Diffusion-weighted MRI derived apparent diffusion coefficient identifies prognostically distinct subgroups of pediatric diffuse intrinsic pontine glioma
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- Lober, R.M., Cho, Y., Tang, Y. et al. J Neurooncol (2014) 117: 175. doi:10.1007/s11060-014-1375-8
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While pediatric diffuse intrinsic pontine gliomas (DIPG) remain fatal, recent data have shown subgroups with distinct molecular biology and clinical behavior. We hypothesized that diffusion-weighted MRI can be used as a prognostic marker to stratify DIPG subsets with distinct clinical behavior. Apparent diffusion coefficient (ADC) values derived from diffusion-weighted MRI were computed in 20 consecutive children with treatment-naïve DIPG tumors. The median ADC for the cohort was used to stratify the tumors into low and high ADC groups. Survival, gender, therapy, and potential steroid effects were compared between the ADC groups. Median age at diagnosis was 6.6 (range 2.3–13.2) years, with median follow-up seven (range 1–36) months. There were 14 boys and six girls. Seventeen patients received radiotherapy, five received chemotherapy, and six underwent cerebrospinal fluid diversion. The median ADC of 1,295 × 10−6 mm2/s for the cohort partitioned tumors into low or high diffusion groups, which had distinct median survivals of 3 and 13 months, respectively (log-rank p < 0.001). Low ADC tumors were found only in boys, whereas high ADC tumors were found in both boys and girls. Available tissue specimens in three low ADC tumors demonstrated high-grade histology, whereas one high ADC tumor demonstrated low-grade histology with a histone H3.1 K27M mutation and high-grade metastatic lesion at autopsy. ADC derived from diffusion-weighted MRI may identify prognostically distinct subgroups of pediatric DIPG.
KeywordsDiffuse intrinsic pontine glioma (DIPG)DiffusionDiffusion-weighted imaging (DWI)Apparent diffusion coefficient (ADC)MRI
Diffuse intrinsic pontine glioma (DIPG) accounts for 10–15 % of all pediatric brain tumors. It affects ~400 children per year in the United States and accounts for the majority of deaths due to childhood brain tumors . It is not surgically cureable and current adjuvant therapies provide limited benefit beyond transient neurological improvement [2, 3].
On MRI, DIPG presents as a heterogeneous mass that enlarges the pons with irregular interfaces with the midbrain and medulla, and ventral extension into the prepontine cistern [4, 5]. These features distinguish uniformly fatal DIPG from the dramatically more favorable focal brainstem gliomas [4, 5]. There are no characteristic enhancement patterns [3, 5] and most studies have shown poor correlation between conventional MRI features and clinical outcome [6, 7]. One large analysis of MRI features across two clinical trials has demonstrated worse outcomes with enhancing tumors .
Recent molecular studies have shown that DIPG subsets with distinct biology exist  and may harbor unique mutations and genetic expressions different from adult high-grade gliomas and pediatric hemispheric glioblastomas [9–11]. DIPG are diagnosed by MRI and currently biopsied at very few institutions, which limits the translational usefulness of these studies and highlights the value of an imaging biomarker predictive of clinical behavior.
Studies have shown utility of diffusion MRI, or diffusion weighted imaging (DWI), in predicting tumor pathology, histological grade, and clinical behavior [12–20]. DWI may even have a role for tumor monitoring, including reports of normalization of apparent diffusion coefficient (ADC) derived from DWI during chemotherapy and return to pre-therapy levels with tumor recurrence [21, 22]. Early work by Chen et al.  demonstrated a positive linear correlation of ADC with survival in DIPG of nine children and described its potential role for DIPG evaluation. More recent work by Poussaint et al.  has shown that diffusion changes after radiotherapy are associated with survival in DIPG patients. However, no diffusion metric has yet been defined that stratifies clinically distinct DIPG subsets. In this study, we hypothesized that diffusion imaging can be used to stratify DIPG subsets with distinct clinical behavior.
