IMAGING BRAIN TUMORS IN NEWBORNS AND EARLY CHILDHOOD: UTILITY OF COMBINING MR TECHNIQUES

1. IMAGING BRAIN TUMORS IN NEWBORNS AND EARLY CHILDHOOD: UTILITY OF COMBINING MR TECHNIQUES M. MORTILLA, M. ANTONELLO, C. CESARINI, L. TASCIOTTI, C. FONDA UNIVERSITY CHILDREN’S HOSPITAL A. MEYER FIRENZE, ITALY

2. INTRODUCTION In the childhood CNS tumors are the leading cause of cancer-related death. In the last 20 years the advances in neuroimaging, neurosurgery, radiation therapy and chemotherapy have considerably improved the long term survival of children with brain tumors. Conventional MR images show definite details of brain tumor location, extension and morphological characteristics. Therefore MR imaging is widely used in the diagnosis and follow-up of pediatric patients with brain tumors.

3. Since conventional MRI does not provide information about tissue chemistry and the interpretation of these images may lead to poor estimation of the extent of active tumor, non conventional techniques, such as diffusion images and proton spectroscopy, may be used contributing to more accurate diagnosis, prognostication and treatment planning. Tissue diagnosis remains the gold standard. The purpose of the study is to find a role of Diffusion-weighted Imaging (DWI) and proton Spectroscopy in characterizing intracerebral masses and finding a correlation between these techniques and histologic analysis of tumors.

4. PATIENTS & METHODS 62 patients affected with brain tumors aged 1 month-6 years, were studied with a 1.5T MR scanner (Eclipse, Philips) operating at 27mT/m gradient strenght and 40 mT/m/ms slew rate. A quadrature head coil was used. T2w SE images, FLAIR, T1w SE pre and post Gadolinium injection and sometimes GE T2* were obtained. Proton Spectroscopy studies included single voxel studies (PRESS TE 40/135/270ms, STEAM TE 20ms) and/or CSI (PRESS TE 135/270.

5. Spectroscopy acquisition were performed before the injection of the contrast media. Eight children were able to undergo to the MR without need of sedation despite the long duration of the exam (50-60 minutes). The other children were sedated with different modalities regarding weight, age and critical conditions: patients up to 18 months of age (if the weight was below 10Kg) were sedated with chlorale hydrate (50-100 mg/Kg), while from 18mo. to 6yrs. patients were sedated using Sevoflurane through laringeal mask or, rarely, with barbiturates i.v.

6. Echo planar Imaging of Diffusion were obtained with sensitization along the slice select, read out and phase encoding axes (b- value of 800/1000/1200) with Echo Planar Single Shot sequences (TR 6450ms, TE 145.8ms, FA 90°, 5mm/1mm slice thickness/gap, 81x81 > 128x 128 reconstruction matrix, 1 NEX, chemical saturation at FA 180°, FAT saturation) with a total acquisition time of 32 seconds/15 slices.

7. In all patients a set of 3 images along the long orthogonal axes of gradient sensitization were obtained and the DWI TRACE images were post-processed. Apparent Diffusion Coefficient (ADC) images along the same orthogonal axes were also obtained and synthetic ADC map was produced (ADC TRACE. ADC values are expressed as a number x 10-3mm2/sec.

8. Multivoxel (CSI) were acquired with PRESS sequence, TR 1500ms, TE 135ms, FA 90°, thickness 1-1.5 cm, FOV 15 to 20cm. Chemical shift imaging matrix size 16x16, signal averages 1/2 with 1 slice per batch. Single voxel were acquired with PRESS sequence, TR 1500/2000ms, TE 270/135/40ms and with STEAM TE 20ms, FA 90°; acquisition volume from 20x20x20mm, signal averages 128 and reference averages 8.

9. Most of the tumors underwent to surgery excision and the specimens were analyzed by an expertise in pathology in order to make diagnosis following the WHO classification and to determine cell counting expressed as mean value (m.v.) over an are of 0.083mm2. Some tumors were biopsied only, such as germinoma because they are highly responsive to therapy. Biopsy or surgical excision was not performed when the tumor arised from non resectable regions (hypotalamus, brainstem,..)

10. OUR CASES • Craniopharingioma 5 • Low grade glioma (WHO I) 11 • Glioma WHO II 2 • High grade glioma 1 • Pylocitic astrocytoma 10 • Medulloblastoma (MB-PNET) 11 • Germinoma 1 • Subependimal gigantic astrocytoma 2 • Ganglioblastoma 1 • Ganglioglioma 1 • Dysplasia 3 • DNET 2 • Teratoma 1 • Metastasi 1 • Altri 10

11. Diffusion-weighted Imaging It has been reported that the ADC characterizes the biophysical characteristics of tissue microstructure and microdynamics and that it provides information (based on pathophysiologic characteristics) that differs from that obtained with contrast-enhancing imaging. It has been suggested that the minimum ADC value of high-grade gliomas is significantly higher than that of low-grade gliomas and that low ADC values were found in areas of increased cellularity.

12. Others have suggested that the ADC may assist in the early detection of responses to anticancer therapy, because an increase in ADC values has been noted after treatment. DWI measures the molecular mobility of extracellular water,alterations in water mobility appear to reflect treatment-induced changes in tissue structure. Current understanding is that water diffusion increases acutely in tumor responsive to therapy. This precede changes in tumor volume.

