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							Free Neuropathology 5:32 (2024)

						
	
							Original Paper

						
	
							Adult glioblastoma with Lynch syndrome-associated mismatch repair deficiency forms a distinct high-risk molecular subgroup

						
	
							Maria-Magdalena Georgescu

						
	

							NeuroMarkers, Houston, Texas, USA


						
	
							Corresponding author:

								Maria-Magdalena Georgescu · NeuroMarkers · 6907 Academy Street, 77025 · Houston · Texas · USA

								mmgeorgescu@yahoo.com
							

						
	
							Additional resources and electronic supplementary
								material: supplementary material

						
	
							
								Submitted: 17 September 2024

								Accepted: 17 November 2024

								Copyedited by: João Gama

								Published: 10 December 2024

						
	
							https://doi.org/10.17879/freeneuropathology-2024-5892

						
	
							Keywords: Glioblastoma, MMR deficiency, Lynch syndrome,
								TP53, PIK3CA, Chromatin remodeling genes, Multinucleated giant cells, p53
								immuno-histochemistry, Immune checkpoint inhibitors, Ipilimumab, Nivolumab

						
	

							Abstract

							
								Glioblastoma is the most frequent and malignant primary brain tumor. Although the
								survival is generally dismal for glioblastoma patients, risk stratification and the
								identification of high-risk subgroups is important for prompt and aggressive management.
								The G1–G7 molecular subgroup classification based on the MAPK pathway activation has
								offered for the first time a non-redundant, all-inclusive classification of adult
								glioblastoma. Five patients from the large, 218-patient, prospective cohort showed
								germline mutations in mismatch repair (MMR) genes (Lynch syndrome) and a significantly
								worse median survival of 3.25 months post-surgery than those from the G1/EGFR and G3/NF1
								major subgroups, or from the rest of the cohort adjusted for age. These rare tumors were
								assigned to a new subgroup, G3/MMR, a G3/NF1 subgroup spin-off, as they generally show
								genomic alterations leading to RAS activation, such as NF1 and PTPN11
								mutations. An integrated clinical, histologic and molecular analysis of the G3/MMR
								tumors showed distinct characteristics as compared to other glioblastomas, including
								those with iatrogenic high tumor mutation burden (TMB), warranting a separate subgroup.
								Prior history of cancer, midline location or multifocality, presence of multinucleated
								giant cells (MGCs), positive p53 and MMR immunohistochemistry, and specific molecular
								characteristics, including high TMB, MSH2/MSH6 alterations, biallelic
								TP53 Arg mutations and co-occurring PIK3CA p.R88Q and PTEN
								alterations, alert to this high-risk G3/MMR subgroup. The MGCs and p53
								immunohistochemistry analysis in G1–G7 subgroups showed that one in 7 tumors with these
								characteristics is a G3/MMR glioblastoma. The FDA-approved first-line therapy for many
								advanced solid tumors consists of nivolumab-ipilimumab immune checkpoint inhibitors. One
								G3/MMR patient received this regimen and survived much longer than the rest, setting a
								proof-of-principle example for the treatment of these very aggressive G3/MMR
								glioblastomas.
							


						
	

							Introduction

							
								Glioblastoma is the most frequent and deadly primary brain tumor [1], assigned by the
								World Health Organization (WHO) Classification of Central Nervous System Tumors the
								highest tumor grade, WHO grade 4 [2]. Its dismal prognosis is mainly due to diffuse
								brain invasion, tumor heterogeneity, but also activation of pathways that impart
								resistance to therapy. Virtually all glioblastomas show activation of the extracellular
								signal-regulated kinase / mitogen-activated protein kinase (ERK/MAPK) and
								phosphatidyl-inositol 3-OH kinase (PI3K) canonical growth pathways [3–5]. Non-redundant
								molecular alterations in the upstream mediators of the ERK/MAPK pathway, including the
								receptor tyrosine kinases (RTKs) triggering pathway activation, allowed an all-inclusive
								molecular classification of glioblastoma into 7 subgroups [4,5]. These subgroups, termed
								after the most upstream gene showing activating RTK or MAPK effector alterations are:
								G1/EGFR, G2/FGFR3, G3/NF1, G4/RAF, G5/PDGFRA, G6/Multi-RTK, and G7/Other, the latter
								showing dominant PI3K pathway activation [4,5]. Of these, the G1/EGFR, G3/NF1 and
								G7/Other are major subgroups, containing together approximately three quarters of all
								glioblastoma cases, and showing distinct risk stratification, with overall poor
								prognosis for the G7/Other subgroup [5].
							

							
								The treatment of glioblastoma relies on surgical resection, the extent of which
								correlates to the survival benefit [6]. Subsequent radiation and temozolomide (TMZ)
								chemotherapy represent the standard adjuvant regimen for the past 20 years [7], with few
								recent trials proven beneficial for the younger glioblastoma patient population [8].
								Beside risk stratification, the aim of the G1–G7 molecular subgroup classification is to
								identify subgroups benefiting of specific targeted therapy [5]. For example, the
								G6/Multi-RTK has been analyzed in detail and shows RTK gene fusions for which addition
								of specific RTK inhibitors improves survival [5,9,10]. A very small but significant
								subgroup of glioblastomas that harbor germline mismatch repair (MMR) gene deficiency
								consistent with Lynch syndrome has been identified in a previous study [5]. This
								subgroup has been termed G3/MMR, as RAS activation appears to be dominant in these
								tumors, similarly to the G3/NF1 tumors. Lynch syndrome is a cancer predisposition
								syndrome caused by germline pathogenic alterations in the MMR genes MLH1,
								PMS2, MSH2 and MSH6, in which various types of tumors, the
								most prevalent being colorectal carcinoma (CRC), arise by inactivation of the second MMR
								allele and a subsequent high rate of repair errors during DNA replication [11]. This MMR
								deficiency is reflected in a high tumor mutation burden (TMB) that has been shown to
								benefit from immune checkpoint inhibitors [12].
							

							
								The current study focuses on the clinical, histologic and molecular characterization of
								the G3/MMR subgroup in comparison to other glioblastoma cases from the same cohort,
								including cases with high TMB due to TMZ therapy. It shows the distinct characteristics
								of G3/MMR tumors occurring in older adults, similar to most glioblastoma cases, and
								reviews few additional adult cases from the literature [13,14], delineating diagnostic
								guidelines for the identification of these rare cases. Importantly, this is the first
								study showing survival advantage in adult G3/MMR glioblastoma by addition of the dual
								nivolumab-ipilimumab regimen of immune checkpoint inhibitors to the standard adjuvant
								regimen.
							