Materials and methods
All patients diagnosed with DIPG at our children’s hospital between 2001 and 2013 were retrospectively reviewed after approval by our Institutional Review Board (protocol IRB-4223). The study cohort was identified on the basis of the following inclusion criteria: the patient presented with a new DIPG diagnosis, obtained a treatment-naïve diffusion MRI, and had follow-up clinical data, including time of death. The diagnosis was made by MRI features of an expansile pontine lesion usually characterized by T1 hypointensity and T2 hyperintensity following presentation with neurologic signs suggestive of a brainstem lesion or hydrocephalus. Medical records were reviewed for steroid administration, surgery, chemotherapy, radiotherapy, and cerebrospinal fluid (CSF) diversion.
All patients were examined with brain MR imaging at 1.5 or 3 Tesla (Signa, Discovery 750; GE Medical Systems, Milwaukee, WI, USA). Echo planar DWI was obtained using the following parameters: DWI ASSET, 4 mm, 0 skip, 3-direction, TR/TE 8300/70 (1.5 T); 10,000/86 (3 T), b = 1,000 mm2/s, acceleration factor = 2, FOV 24 cm, matrix 128 × 128. Generation of ADC maps was performed with commercially available software (Functool; GE Medical Systems). As part of our routine tumor brain protocol, additional MR sequences included T1 FLAIR, T2 fast spin echo, FLAIR, GRE, T1 spoiled gradient recalled pre and post contrast, and two additional plane contrast-enhanced T1 spin echo sequences.
Autopsy and histological preparations
For the three patients who underwent autopsy, brains were harvested and examined after formalin fixation, overseen by HV. Sequential coronal sections of the cerebrum and transverse sections of the brainstem and cerebellum perpendicular to their long axes were made, and representative sections were taken for histological evaluation. Formalin-stained sections were stained with standard hematoxylin and eosin preparations.
Screening for K27M mutation
Tissue specimens from two cases were prepared and analyzed by YT, YJC, and MM. Genomic DNA was extracted from available frozen post-mortem tumor tissues or control cortex tissues of DIPG patients using DNeasy Blood & Tissue Kit (Qiagen). Extracted DNA used for polymerase chain reaction (PCR) amplification of the genomic regions for targeted Sanger sequencing of the Histone 3.3 (H3F3A) and Histone 3.1 (HIST1H3B) genes. The PCR amplicons were then extracted using QIAquick Gel Extraction Kit (Qiagen) and underwent Sanger sequencing with the primer sequences H3F3A_gDNA-F (aggccgttcgaggtattttt) and H3F3A_gDNA-R (aaatgagggggtaggaacttt). Histone 3 gene analysis was targeted since recent studies have found that somatic mutation of the Histone 3.3 or 3.1 genes are common in pediatric high-grade gliomas, particularly midline tumors [10, 11, 24].
All statistical analyses were performed by RL using SPSS Statistics version 21.0 (IBM) with an a priori significance level of 0.05, corrected to 0.003 by the Bonferroni method for 16 statistical tests applied to the same patient sample. Kaplan–Meier curves for survival were compared using the Mantel-Cox log-rank and Breslow tests. Differences in distributions of ADC among gender were assessed by the Kruskal–Wallis test for independent samples. Differences in treatment characteristics (surgery, radiotherapy, chemotherapy, CSF diversion, and steroid administration), among ADC groups and genders were assessed with the Fisher’s exact test. Mean ADC was compared between patients with and without steroid administration using the Mann–Whitney U Test.
Clinical and diffusion MRI data
Twenty children met the inclusion criteria and were included in the study. Median age at diagnosis was 6.6 (range 2.3–13.2) years, with median survival seven (range 1–36) months. There were 14 boys and six girls. Seventeen patients received radiotherapy, five received chemotherapy, and six underwent CSF diversion. All but one patient succumbed to the disease at the time of study completion.
Baseline ADC of DIPG stratified by gender (n = 20)
Boys (n = 14)
Girls (n = 6)
Low ADC group
High ADC group
Fisher’s exact test p = 0.011
Mean ADC (10−6 mm2/s)
1,218 ± 188
1,481 ± 117
There was no difference in treatment characteristics (surgery, radiotherapy, chemotherapy, and CSF diversion) between ADC groups (p = 1.000) or genders (p = 1.000), although three of the eight patients with low ADC tumors did not complete radiotherapy because of worsening symptoms or a parental decision to switch to palliative care. Of the 20 patients, 16 had detailed information in their medical records regarding steroid administration, which was not different between ADC groups (p = 0.608) and did not affect mean ADC (1,327 ± 196 for steroid administration and 1,230 ± 229 for no steroids; p = 0.428). The feature of contrast enhancement was present in six patients, and was equally distributed between ADC groups.
