MSH2, MSH6, MLH1, and PMS2 immunohistochemistry as highly sensitive screening method for DNA mismatch repair deficiency syndromes in pediatric high-grade glioma

Abstract

Pediatric high-grade glioma (pedHGG) can occur as first manifestation of cancer predisposition syndromes resulting from pathogenic germline variants in the DNA mismatch repair (MMR) genes MSH2, MSH6, MLH1, and PMS2. The aim of this study was to establish a generalized screening for Lynch syndrome and constitutional MMR deficiency (CMMRD) in pedHGG patients, as the detection of MMR deficiencies (MMRD) may enable the upfront therapeutic use of checkpoint inhibitors and identification of variant carriers in the patients' families. We prospectively enrolled 155 centrally reviewed primary pedHGG patients for MMR-immunohistochemistry (IHC) as part of the HIT-HGG-2013 trial protocol. MMR-IHC results were subsequently compared to independently collected germline sequencing data (whole exome sequencing or pan-cancer DNA panel next-generation sequencing) available in the HIT-HGG-2013, INFORM, and MNP2.0 trials. MMR-IHC could be successfully performed in 127/155 tumor tissues. The screening identified all present cases with Lynch syndrome or CMMRD (5.5%). In addition, MMR-IHC also detected cases with exclusive somatic MMR gene alterations (2.3%), including MSH2 hypermethylation as an alternative epigenetic silencing mechanism. Most of the identified pedHGG MMRD patients had no family history of MMRD, and thus, they represented index patients in their families. Cases with regular protein expression in MMR-IHC never showed evidence for MMRD in DNA sequencing. In conclusion, MMR-IHC presents a cost-effective, relatively widely available, and fast screening method for germline MMRD in pedHGG with high sensitivity (100%) and specificity (96%). Given the relatively high prevalence of previously undetected MMRD cases among pedHGG patients, we strongly recommend incorporating MMR-IHC into routine diagnostics.

Keywords: Constitutional mismatch repair deficiency; Immunohistochemistry; Lynch syndrome; Pediatric high-grade glioma.

Fig. 1

Study design and composition of the cohort. a Flowchart of study design. b Composition of the cohort displayed by grading (inner circle) and tumor type (outer circle) of all included patients (n = 155; left) and all patients with loss of MMR protein expression in MMR-IHC (n = 10; right). c Heat map of MMR-IHC results of control cases (left; green) and all cases suspicious of MMRD in the screening group (right; blue). The control group includes tumor samples of nine patients with initial pedHGG diagnosis outside the screening period and confirmed germline MMRD. This group was assembled for antibody testing and IHC protocol optimization. Case IDs are displayed on the y-axis. A IDHmut °3/4 astrocytoma, IDH-mutant (CNS WHO grade 3/4), CTRL control, DAG °3/4 diffuse astrocytic glioma (CNS WHO grade 3/4), DHG H3 G34mut °4 diffuse hemispheric glioma, H3 G34-mutant (CNS WHO grade 4), DMG H3 K27alt °4 diffuse midline glioma, H3 K27-altered (CNS WHO grade 4), MMR(D) mismatch repair (deficiency), IDH isocitrate dehydrogenase, IHC immunohistochemistry, infant-type HG infant-type hemispheric glioma, NOS not otherwise specified, pedHGG pediatric high-grade glioma, ped-type HGG H3wt IDHwt °4 pediatric-type high-grade glioma H3-wildtype and IDH-wildtype (CNS WHO grade 4)

Fig. 2

Immunohistochemical screening for mismatch repair deficiency in pediatric high-grade glioma. a–d Two control cases (a, b) and two HIT-HGG-2013 cases (c, d) with lost MMR protein expression are representatively displayed. a, b Both control cases are diagnosed with CMMRD. a Case CTRL 6 shows a complete loss of PMS2 expression, whereas the expression of MSH2, MSH6, and MLH1 is preserved. The underlying mechanism is a homozygous PMS2 alteration. b In case CTRL 1, the expression of MSH2 and MSH6 is retained. MLH1 and PMS2 protein cannot be detected due to a homozygous MLH1 alteration. c In case ID 90, a patient with confirmed LS, a heterozygous MSH2 alteration causes a loss of expression of MSH2 and MSH6 in the tumor cells but not in the endothelial cells. MLH1 and PMS2 IHC are retained. d Patient ID 29 resembles CTRL 1 (in b) but here the loss of MLH1 and PMS2 is restricted to the tumor cells; endothelial cells are positive for all four MMR proteins. The underlying pathomechanism is a heterozygous MLH1 alteration in the context of LS. e Variances in the appearance of giant cell morphology in four different H&E stained tissue samples. Case IDs in order from left to right: 26 (InDel in MSH6; CMMRD), 29 (SNV in MLH1, LS), 84 (InDel in MSH2; LS) and 137 (hypermethylation of MSH2; no germline affection). Cases are selected due to their different underlying genetic alterations and germline affections. Scale bars equate 50 µm. CMMRD constitutional mismatch repair deficiency, CTRL control, MMRD mismatch repair deficiency, H&E hematoxylin and eosin, IHC immunohistochemistry, InDel insertion/deletion, LS Lynch syndrome, SNV single-nucleotide variant

Fig. 3

Oncoplot of cases with mismatch repair deficiency. A detailed case-related listing of clinical and histo-morphological characteristics and summary of most frequent molecular genetic findings. The latter includes results from tumor and germline analyses. Somatic alterations in genes other than MSH2, MSH6, MLH1, and PMS2 are displayed when detected in three or more MMRD cases. A IDHmut °4 astrocytoma, IDH-mutant (CNS WHO grade 4), ALT alternative lengthening of telomeres, CMMRD constitutional mismatch repair deficiency, DHG H3 G34mut °4 diffuse hemispheric glioma, H3 G34-mutant (CNS WHO grade 4), IDH isocitrate dehydrogenase, IHC immunohistochemistry, InDel insertion/deletion, MS microsatellite, NA not analyzed, ped-type HGG H3wt IDHwt °4 pediatric-type high-grade glioma, H3-wildtype and IDH-wildtype (CNS WHO grade 4), Seq sequencing, SNV single-nucleotide variant, TMB tumor mutational burden, TSO 500 TruSight Oncology 500, WES whole exome sequencing, wt wildtype

Fig. 4

Hypermethylation of MSH2 as alternative epigenetic silencing. a Diagram of MSH2 gene pyrosequencing in tumor tissue of case ID 137. The first eight analyzed CpG dinucleotide positions are displayed. The average methylation of all eight positions was 63.6%. As bisulfite conversion control, the DNA nucleobase cytosine at position 47 is highlighted in green. b Immunohistochemistry reveals a loss of MSH2 and MSH6 protein expression in the tumor cells. Intratumoral vessels show preserved MSH2 and MSH6 protein expression

Fig. 5

Adapted diagnostic workflow for routine MMRD screening in pedHGG. We would like to recommend performing MMR-IHC in all cases of pedHGG. In cases with unavailable MMR-IHC, or MMRD-associated tumor cell morphology (severe pleomorphism and/or giant cell features), molecular testing should be performed—if feasible—to identify any underlying MMR gene alteration with the need for subsequent MMR gene germline testing. Based on the present findings, MMR gene germline testing may be promptly initiated in cases of MMR-IHC loss; however, prior molecular analysis of tumor tissue for MMR gene alterations can be performed at the investigator’s discretion. Ideally, a combination of molecular assays covering all potential types of alterations including hypermethylation is implemented. Germline testing is recommended for all MMRD patients. MMR(D) mismatch repair (deficiency), IHC immunohistochemistry