							Methods

							
								Tumor specimens, histology and immunohistochemistry (IHC). Surgical
								specimens were obtained from patients, as previously described [15–17]. Formalin-fixed
								paraffin-embedded (FFPE) sections from glioblastoma biopsies or surgical resections were
								stained with hematoxylin and eosin (H&E) or various antibodies for protein
								expression by IHC: p53 (DO-7), p16 (E6H4), Ki-67 (30-9), MLH1 (M1), PMS2 (A16-4), MSH2
								(G219) and MSH6 (SP93) (Roche / Ventana Medical Systems Inc., Tucson, AZ), and GFAP
								(EP672Y) (Ventana / Cell Marque, Rocklin, CA), as described [5,16]. Images were acquired
								with Nikon Eclipse Ci microscope equipped with Nikon Digital Sight DS-Fi2 camera (Nikon
								Instruments Inc., Melville, NY) [4]. An integrated histologic and molecular diagnosis,
								according to the most recent WHO guidelines [2] was obtained for all cases, prior to
								classifying them in the G1–G7 molecular subgroups [4,5].
							

							
								Next generation sequencing (NGS), copy number variation (CNV), and
									transcriptomics. The DNA and RNA NGS analyses were performed from FFPE
								samples by using the xT-648-gene panel (Tempus Labs, Chicago, IL), as previously
								described [15,17,18]. For each case, the same FFPE block was used for DNA and RNA
								extraction to allow direct comparison of results. Variant and CNV assessment and
								interpretation, and the RNA expression analysis were performed as extensively described
								elsewhere [15,18].
							

							
								Assessment of mutation germline origin. Genetic testing from blood was
								performed for patient F70. For patients M62, M68 and M72, the germline origin was
								inferred by comparing the variant allele fraction (VAF) for the MMR mutations and the
								VAF for somatic heterozygous mutations (e.g. PTEN, PIK3CA,
								TP53), in parallel with CNV analysis. A clear distinction between the VAF in
								the germline versus somatic range was present in each of these cases (see Suppl.
								Table S1). When possible, this method was extended to the rest of MMR mutations from
								tumors not mapping to the G3/MMR subgroup (see Suppl. Table S2). This assessment based
								on VAF showed reliable somatic versus germline prediction on the NGS platform used in
								the cases for which genetic testing was performed in parallel [15,17–19].
							

							
								Statistical analysis. Survival Kaplan-Meyer curves were analyzed by
								Log-rank (Mantel-Cox) and Gehan-Breslow-Wilcoxon tests for statistical significance, as
								described [4,5]. Parametric unpaired t-test with Welch’s correction was performed by
								using GraphPad Prism (Version 10.2.3, GraphPad Software, La Jolla, CA). Statistical
								significance was considered for p < 0.05, and confidence intervals were 95 % for all
								tests. Data were analyzed and plotted by using Microsoft Excel (Microsoft Corp.,
								Redmond, WA), and GraphPad Prism.
							

							




							Results

							
								Dismal prognosis of G3/MMR glioblastoma patients: treatment of one patient with
									nivolumab-ipilimumab immune checkpoint inhibitors improves outcome.
							

							
								In a prospectively assembled cohort of 218 glioblastoma patients where all the tumors
								were subjected to DNA and RNA NGS, and classified in molecular subgroups according to
								the MAPK-based G1–G7 subgroup classification [4,5], five cases with germline mutations
								in MMR genes stood apart as a separate subset, G3/MMR (Fig. 1A). The G3/MMR subgroup
								represented approximately 2 % of the glioblastoma cohort, and showed distinctive
								clinical, histologic and molecular characteristics, summarized in Table 1. The G3/MMR
								patients frequently had a clinical history of prior cancers, sometimes with multiple
								different malignancies, the most commonly reported being CRC in two of the five
								patients. Skin cancer, either melanoma or basal cell carcinoma, was also relatively
								common in these patients, and lymphoma and thyroid carcinoma less common (Table 1).
							


							
								Figure 1: Location of G3/MMR molecular subgroup tumors

								

								A. Pie chart distribution (%) of 218 prospective glioblastoma cases in G1–G7 molecular subgroups. Purple arrow shows the G3/MMR subgroup. GBM, glioblastoma; EGFR↑, EGFR-amplified; EGFRm, EGFR-mutated. B. Regional distribution of the glioblastomas from the G3/MMR, as compared to the rest of the cohort (GBM). CC, corpus callosum. C. Selected MRI images showing multifocality (red arrows) and brainstem infiltration (blue arrow) in three G3/MMR cases.

							




							
										*GBM-MMR n=213 1(192)	G3/MMR n=5	M62	M68	F70	F72	F79
	Median age: years	65 1 (66)	70	62	68	70	72	79
	Sex M:F ratio	1.5 : 1	4 : 1	Male	Male	Female	Male	Male
	Race W:H ratio	53 : 1	4 : 1	White	Hispanic	White	White	White
	History of prior cancer	ND	60 %	No	No	CRC	BCC, lymphoma	Melanoma, CRC, Thyroid
	2Location	22 % midline	40 % midline	R BG	L frontal	R temporal	L BG	R frontal
	2Midline/Crosses midline	22 %	60 %	Yes	No	No	Yes	Yes
	2Multifocal	ND	60 %	Yes	Yes	No	Yes	No
	2Tumor size: cm	Variable	Large	4.9 x 4.3 x 4.2	4.3 x 4.1 x 3.1	7.7	2.8 x 1.8	7 x 5
	3Survival: months	91 (8)	3.25	2	3.25	10	1.7	4.5
	Surgery	24 % Biopsy	40 % Biopsy	Biopsy	STR	STR	Biopsy	GTR
	Adjuvant treatment	-	-	None	RT/ICPI4	RT/TMZ/ICPI	None	RT/TMZ
	MGMT promoter methylation	-	-	Negative	Positive	ND	Positive	ND
	5Histologic clusters	All 5	#3 and #5	Mix #3 + #5	#3/Ana	Mix #3 + #5	#5/Epi	Mix #3 + #5
	Multinucleated giant cells	18 %	100 %	Yes	Yes	Yes	Giant cell GBM	Yes
	MMR IHC	Retained	Loss	MSH2/MSH6	MSH6	MSH2/MSH6	ND	MSH2/MSH6
	Germline mutation	No MSH2/6	MSH2/MSH6	MSH6	MSH6	MSH2	MSH6	MSH2/MSH6
	Detected at GBM workup	NA	80 %	Yes	Yes	Prior	Yes	Yes
	TMB: mutations/MB	4.3 ± 0.1	24.3 ± 5.1	9	29.5	38.9	17.6	26.3
	MSI status	Stable	Mixed	Stable	Stable	High	ND	Stable
	6DNA mutation signature	None	MMR	MMR	MMR	MMR	MMR	MMR


