Indian Journal of Pathology and Microbiology

: 2020  |  Volume : 63  |  Issue : 2  |  Page : 262--266

Cerebral hemispheric glioblastoma with PNET-like morphology and histone H3.3 G34 mutation in younger patients: Report of three rare cases and diagnostic pitfalls

Yuanyuan Cheng1, Weiqi Bao2, Qian Wu1,  
1 Department of Pathology, Huashan Hospital, Fudan University, Shanghai, China
2 PET Center, Huashan Hospital, Fudan University, Shanghai, China

Correspondence Address:
Yuanyuan Cheng
Department of Pathology, Huashan Hospital, Fudan University, Shanghai - 200040


Recurrent mutations in H3F3A that encodes the histone 3 variant H3.3, lead to amino acid substitutions including K27M and G34R/V-which are observed in high-grade gliomas (HGGs) of children and young adults. Previous studies have focused on gliomas with K27M mutation, whereas gliomas with G34R/V mutation have received little attention. Herein, we report three rare cases of glioblastoma (GBM) with H3.3 G34 mutation arising from a cerebral hemisphere in two children and one young adult. All three cases showed microscopic characteristics of central nervous system primitive neuroectodermal tumor (CNS-PNET, called CNS embryonal tumors in WHO 2016 Revised 4th Edition) and presented H3.3 G34 mutation. H3.3 G34-mutant brain tumors were formerly a group of histopathologically distinct neoplasms, involved in GBM, CNS-PNET, and astroblastoma. However, recent studies have demonstrated that different CNS tumors with H3.3 G34 mutation display coherent epigenetic signatures, implying a single biological origin. Correspondingly, our three cases showed high consistency in tumor location, histological morphology, and molecular phenotype. Their immunophenotypes are similar to astrocytoma, with ATRX loss and TP53 mutation. Therefore it suggests that these H3.3 G34-mutant brain tumors may be a rare entity of HGG.

How to cite this article:
Cheng Y, Bao W, Wu Q. Cerebral hemispheric glioblastoma with PNET-like morphology and histone H3.3 G34 mutation in younger patients: Report of three rare cases and diagnostic pitfalls.Indian J Pathol Microbiol 2020;63:262-266

How to cite this URL:
Cheng Y, Bao W, Wu Q. Cerebral hemispheric glioblastoma with PNET-like morphology and histone H3.3 G34 mutation in younger patients: Report of three rare cases and diagnostic pitfalls. Indian J Pathol Microbiol [serial online] 2020 [cited 2020 Jul 7 ];63:262-266
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Glioblastoma (GBM) is a brain tumor that has a dismal prognosis and presents considerable heterogeneity. GBMs in adults and children indicate different molecular characteristics by the application of sequencing methods and other molecular techniques.[1],[2] Notably, histone H3.3 (H3F3A) mutations in the codon for lysine 27 (K27M) and glycine 34 (G34R/V) at two critical positions within the histone tail have been identified as driver mutations in pediatric GBMs.[3] Especially, diffuse midline glioma with H3 K27M mutation has been defined as a novel tumor entity in the 2016 edition of the World Health Organization (WHO) Classification of Tumors of the central nervous system (CNS).[4] In contrast to H3 K27M mutant gliomas in the midline region, H3.3G34R/V mutant brain tumors are less clear in the clinicopathological and biological significances. H3.3 G34-mutant brain tumors are usually located in the hemispheric region with variable extents of poorly differentiated/PNET-like morphology.[5] Recently, Andrey et al.[6] have investigated a cohort of primary H3.3 G34-mutant tumors including GBM and CNS-PNET using genome-wide methylation profiling and other molecular technologies. They have revealed that tumors with H3.3 G34 mutation display uniform molecular signatures in spite of their histopathological heterogeneity, suggesting a single biological origin. Hence, they propose that these tumors should be defined as HGGs with H3.3 G34 mutation. However, until now, there have been very few case reports of H3.3 G34-mutant brain tumors.[5],[6],[7],[8] In this study, we report three PNET-like GBMs of younger patients with H3.3 G34 mutations that all occurred in the cerebral hemisphere.