Cases with tissue correlation
DIPG has a dismal prognosis with a median survival under 1 year [2, 25]. However, among DIPG some tumors are unpredictably aggressive, resulting in death within a few months. Recently, gene expression profiles have revealed at least two biological subtypes of DIPG with distinct survival and molecular characteristics , and attempts have been made to stratify tumors based on specific mutations, such as K27M in the histone H3.3 or 3.1 genes [11, 26].
Aside from imaging features that distinguish uniformly fatal DIPG and more favorable focal brainstem gliomas, no conventional MRI features have shown to correlate with clinical outcome [4–7]. Several recent studies have found that MR perfusion and spectroscopy may help assess clinical course in DIPG [27, 28]. Here, we investigated diffusion MRI as a prognostic imaging marker that could predict clinical behavior and thereby help stratify patients for prospective studies and clinical trials.
Diffusion MRI measures the movement of both extracellular and intracellular water within an image voxel [18, 29] and has increasingly been applied in brain tumors and, in some cases, for therapeutic monitoring and prognosis [12–16, 19, 21, 22, 29–31]. A recent study has inferred based on correlation with perfusion that focal anaplasia in DIPG can be identified with diffusion imaging , and that diffusion changes after radiotherapy may correlate with survival . Our results suggest diffusion metrics can define clinically distinct subgroups of pediatric DIPG, with lower tumor ADC at initial presentation predictive of aggressive clinical behavior. Low ADC in clinically more aggressive tumors suggest these may represent higher grade tumors at presentation, similar to prior studies of adult glioma in which ADC values inversely correlated to cell density and nuclear area to total cytoplasm ratio [12, 14, 15]. This is also supported by tissue specimens available in select patients in our cohort that showed grade III or IV histology in low ADC tumors and grade II histology in a high ADC tumor. It is noteworthy, that while the latter patient showed grade II histological features within pons at autopsy, the distant tumor nodules displayed grade IV features, suggesting diverse biology and evolution pattern.
This is also the first study to note potential gender differences in survival of DIPG. There was a trend toward worse survival in boys. Interestingly, low ADC tumors were found only in boys, whereas high ADC tumors were found in both boys and girls (Table 1). The low ADC tumors appeared to account entirely for the trend toward worse survival in the boys of this cohort.
Recent studies have identified somatic mutation of the H3F3A gene encoding the histone 3.3 or HIST1H3B gene encoding Histone 3.1 that results specifically in lysine 27-to-methionine (K27M) substitution in over 30 % of pediatric high-grade gliomas  and 70 % of DIPG cases, [24, 26]. Interestingly, K27M gliomas often have distinct midline location pattern, including thalamus and pons with K27M molecular signatures closely resembling mid to late fetal stages of striatum and thalamus that might suggest potential tumor cellular origins . Given frequent K27M mutation in midline pediatric gliomas, we were curious regarding the K27M status in our autopsy samples. Here, we observed K27M mutation in a high ADC tumor but not in low ADC tumor. The significance of this may be better understood with a larger cohort.
This study is limited by its low sample size and retrospective nature, as treatment decisions are often based on many factors, including patient quality of life and parental input regarding goals of care. Although we found that therapies were not different between low and high ADC groups, three of eight patients initiated on with low ADC tumors were unable to complete radiotherapy because of poor quality of life and the wishes of their parents. Steroid administration could potentially alter tumor ADC, but this was not different between low and high ADC groups. Another limitation is the paucity of histological data and the nature of the autopsy specimens that reflects the disease state at the end of the patient’s course, rather than what would potentially be found at the time of diagnosis.
ADC may identify distinct subgroups of pediatric DIPG and may be a useful noninvasive marker for patient selection in clinical trials.
The authors express our sincere gratitude to Dr. Terri Haddix for her assistance in histologic preparations and photography of specimens.
Conflict of interest
There are no conflicts of interest or funding source to disclose.