							



							
									Table 1: Demographic, clinical, radiologic, histologic and molecular characteristics of G3/MMR patients.
GBM, glioblastoma; M, male; F, female; W, White / Caucasian; H, Hispanic; R, right; L, left; BG, basal ganglia; CRC, colorectal cancer; BCC, basal cell carcinoma; GTR, gross total resection; STR, subtotal resection; RT/TMZ, radiation / temozolomide adjuvant therapy; ICPI, 
												immune checkpoint inhibitors; ND, not determined; NA, not applicable.
												*GBM cohort without the G3/MMR cases.

												1Numbers in parenthesis apply to the age-corrected cohort excluding less-than-50 year-old patients.

												2Parameters determined by pre-operative MRI. Size of enhancement measurements: M62, the whole enhancing area; M68 and M72, the largest focus. 

												3Median survival is indicated for the G3/MMR subgroup and for the rest of the cohort (GBM–MMR) with or without age correction.

												4RT/ICPI for patient M68 was started 1.5 months post-surgery and stopped 3 weeks later as the tumor progressed during therapy.

												5Histologic clusters include the #3/Ana (anaplastic) and #5/Epi (epithelioid) clusters.

												6The MMR-type DNA mutation signature consists in a predominance of C:G to T:A transitions.
												


							


							
								The survival in glioblastoma depends on many factors, and these include tumor location
								and subsequent extent of surgical resection, age and comorbidities, accessibility to and
								effectiveness of adjuvant treatment [20,21]. The intrinsic aggressivity of the tumor may
								also influence survival [5,21].
							

							
								The location of the tumors varied, with a high percentage of tumors showing midline
								location and multifocality, the latter often interpreted as metastatic disease (Table 1,
								Fig. 1B–C). The two multifocal tumors located in the midline that were only biopsied
								associated with the worst survival in patients M62 and M72 (Table 1, Fig. 1C). Patient
								M79, with peripherally located tumor crossing the midline, and receiving gross total
								resection and standard radiation-TMZ adjuvant therapy, marginally survived longer.
								Patient M68 underwent subtotal resection of the larger right frontal focus (Fig. 1C),
								and had slow post-surgical recovery, during which the residual tumor focus progressed.
								The radiation and immune checkpoint inhibitor therapy with pembrolizumab was initiated
								1.5 months post-surgery and did not stop tumor growth, being aborted after 3 weeks.
							

							
							
								The median survival calculated from the date of the surgery was 3.25 months for the
								G3/MMR subgroup patients, and 9 months for the rest of the cohort (Table 1). The
								survival was equally dismal for four out of the 5 G3/MMR patients, within a range
								between 1.7 and 4.5 months, with only F70 surviving to 10 months. The age range between
								62 and 79 years within the G3/MMR subgroup did not appear to influence the survival, as
								patients in their 60s or 70s performed the same in this subgroup (Table 1). However,
								age-adjusted survival showed a highly significant difference between patients older or
								younger than 50 years in the rest of the cohort, with 8 versus 20 months median survival
								values, respectively (Fig. 2A). The median age of the G3/MMR patients was comparable to
								the ≥  50-years age-adjusted cohort, but the median survival was significantly lower, at
								3.25 months versus 8 months, respectively (Fig. 2A). Compared to the three major
								glioblastoma subgroups, a statistically significant difference was apparent between
								G3/MMR patients and the G1/EGFR and G3/NF1 subgroup patients that showed 11.3- and
								8.5-month median survival, respectively, but not the G7/Other subgroup patients that
								showed a lower median survival of 7 months (Fig. 2B).
							

							
								Figure 2: Survival of G3/MMR subgroup patients

								

								A-B. Survival curves for G3/MMR patients in comparison to patients from the rest of the cohort: older than 50 years (≥50 yo) or younger than 50 years (< 50 yo) (A), and from other G1–G7 subgroups (B). Statistical significance: *, p < 0.05; **, p < 0.01; ***, p < 0.001. N, number of deaths; n, number of patients in follow-up. The median survival in months, and the median age in years, are indicated. C. Timeline of disease progression and treatment for the G3/MMR patient F70. GBM, glioblastoma; STR, subtotal resection; ICPI, immune checkpoint inhibitors. D. Expression of the CTLA4, PD-L1, PD-1 and IDO1 immune therapy response targets in the 5 G3/MMR tumors. Levels were normalized to the lower expression values for the respective immune target. Note very high and high expression levels for IDO1 and CTLA4, respectively, in M62 and M72 tumors.
								


							
								F70 was the only patient from the G3/MMR subgroup with significantly longer survival to
								10 months (Table 1, Fig. 2C). She had family history of first-degree cousin with
								glioblastoma, and history of colorectal cancer 10 years prior, status post resection and
								5-flurouracil chemotherapy. She presented with dizziness, unsteady gait, sleepiness and
								confusion and received subtotal resection of the 7.7 cm, right temporal lobe
								glioblastoma, leaving residual enhancing and non-enhancing tumor in the anterior mesial
								temporal region. Surgery was followed by the standard radiation-TMZ regimen consisting
								of radiation with concurrent TMZ. A regimen of immune checkpoint inhibitors consisting
								of combination nivolumab-ipilimumab (Opdivo-Yervoy), in the dosing and schedule as for
								metastatic non-small cell lung cancer (NSCLC) [22], was then added for the following 4
								months and interrupted when the patient showed decline of cognitive skills. The patient
								survived an additional 4 months post-therapy.
							