 Case History

Case 1

The patient, a 15-year-old boy, visited our hospital owing to seizures 1 month ago. Magnetic resonance imaging (MRI) showed a left frontal lobe tumor [Figure 1]a. The tumor was completely excised and the patient underwent radiotherapy with concomitant temozolomide after the operation. He remained well until 10 months after the operation when a PET/CT examination revealed recurrent tumors in multiple regions including the left temporal lobe and cerebellar hemisphere. The patient then underwent a second round of radiotherapy with concomitant temozolomide. The patient was alive but in a coma after 31 months of close follow-up.{Figure 1}

Case 2

The patient, a 15-year-old boy, presented to our hospital with a complaint of irregular dizziness and headache for half a month before. A left frontal lobe tumor was found by MRI examination [Figure 2]a. Then the tumor was completely excised. Postoperatively, the patient received radiotherapy with concomitant temozolomide. However, he had recurrent lesion 5 months after the first operationand died of progressive disease 11 months after the first operation.{Figure 2}

Case 3

The patient, a 28-year-old female, presented with progressive aggravation of hypomnesia for 5 months, headache and blurred vision for 1 month, and accompanied by the right limb weakness and dysphasia. MRI examination revealed a tumor in the left frontal lobe [Figure 3]a. The tumor was partially removed. Following the operation, the patient underwent radiotherapy with concomitant temozolomide. She remained well without signs of recurrence until 5 months after the operation when an MRI examination revealed a tumor at the resection site. The patient died of progressive disease 7 months after initial treatment.{Figure 3}

Clinical information for these three patients is provided in [Table 1].{Table 1}

Microscopic examination revealed that these three cases had consistent histological features, exhibiting PNET-like appearance. The cellular areas were composed of densely packed, primitive, poorly differentiated small neoplastic cells with scant cytoplasm and round-to-oval hyperchromatic nuclei. Brisk mitotic activity and microvascular proliferation were observed [Figures 1]b, [Figures 2]b, and 3b]. Moreover, the tumor cells disseminated to the subarachnoid spaces in all three cases [Figures 1c, [Figures 2]c, and [Figures 3]c. However, these three cases were absent of astrocytic morphology. They showed small blue cells with sparse cytoplasm but not giant cells. Microcysts were not seen either.

Immunohistochemically, these three cases were also surprisingly accordant. The tumor cells were positive for GFAP [Figure 1]d, [Figure 2]d, and [Figure 3]d but negative for Olig2 [Figure 1]g, [Figure 2]g, and [Figure 3]g. The nuclear staining of ATRX was completely lost, while residual normal glial cells showed internal positive control reactivity [Figure 1]h, [Figure 2]h, and [Figure 3]h. The Ki-67 index was 50% in case 1 and 20% in case 2 and case 3. P53 was positive in 80% in case 1, 90% in case 2, and 40% in case 3 [Figure 1] e, [Figure 2]e and [Figure 3]e. Particularly, the tumor cells were immunopositive for H3.3 G34V in case 1 [Figure 1]f and case 3 [Figure 3]f and H3.3 G34R in case 2 [Figure 2]f. The positive patterns of H3.3G34R and H3.3G34V were in the nuclear of the tumor cells. The tumor cells were negative for H3K27M [Figures 1i, 2i and 3i] and IDH1 R132H (data not shown). Although these three cases presented PNET-like morphology, the neuronal marker synaptophysin (Syn) was shown negatively in these tumor cells (data not shown), indicating that they are different cell origin from CNS-PNET.

After performance of DNA extraction, polymerase chain reaction, Sanger sequencing, and high-resolution melting analyses of mutation hotspots on H3F3A, a G34V mutation was identified in case 1 and case 3, and a G34R mutation in case 2 (data not shown). In addition, no IDH1 or IDH2 mutation was detected by sequencing (data not shown).

In summary, on the basis of pathological features, immunophenotype and molecular signatures, these three cases were diagnosed as “Glioblastoma, NEC (not elsewhere classified), with H3.3 G34R/V mutation.”


