could be expected to respond as a methylated tumour with a better response to temozolomide. Unfortunately, the patient died prior to initiation of therapy; correlation with clinical management and response would have been informative in this case. This case also raises issues with specific genetic testing: genome wide molecular cytogenetic karyotyping (e.g., for detection of chromosome 7 gain/10 loss), versus sequencing technologies (e.g., for MGMT promoter methylation, IDH1/2 status). There are practical deliberations to consider. SNP-CMA works best on fresh tissue, so there are limitations for specimen handling and transportation. There is also a higher cost associated with this test in Australia, in terms of specialised processing, laboratory expertise and monetary cost. Pyrosequencing is performed on FFPE tissue meaning this technique can be embedded into current pathology workflow, similar to next generation sequencing. There is also an associated lower cost compared to cytogenetics. Our case demonstrates that any molecular test has both advantages and disadvantages, and anatomical pathologists are best placed to balance the tissue requirements necessary for diagnosis. There is a growing recognition of a need to test for epigenetic changes as well as genetic changes in gliomas, and technologies such as methylation SNP microarrays, may find a more routine place in tumour molecular pathology. The case illustrates biallelic loss of MGMT in glioblastoma, a novel finding in literature with potential management ramifications, and has demonstrated the need for complementary molecular testing in glioma with regards to tumour prognostication and management determination. The authors state that there are no conflicts of interest to disclose.