							
								The gene expression for various immune checkpoint inhibitor targets is one of the
								modalities used for assessing immune checkpoint inhibitor therapies [23]. The expression
								of CTLA4, the CD274/PD-L1 and PDCD1/PD-1 interactors, and
								IDO1, a negative immunomodulator in glioblastoma [24,25], showed variable
								levels in the G3/MMR tumors (Fig. 2D). All markers were low in the M68 tumor.
								IDO1 showed very high levels in the M62 and M72 tumors and low levels in the
								rest. The CTLA4 levels were also slightly elevated in the M62 and M72 tumors.
								The PD-L1 and PD-1 levels were variable, with at least one slightly elevated in the M62,
								M72 and M79 cases.
							

							
							
								MMR glioblastoma shows multinucleated giant cells (MGCs) and IHC compatible with
									p53 mutation and MMR deficiency.
							

							
								Histologic examination of the G3/MMR tumors showed presence of MGCs in all cases
								(Table 1, Fig. 3). However, except for case M72 that was diagnosed as giant cell
								glioblastoma, the MGCs were either scattered or focal in the other cases and admixed
								with neoplastic cells with high-grade neuroendocrine (HGNE) or gemistocytic morphology,
								which were the predominant histologic patterns. Moreover, myxoid extracellular matrix, a
								feature present in many tumors with HGNE morphology from the G7 / Other molecular
								subgroup [4], was present in three cases, M62, M68 and F70. The GFAP expression was
								variable, being lost in subsets of neoplastic cells, in correlation with the HGNE
								morphology [4].
							

							
								Figure 3: Histology and IHC of G3/MMR molecular subgroup tumors

								

								H&E panels showing tumor morphology with presence of MGCs for all 5 cases, as indicated in the upper left corner. IHC with color-coded antibodies: yellow for MMR, green for GFAP, blue for p53, and pink for p16. The MMR IHC is shown only for the F70 case and is representative for the other G3/MMR cases. All panels are shown at 200x magnification, and the scale bars are all the same, at 100 μm. Note gigantic cells in the M72 tumor, the only of the 5 G3/MMR tumors diagnosed as giant cell glioblastoma.

							

							
								IHC for the MMR proteins MLH1, PMS2, MSH2 and MSH6 consistently showed preserved nuclear
								staining for the MLH1-PMS2 pair and loss of nuclear staining for the MSH2-MSH6 pair, or
								just MSH6 for M68 (Table 1, Fig. 3). NGS confirmed germline MSH2 or MSH6 alterations,
								showing perfect correlation with MMR IHC. Importantly, the combined IHC and NGS workup
								revealed the MMR deficiency for the first time in 4 of 5 patients, including in patients
								with prior history of cancer (Table 1), underscoring the importance of combined IHC and
								NGS workup for glioblastoma.
							

							
								All the tumors showed diffuse nuclear p53 immunostaining, suggestive of TP53
								mutation. The Ki-67 proliferation index was variable, showing moderate elevation of
								approximately 20 % in M62 and M79 tumors and high rate of approximately 50 % in the M68
								and F70 tumors. IHC with p16 antibody, performed in the M62, M68 and M79 tumors, showed
								a different expression pattern for each case (Fig. 3). This ranged between strong
								expression for M79, lack of expression for M68, and mixed expression for M62,
								correlating with the intact, deleted, and mutant status of the CDKN2A gene,
								respectively (Suppl Table S1).
							

							
							
								MGC histology associates with TP53 mutation in multiple glioblastoma
									subgroups.
							

							
								The morphologic classification of glioblastoma in 12 patterns assigned to 5 histologic
								clusters, #1/Astrocytic (previously called EGFR-like), #2/Small neuronal, #3/Anaplastic,
								#4/Spindle and #5/Epithelioid, has been previously described [4]. Histologic examination
								of the entire cohort showed that MGCs were also present in tumors from other molecular
								subgroups, making up approximately one fifth (18 %) of the total glioblastoma cases
								other than G3/MMR ones (Fig. 4A). Tumors containing scattered MGCs resembling the G3/MMR
								tumors were especially enriched in the G1/EGFR-mutant subgroup (50 %), and less in the
								G6/Multi-RTK (25 %), G7/Other (24 %), G3/NF1 (20 %) and G5/PDGFRA (20 %) subgroups, with
								the major G1/EGFR-amplified subgroup showing only few cases (8 %). In total, 43 primary
								glioblastoma cases featured MGCs, with the G7/Other and G3/NF1 subgroups contributing 10
								cases each, and the G3/MMR subgroup, G1/EGFR-amplified, G1/EGFR-mutant and G6/Multi-RTK
								subgroups contributing 5 cases each (Fig. 4B). These data indicated that the presence
								MGCs in a tumor is not specific for the G3/MMR subgroup.
							

							
								Figure 4: MGCs in glioblastoma: distribution in G1–G7 molecular subgroups, histologic profiles, and p53 pathway mutations

								

								A. Incidence of cases with MGCs in the G1–G7 glioblastoma subgroups. GBM, glioblastoma; EGFR↑, EGFR-amplified; EGFRm, EGFR-mutated; Total GBM-MMR, GBM cases without the G3/MMR cases. B. Pie-chart distribution (%) of the 43 cases with MGCs in G1–G7 molecular subgroups. C. Distribution of the 43 cases with MGCs into histologic clusters in the G1–G7 molecular subgroups (upper bar graph), or as % (lower pie chart). #, number of cases; Ana, anaplastic; Epi, epithelioid. D. Distribution of p53 pathway alterations in the 43 tumors containing MGCs. TP53/IHC+ and TP53/IHC-, MGC cases with TP53 mutations and positive or negative p53 IHC, respectively; Other (than TP53 or MDM2/4) alterations include one case each with RPL5 and EBF1 mutations. E. Bar graph showing the % of cases with or without MGCs from the total GBM cases with TP53 mutations (N) and per G1–G7 molecular subgroup. Total GMB-MMR, TP53-mutant cases without the G3/MMR cases. Note that overall, only 45 % of the TP53-mutant tumors contain MGCs. F. Bar graph showing the number of allelic TP53 mutations. GBM, TP53-mutant glioblastoma cases without the G3/MMR cases.