Owing to the use of molecular biological techniques, the pathological diagnosis of brain tumors transfers from a simple morphological diagnosis to a combination diagnosis with morphological and molecular characteristics. Therefore, according to the 2016 WHO classification of brain tumors,[4] high-grade gliomas include “GBM, IDH-wild type,” “GBM, IDH-mutant,” “GBM, NOS,” and “Diffuse midline glioma, H3 K27M-mutant,” which are classified as grade IV tumors. However, H3.3 G34-mutant GBM has not been absorbed in the new edition of WHO. Partly because the origin of H3.3 G34-mutant brain tumors is still controversial and the past diagnosis of H3.3 G34-mutant brain tumors was confusing. Previously, H3.3 G34-mutant brain tumors were diagnosed as GBM or CNS-PNET depending on their distinct histological morphologies,[7] which also received different treatment strategies.[9] Histologically, when the H3.3 G34-mutant tumors presented cellular polymorphism, high mitotic index, vascular proliferation, and/or areas of necrosis, GBM would be diagnosed. While higher cell densities, small round hyperchromatic nuclei with sparse cytoplasm and brisk mitotic activity existed in H3.3 G34-mutant tumors, CNS-PNET may be diagnosed. Japanese scholars Yoshimoto K et al.[8] have reported four H3.3 G34R-mutant cases including three GBMs and one astroblastoma among Japanese patients.

Korshunov A et al.[6] have performed an integrative clinical, histopathological, and molecular analysis of H3.3 G34-mutant CNS tumors including GBMs and CNS-PNETs. They demonstrated that these brain tumors with H3.3 G34 mutations displayed uniform epigenetic signatures, suggesting a single biological origin. Therefore, they have recommended that these tumors should be termed “high-grade gliomas with H3.3 G34 mutation.” Furthermore, H3.3 G34-mutant tumors have almost arised from the cerebral hemisphere predominately in young patients under the age of 30,[10],[11] and shown ATRX loss and p53 accumulation, which is similar to the immunophenotype of astrocytoma.

Because H3.3 G34-mutant GBMs often present high-cell density, small round blue cells, and brisk mitotic activity, the differential diagnosis should be made between H3.3 G34-mutant GBM and supratentorial PNET or small cell glioblastoma (SCGBM). H3.3 G34-mutant GBMs that occur predominately in young adults with a median age of 19 years,[6] present ATRX loss and TP53 mutation. Supratentorial PNETs particularly in patients younger than 10 years of age are now collectively referred to as CNS embryonal tumors according to the WHO 2016 classification.[4] Supratentorial PNETs harbor differ molecular alterations according to their histological features, such as C19MC amplification in embryonic tumor with multilayered rosettes,[12] SMARCB1, or SMARCA4 loss in atypical teratoid/rhabdoid tumor.[13] SCGBMs often occur in older patients and are strongly associated with EGFR amplifications.[14],[15]

Although both G34 and K27 mutations happen in histone H3.3, G34-mutant, and H3 K27M-mutant, HGGs likely arise from different cells of origin and are principally distinct disease. A comparison of the H3.3 G34-mutant GBM and H3 K27M-mutant HGG is shown in [Table 2]. Notably, H3 G34 mutant GBM patients show a better overall survival than H3 K27M mutant GBM patients. However, the tumors in case 2 and case 3 showed very early recurrence. We speculate that the tumors of case 2 and case 3 were discovered late and grew much big, according to the tumor size showing on MRI. In addition, the tumor of case 3 was not completely excised on operation, owing to protection of the cerebrum function area. Hence, case 2 and case 3 showed very early recurrence and short overall survival.{Table 2}

Up to now, H3.3 G34-mutant GBMs have not been reported in tumor samples from Chinese patients. Herein, we report three cases of H3.3 G34-mutant GBMs with PNET-like appearance in younger patients with the median age of 19 years. All of the three cases were located in the hemisphere region, presenting positive for GFAP and p53, negative for ATRX and Olig2. Further, we detected H3.3 G34R/V by immunohistochemistry and H3F3A gene mutation by sequencing methods and discovered G34V mutation in case 1 and case 3, and G34R mutation in case 2. Therefore, we speculate that there may exist more H3.3 G34-mutant cases, especially in younger patients with an immunophenotype of ATRX and Olig2 negative, GFAP and p53 positive. The investigation of H3.3 G34-mutant tumors and its underlying molecular mechanisms in these cases is our future research direction.

In conclusion, our three cases showed high consistency in tumor location, histological morphology, and molecular phenotype, suggesting a single biological origin of H3.3 G34-mutant brain tumors. In daily pathological diagnosis, we could routinely detect H3.3 G34 mutation by immunohistochemical and sequencing methods in HGGs that occur in the cerebral hemisphere of younger patients, especially with poorly differentiated/PNET-like morphology. This is of great significance for pathological diagnosis, follow-up treatment, and judging the prognosis of these patients.

Financial support and sponsorship

This work was supported by grants from the National Nature Science Foundation for Young Scientists of China (No. 81502273).

Conflicts of interest

There are no conflicts of interest.


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