							

							
								Examination of the associated morphologies in these cases revealed that they either fell
								into the #3/Anaplastic, #5/Epithelioid, mixed Anaplastic/Epithelioid or mixed
								Spindle/Epithelioid clusters, indicating that anaplastic and epithelioid morphologies
								are dominant in the cases with MGCs (Fig. 4C). Moreover, the mixed Spindle/Epithelioid
								morphology was limited to G3/NF1 subgroup tumors that may show a SEGA-like histologic
								profile [16]. Other than this SEGA-like morphology that was noted only in the G3/NF1
								subgroup, and the giant cell glioblastoma case M72 that was unique to the cohort, the
								other cases showed scattered MGCs, significantly smaller than in the M72 case (Fig. 3),
								regardless of the molecular subgroup. No giant cells were recorded in cases from the
								minor subgroups G2/FGFR3 and G4/RAF, and only 5 cases mapped to the largest molecular
								subgroup G1/EGFR-amplified, as these three subgroups predominantly display morphologies
								in the #1/Astrocytic or #2/Small neuronal clusters that were virtually exclusive of
								MGCs.
							

							
								All the cases in the G3/MMR subgroup showed IHC positive for p53 (Fig. 3 and 4D), and
								carried TP53 mutations (Suppl. Table S1). Analysis of the other cases with MGCs
								showed that the large majority also showed p53 IHC positivity and carried TP53
								mutations, regardless of the molecular subgroup (Fig. 4D). One tumor occurring in a
								patient with Li-Fraumeni syndrome showed frameshift mutation in the beginning of
								TP53 gene with lack of p53 protein expression, and another 12 % of cases with
								MGCs showed alterations in mediators of the p53 pathway, such as MDM2,
								MDM4, RPL5 or EBF1 [5,26,27] (Fig. 4D). Only 7 % of the cases
								with MGCs did not show alterations in the p53 pathway, and they all clustered in the
								G3/NF1 subgroup. In total, approximately one fifth (21.3 %) of the cases with MGCs were
								negative for p53 IHC (Fig. 4D, pie chart).


							
								Examination of the TP53 mutations in the glioblastoma cohort showed some
								distinctive features of the G3/MMR cases. Whereas all the cases in the G3/MMR subgroup
								showed TP53 mutations and MGCs, only approximately half of the cases with
								TP53 mutations from other molecular subgroups showed an MGC phenotype, except
								for the G1/EGFR-mutant subgroup that showed 83 % of TP53 mutant cases
								displaying an MGCs (Fig. 4E). Overall, the G3/MMR cases represented one seventh (14.3 %)
								of the cases showing both MGCs and p53 immunopositivity. A major difference also
								consisted in the number of the TP53 mutations: whereas in G3/MMR cases,
								TP53 was inactivated by different missense mutations most likely targeting both
								alleles (Suppl. Table 1), 83 % of the tumors in the rest of the cohort showed only one
								TP53 mutation, with the second allele inactivated by loss or neutral loss of
								heterozygosity (Fig. 4F).
							


							

							
								Molecular landscape of the G3/MMR glioblastoma subgroup tumors.
							

							
								The TMB of the G3/MMR cases was significantly higher than that of the other glioblastoma
								cases, with or without high MSI (Table 1 and Fig. 5A). For comparison, three additional
								cases from the glioblastoma cohort showed very high TMB (Fig. 5A; Suppl. Table S2). One
								was a tumor at initial presentation in a 53-years-old male (M53/SC) that showed clonal
								VAF in few mutations common to G1/EGFR-amplified tumors, and subclonal VAF in a
								multitude of less common mutations indicative of a subclonal population of cells with
								very high TMB. Two other cases were recurrent, G1/EGFR-amplified subgroup tumors in male
								and female patients aged 66 and 58 years, respectively (M66/TMZ and F58/TMZ), previously
								treated with TMZ, a known hypermutator phenotype inducer [28]. Interestingly, of the 20
								post-TMZ, recurrent tumors subjected to NGS in this cohort, of which only 5 mapped to
								the G1/EGFR-amplified subgroup, both tumors with hypermutator phenotype belonged to the
								G1/EGFR-amplified subgroup. All these three G1/EGFR-amplified tumors harbored very high
								TMB, stable MSI, but lacked MGC histology. Their immunohistochemical profiles were
								undistinguishable from those of G1/EGFR-amplified tumors, showing negative p53 and p16
								IHC.
							

							
								Figure 5: Molecular profiling of the G3/MMR subgroup

								

								A. TMB mean±SEM graph of individual case values from indicated cases. Post-TMZ, post TMZ chemotherapy; Total GBM-MMR, GBM cases without the G3/MMR cases. Statistical significance: *, p <  0.05. B. Genomic alterations in effectors of the pathways indicated on the left are shown by color-coded squares. Telom, telomere maintenance; CC G1, cell cycle G1 phase; DDR, DNA damage response; ChRm, chromatin remodeling; Chrom, chromosomes; expr, RNA expression. C–D. Line graphs tracing the incidence of the indicated alterations in the G3/MMR subgroup in comparison with the rest of the G1–G7 molecular subgroups. E. RNA expression heatmaps for the indicated RTKs, transcription factor AP-1 complex and MAPK pathway feedback effectors.

							

							
								A comparison of the molecular landscape of the G3/MMR cases with the 3 cases with very
								high TMB and, in general, with the distribution of mutations from the rest of the G1–G7
								molecular subgroups (Suppl. Tables S2-S3), showed distinctive features for the G3/MMR
								subgroup (Table 1; Fig. 5B–D; Suppl. Table S1).
							


							
								Germline pathogenic alterations in MSH2 or MSH6 MMR genes were present
								in all G3/MMR cases but absent in the rest of the cohort. These were either splice site
								or frameshift mutations, resulting in truncation of the protein, or loss of the
								MSH2/MSH6 locus on chromosome 2p16.3, an exceedingly rare event in glioblastoma
								present only in the M79 G3/MMR case from the entire cohort. Interestingly, the
								MSH6 p.F1088fs mutation was recurrent, either as germline or as second hit
								somatic mutation (Suppl. Table S1). In comparison, the 3 cases with very high TMB showed
								pathogenic or likely pathogenic MSH2 or MSH6 mutations with VAF in the
								somatic range, of which only one resulted in protein truncation, in M66/TMZ, the other
								being missense mutations, with subclonal VAF in the M53/SC tumor (Suppl. Table S2). In
								comparison, a paucity of pathogenic mutations in MMR, POLE and MUTYH
								genes, the latter two also known to induce hypermutator phenotype [29,30], was apparent
								in the cohort, without high TMB in any of these mutant cases (Suppl. Table S2). In
								particular, the G2/FGFR3 molecular subgroup displayed higher incidence of MMR/MUTYH
								mutations, with the M60 case showing the only MSH6 truncating mutation with VAF
								in the somatic range from the cohort. As previously described, this tumor showed a
								typical G2/FGFR3 molecular profile and lacked MGCs [15] (Suppl. Table S2). A truncating
								mutation in MLH3, a paralog of MLH1, with VAF in the germline range,
								in the G1/EGFR-amplified M31 tumor, was the only other germline MMR truncating mutation
								from the cohort, and did not show high TMB, most likely due to compensation by the
								MLH1-PMS2 MMR pair. The only two tumors that showed concomitant MGCs, TP53
								mutations and mutations in either MUTYH or MSH3 but without hypermutator phenotype
								belonged to the G5/PDGFRA subgroup (Suppl. Table S2).
							

							
								It has been shown that deficiency in DNA repair results in a specific DNA mutation
								signature in tumors, represented by predominance of C:G to T:A transitions in
								MMR-deficient tumors, and C:G to A:T transversions in POLE, POLD1 and
								MUTYH mutant tumors [31,32]. All G3/MMR tumors, as well as the M66/TMZ and
								F58/TMZ tumors, showed an MMR-type DNA mutation signature (Table 1). Surprisingly, the
								M53/SC tumor showed a POLE-type DNA mutation signature, and mutation analysis revealed
								two subclonal POLH mutations, including one pathogenic (Fig. 5B, Suppl.
								Table S2). Since no report is available in the literature for association of
								POLH mutations with high TMB, the latter may be due to the MSH2
								subclonal mutations but possibly fine-tuned by the POLH deficiency.
							

							
								The G3/MMR subgroup tumors lacked or showed low incidence for many of the common
								glioblastoma molecular alterations, such as simultaneous chromosome 7 gain and 10 loss
								(see also [5]), TERT promoter mutations and CDKN2A homozygous loss (Fig. 5B–C,
								Suppl. Table S3). Conversely, they showed 100 % TP53 mutation rate and high incidence of
								mutations in genes from DNA damage response (DDR) and chromatin remodeling pathways,
								especially from the SWI/SNF complex (Fig 5B–C, Suppl. Tables S1, S3). Interestingly,
								likely pathogenic NOTCH1 missense mutations were detected in three of the five
								G3/MMR cases. 100 % of G3/MMR TP53 mutations were biallelic (Fig. 4F) and
								targeted Arg residues (Suppl. Table S3). In the rest of the cohort, TP53 Arg
								mutations were seen in a quarter of cases, the highest rate of 40 % being noted in the
								G7 / Other subgroup.
							

							
								The PI3K pathway was strongly activated by mutations in PIK3CA and PTEN
								upstream effectors, but also by TSC2 mutations (Fig. 5B–D, Suppl.
								Table S1). The 80 % mutation rates for PIK3CA and PTEN were the
								highest in the cohort, the PIK3CA mutation rate being three-fold higher than
								the next highest rate from the G5/PDGFRA subgroup (Suppl. Table S3). Three G3/MMR cases
								showed overlapping PIK3CA and PTEN mutations, and one of these showed
								additional PIK3R1 subclonal mutation (Fig. 5B). The G3/MMR 60 % rate of
								overlapping mutations in PI3K pathway upstream effectors was 10-fold higher than the
								5.6 % rate in the rest of the cohort (Fig. 5D). Strikingly, PIK3CA p.R88Q was
								the activating mutation in all four G3/MMR mutant cases, whereas it was a single event
								in the 34 PIK3CA-mutant cases from the rest of the cohort (Fig. 5B, D; Suppl.
								Table S3). Similar to TP53, PIK3CA mutations on Arg residues were
								scarce in the rest of the cohort, numbering one p.R88Q and one p.R38L in the G7 / Other
								and G3/NF1 subgroups, respectively. PTEN mutations were biallelic in two G3/MMR
								cases (40 %), whereas two concomitant PTEN mutations were noted only in 3 cases
								in the rest of the cohort (2.5 %), all showing subclonal VAF, suggestive of
								heterogeneity rather than of biallelic hit. Double hit mutations were also detected in
								TSC2 tumor suppressor gene, in two out of three mutant G3/MMR cases (Fig. 5B,
								Suppl. Table S1). TSC1/2 mutations are relatively uncommon in glioblastoma,
								numbering 8 cases in the rest of the cohort (3.8 %), without double hit (Fig. 5D).
							

							
								The MAPK pathways was activated by mutations in many effectors, some overlapping, such
								as PTPN11 and NF1 or KRAS (Fig. 5B). None of the major RTKs
								defining the G1/EGFR, G2/FGFR3, G5/PDGFRA or G6/Multi-RTK subgroups was amplified or
								mutated. Likely pathogenic missense mutations in RET in two cases, and
								FLT1 and CSF1R in one case each, were detected (Suppl. Table S1), of
								which the RET mutation in case F70 was accompanied by overexpression (Fig. 5E,
								Suppl. Table S4). Virtually all the G3/MMR cases showed moderate overexpression of at
								least one RTK except for EGFR, and the M72 tumor showed high MET
								overexpression close to the range of the MET-amplified tumors from G6/Multi-RTK subgroup
								(Fig. 5E, Suppl. Table S4). FOS and JUN families of transcription factors heterodimerize
								to form the activator protein-1 (AP-1) complex that is phosphorylated and activated by
								MAPK [33]. The four members of the FOS family are transcription activators, whereas JUND
								from the JUN family acts mostly as repressor. The expression of the AP-1 complex
								transcription factors in G3/MMR cases showed overexpression of one or more activating
								transcription factors, with the M72 and M79 tumors highly overexpressing FOSB, and
								additionally FOSL1 for M72 (Fig. 5E, Suppl. Table S5). Consistent with its role
								as suppressor, JUND showed no overexpression and was decreased in the M68 and
								F70 tumors that showed lesser overexpression of the transcription activator family
								members (Suppl. Table S5). The activation of the MAPK and PI3K canonical growth pathways
								has been shown to result in an inhibitory feedback response in glioblastoma [15]. The
								dual-specificity phosphatases (DUSP) directly dephosphorylate and inactivate MAPK, with
								DUSP5 and DUSP1/6 specifically dephosphorylating ERK1 and ERK2, respectively, and DUSP4
								dephosphorylating ERK1, ERK2 and JUNK [34]. Of these, DUSP5 was upregulated in
								all five G3/MMR tumors, and DUSP1 in three tumors (Fig. 5E and Suppl.
								Table S6), suggesting MAPK activation in all tumors. Moreover, the more specific
								inhibitors ERRFI1 and SPRY1/4 targeting downregulation of growth
								signaling from the EGFR and FGFR RTK families, respectively [35,36], were strongly
								upregulated in M72 and F70 tumors, respectively (Fig. 5E and Suppl. Table S6).
							

							Discussion

							
								The identification of molecular subgroups susceptible to targeted therapies is the
								ultimate goal of tumor classifications. The MAPK pathway-based G1–G7 molecular
								classification of glioblastoma allowed subclassification of the small number of cases
								with genetic MMR deficiency in the G3/MMR molecular subgroup [5]. The incidence of these
								tumors in the prospective cohort of 218 glioblastoma cases is low, approximately 2 %.
								Unlike three recent studies that have characterized few glioblastoma cases with MMR
								deficiency mainly in younger adults [13,14,37], this is the first study assembling five
								cases of glioblastoma with germline MMR deficiency in adults over 60 years of age, who
								represent the large majority of patients with glioblastoma. Of the studies in younger
								MMR patients, the only one showing survival data concluded that these MMR patients
								exhibit better survival by comparing them to a standard glioblastoma cohort
								significantly much older, with a difference of 13 years in median age [13]. Studies,
								including the current one, have clearly shown significant age-dependent differences for
								glioblastoma patient survival [20,21], and therefore the conclusion of a longer survival
								even for younger adult MMR patients is inaccurate methodologically in the afore
								mentioned study [13]. The current study unequivocally showed dismal survival for the
								adult G3/MMR glioblastoma patients, significantly shorter than for the rest of the
								glioblastoma cohort adjusted for age. This finding stratifies the G3/MMR as a high-risk
								glioblastoma subgroup and aligns it with a recent study reporting poor prognosis for
								Lynch-syndrome patients with IDH-mutant astrocytoma [38].
							

							
								The speedy identification of the G3/MMR patients is peremptory for prompt therapy with
								immune checkpoint inhibitors. The only controlled study to date of immune checkpoint
								therapy for recurrent MMR-deficient glioblastoma has not found a significant difference
								to control when using pembrolizumab, a PD-1 inhibitor [39]. In addition, a recent case
								report has shown lack of effectiveness of pembrolizumab for controlling
								Lynch-syndrome-associated glioblastoma in a young adult patient [40]. In agreement with
								these data, the M68 patient from the G3/MMR subgroup was briefly treated with
								pembrolizumab without response, although it is not clear if the lack of immune
								checkpoint marker expression may have contributed to the inhibitor resistance. In
								contrast, the F70 patient who was treated with the dual regimen of nivolumab-ipilimumab
								after standard concurrent radiation-TMZ adjuvant therapy, showed much better survival
								than the other patients from the G3/MMR subgroup. This result, even if unique to this
								adult G3/MMR patient, shows that the combination nivolumab-ipilimumab targeting both
								PD-1 and CTLA-4, respectively, may be effective on adult G3/MMR glioblastoma. Few
								pediatric examples of MMR/Lynch syndrome glioblastoma were successfully treated with
								PD-1 inhibitors [41,42], but in one case, a sustained response could only be achieved
								after addition of anti-CTLA-4 ipilimumab to the initial anti-PD-1 nivolumab regimen
								[43]. This dual regimen is also used in metastatic CRC associated to Lynch syndrome
								[44], and as first-line therapy for metastatic or advanced NSCLC with TMB ≥  10
								mutations / MB, regardless of the PD-L1 expression levels [22,45,46].
							






							
								The identification of the G3/MMR patients relies on clinical, histologic and molecular
								clues. A review of the literature identified 5 additional adult (≥ 50-years) G3/MMR
								cases with clinical, histologic and molecular data [13,14], and the integrated findings
								from the total of 10 G3/MMR adult glioblastoma patients is summarized in Table 2.
								Clinical history of prior cancers pertains to half of the patients, with highest
								frequency of CRC, in 4 of the 10 cases. Older G3/MMR patients appear to be prone to
								multifocal or deep-seated tumors that warrant subtotal resections or only biopsies.
								Histologically, 100 % of these tumors contain MGCs. As shown in this study, MGCs are not
								specific for G3/MMR tumors, and other G1–G7 subgroups, especially the ones with tumors
								displaying morphology classified in the anaplastic and epithelioid clusters, may show a
								variable number of tumors with MGCs (see Fig. 4). In addition to MGCs, all the G3/MMR
								tumors also show positive p53 IHC. However, as shown in this study, nearly 80 % of the
								tumors with MGCs showed p53 immunopositivity, with the G1/EGFR-mutant molecular subgroup
								mimicking the closest the G3/MMR subgroup in terms of morphology and p53
								immunopositivity (see Fig. 4). Overall, the G3/MMR subgroup cases represented 14.3 %
								(one seventh) of the total glioblastoma cases showing both MGCs and p53
								immunopositivity. The IHC with the MMR panel appears to be specific for G3/MMR cases
								(Table 2), but will only be yielding positive results in 1 out of 7 glioblastoma cases
								with concomitant MGCs and p53 immunopositivity.
							

							
							
										Current study	Kim et al. [14]	Hadad et al. [13]	Total 3 studies
	Number of patients	5	3	2	10
	Median age (range) in years	70 (62 to 79)	69 (50, 69 and 75)	53.5 (50 and 57)	68.5 (50 to 79)
	Sex M:F ratio	4 : 1	1 : 2	1 : 1	1.5 : 1
	History of prior cancer	60 %	67 %	0 %	50 %
	Location: multifocal or midline	60 %	67 %	0 %	50 %
	Survival: months	3.25 (median)	ND	6.4 and 50.5	4.5 (median)
	Surgery	40 % Biopsy	100 % GTR	100 % GTR	60 % GTR
	Adjuvant treatment (RT/TMZ)	67 %	100 %	100 %	80 %
	Multinucleated giant cells	100 %	100 %	100 %	100 %
	IHC MSH2/MSH6	100 %	100 %	100 %	100 %
	MMR mutation frequency 	MSH6 > MSH2	MSH6 > MSH2	MSH2	MSH6 ≥ MSH2
	IHC p53	100 %	100 %	100 %	100 %
	TP53 mutations (biallelic/R mutations %)	100 % (100/100)	100 % (67/100)	100 %(50/100)	100 % (80/100)
	MAPK pathway (PTPN11/RAS/NF1 %)	60 % (40/20/40)	100 % (67/0//67)	100 % (0/0/100)	80 % (40/10/60)
	PI3K pathway (PIK3CA/PIK3CA R88Q/PTEN %)	100 % (80/100/80)	100 % (67/0/33)	100 % (50/100/100)	100 % (70/71/70)
	DDR pathway	80 %	67 %	50 %	70 %
	Chromatin Remodeling (DNMT3A/ SETD2/ SWI/SNF %)	100 % (40/20/80)	100 % (0/33/100)	100 % (100/100/100)	100 % (40/40/90)
	Common GBM alterations (TERT; chr 7gain&10 loss)	0 %	0 %	0 %	0 %
	Average TMB (mutations/MB)	24.3 	23	35	27.3
	MSI-high	25 %	100 %	100 %	67 %


							



							
									Table 2: Review: integrated characteristics of G3/MMR subgroup glioblastoma in ≥ 50-year-old patients.
GBM, glioblastoma; M, male; F, female; GTR, gross total resection; STR, subtotal resection; RT/TMZ, radiation/temozolomide; ND, not determined; chr, chromosome.
						
												


							

							
								The NGS molecular analysis with TMB inclusion is necessary for detecting and/or
								confirming the presence of G3/MMR cases. In general, high TMB is a relatively rare
								occurrence in untreated, first-presentation glioblastoma, and is more often seen in
								recurrences with TMZ-related hypermutator phenotype [28]. Noteworthy, both post-TMZ
								recurrences described here mapped to the G1/EGFR-amplified subgroup, suggesting an
								association worth looking into. A hypermutator phenotype in untreated tumors may be the
								result of germline and/or somatic mutations in MMR, POLE or MUTYH
								genes. Germline MMR mutations were found only in MSH2 or MSH6, with
								the MSH6 p.F1088fs mutation recurrent in 2 studies, the current one and that by
								Kim et al. [14]. In the current study, somatic MMR mutations did not result in
								hypermutator phenotype in untreated, first-presentation glioblastomas (see Suppl.
								Table S2). However, at least three ≥ 50-year-old patients in the studies by Hadad et al.
								and Kim et al. presented with hypermutator phenotype in sporadic glioblastomas with
								somatic MMR alterations [13,14]. The question arises if these tumors should be included
								in the G3/MMR high-risk subgroup where MMR mutations are causative, or if their tumors,
								like the M53/SC case, show only added hypermutator phenotype to a baseline mutation core
								that fits another G1–G7 subgroup and show therefore better prognosis. More studies are
								necessary to clarify this issue, and treatment with immune checkpoint inhibitors may
								prove beneficial in glioblastomas with both germline and somatic MMR mutations.


							
								For MUTYH, a pan-cancer recent study has shown that biallelic mutations and not
								heterozygous germline variants result in high TMB [30]. Glioblastoma cases with high TMB
								due to MUTYH mutations have not been reported in the literature, and in our
								series, the two tumors with heterozygous germline MUTYH mutations did not show
								high TMB (see Suppl. Table S2). POLE mutations in glioblastoma are equally
								rare, with only one in this cohort, without high TMB. However, POLE somatic mutations
								with hypermutator phenotype and usually accompanied by germline or somatic MMR mutations
								have been reported mainly in young glioblastoma patients [13,29].
							

							
								Beside high TMB, the G3/MMR subgroup tumors showed a common signature represented by mutations resulting in RAS activation, with high incidence of PTPN11 and NF1 mutations (Table 2), an association also noted in the G3/NF1 subgroup [4]. The resulting MAPK activation was accompanied by high levels of the transcription factors from the AP-1 effector complex and by upregulation of a negative feedback, of which DUSP5 was the most consistently upregulated. The activation of the PI3K pathway was present in all the cases, with high incidence of PIK3CA mutations, especially of p.R88Q, of overlapping PIK3CA and PTEN mutations, and of TSC2 mutations (Table 2 and Fig. 5D). As shown, TP53 was mutant in all the cases, with predominance of biallelic Arg mutations. Very high incidence of mutations in the DDR and chromatin remodeling pathways, particularly in DNMT3A, SETD2 and the SWI/SNF complex, coupled to absence of the characteristic TERT promoter mutations or concomitant chromosome 7 gain and 10 loss, distinguished the G3/MMR subgroup from the other G1–G7 subgroups (Table 2 and Fig. 5C) [5]. Mutations in APC and NOTCH1/2 genes were also overrepresented in G3/MMR patients from this study and that by Kim et al. [14]. In general, the G3/MMR molecular profile resembled closer the profiles of other solid cancers and was highly divergent from that of non-MMR glioblastoma [47].
							

							Conclusions

							
								In conclusion, this study characterizes rare cases of adult glioblastoma associated to Lynch syndrome that appear to represent a high-risk G3/MMR subgroup showing distinctive clinical, histologic and molecular characteristics. It reviews the available cases in the literature, and proposes immune checkpoint inhibitors as adjuvant treatment, particularly the dual nivolumab-ipilimumab regimen that showed net survival benefit in the G3/MMR patient receiving it. More studies are necessary to validate this regimen and correlate it with the molecular parameters usually used in CRC associated to Lynch syndrome, such as TMB, MSI status and immune checkpoint markers expression levels.
							

		
							Ethics, Consent and Permissions

							This study was conducted in accordance to the Declaration of Helsinki, and approved by the Ethics Committee of NeuroMarkers (Protocol code NM-REC-2 on 9 January 2020 and 30 January 2023) for studies involving human subjects. Informed consent was obtained for all subjects.

							Consent for Publication

							Patient consent for publication was waived due to the study not identifying the patients.
								

								Availability of Data and Materials

								The genomic and transcriptomic datasets supporting the conclusions of this article are de-posited in the public repository NeuroMarkers Open Research Database https://neuromarkers.org/database under the accession numbers G-NM/24-1 and T-NM/24-1, respectively.
									

									Conflicts of Interest Statement

									The author declares no conflicts of interest.
										


										Funding Statement

							This work was supported by an award from NeuroMarkers [NM2024-1] to M.-M.G.
								

								Acknowledgements

							This paper is dedicated to the patients and their families.
								








							




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