13. 1H-MRS (proton magnetic resonance spectroscopy) • 1H-MRS (Magnetic Resonance Spectroscopy) provides a qualitative and quantitative evaluation of brain chemistry • In a proton spectrum at 1.5T the metabolites are spread out between 63 and 64 MHz, and near 300 MHz at 7T • The resonant frequencies are expressed in part per million (ppm), and are read from right to left

14. 1H-MRS • Fat and water are eliminated, because their peaks are to high and in spectrum scaling the brain metabolites would be invisible • The water suppression is obtained with CHESS (Chemical Shift Selective) or IR (Inversion Recovery) techniques • The peaks are separated into the individual frequencies through a Fourier Transform • The magnetic field felt by the Protons in different molecules depends on electron clouds related to their different molecular position -> different chemical shift -> spread of single peaks over the ppm or hertz scale

15. Sequences in 1H-MRS • STEAM (Stimulated Echo Acquisition Mode): 90o refocusing pulse, short echo time, less signal-to-noise ratio • PRESS (Point Resolved Spectroscopy): 180o refocusing pulse, short and long echo time • With short echo time (TE 20-40 ms) metabolites of both short and long T2 are visualized • With long echo time (TE 270 ms) only metabolites with long T2 are seen . Echo Time of 135 ms allows the separation of lactate doublet from lipids peaks with phase inversion. • TE 65 ms increases sensitivity in lipid detection nulling lactate

16. voxel • MR Spectra may be acquired with a single voxel localized in region of interest (normal or pathological) with variable volume (usually of 2x2x2 cm or more). Small volume are characterized by less signal to noise ratio. Large volumes experience higher averaging and are not indicated for higher resolution data collection, but only for mean value in a defined area

17. multivoxel • MR Spectra may be acquired within a brain slice in 2D acquisition or more slices in 3D acquisition) with variable matrix and variable volume (usually of 1x1x1 cm or more) Small volume of voxels experience lower averaging than single voxel with larger volume. Chemical Shift Imaging (CSI) may create the metabolite maps with direct visualization of peak concentration • Magnetic field inhomogeneity, insufficient shimming and lipids contamination frequently alter the quality of multivoxel spectra.

18. Many metabolites may be identified in the proton magnetic resonance at 1.5 tesla, NAA, Cho, Cr, Lactate, myo-Inositol, Lipids are currently evaluated • In the following slides there is a list brain peaks:

19. List of metabolites that can be individualizedby 1H-MRS • N-acetyl methylgroups • (NAA –N-acetylaspartate and NAAG – N-acetylaspartylglutamate) • Methyl and Methylene protons of total creatine (Cr +PCr) • Trimethylammonium groups –Choline containing (Cho) • Myo-Inositol (mI) • Glycine co-resonating with main mI peak (Gly) • Glutamate & Glutamine with a and b-/g- protons (Glu/Gln) • Glucose • Scyllo-inositol • Lactate • GABA • Glutathione • Taurine • Homo-carnosine • Phospho-ethanolamine • Macromolecules • Lipids

20. Resonance intensitiesexpressed in ppm at 1.5T • Lactate/lipids 1.33 • NAA 2.02 • Glx 2.2-2.4 • Cr/PCr 3.02 • Cho 3.22 • mI 3.56

21. Control 5 yrs old: CSI PRESS TE 135ms

22. NAA(N-acetyl aspartate): free aminoacid, high CNS concentration (just less to glutamate) • in adults in neural tissue, axons and dendrites • in brain maturation also in oligodendrocytes type 2 and in non neuronal cells(mast cells) • used as neuronal marker

23. Cho(choline - N(CH3)3 GPC,PC)cellularmembrane turnover marker • High in tumors, demyelinating processes, inflammation • Cr( creatine - Cr + PCr = k),reference internaldue to its stability,marker of byproducts of energy chains >ATP • Lac (Lactate) expresses the anaerobic metabolism

24. mI (myo inositol)glial pool marker. Small amounts from glycine • Glu or Glx (glutamate) neurotransmitter, intermediate in aminoacid catabolism • Gln (glutamine) metabolism of glutamate glial marker

25. PRESS TE 135ms STEAM TE 20ms  Cho  Lac  NAA 4 years old girl: pilocytic astrocytoma Lactate in solid nodule High choline peak In P.A. the Choline Levels are usually below 3.0, while in MB-PNET are usually higher b a c

26. Mean ADC value 1.68 10-3 mm2 /sec DWI TRACE ADC TRACE 215 m.v. 4y.o. girl: pilocytic astrocytoma. Histologic surgical specimen and cell density counting over mixoid (185 cells) and more compact (215 cells) portion of the tumor. 185 m.v. Nr. of cell: 0.083mm2

27. PRESS TE 270ms PRESS TE 135ms Boy, 13 months BRAINSTEM GLIOMA Cho/Cr NAA/Cr Lac : MRS data indicates that it could be a pilocytic astrocytoma

28. b= 800 6 years old boy: Brainstem glioma 1st MRI: diagnosis (WHO I) 2nd:after chemo High Cho 3rd:6mo. after Stop Therapy Higher Cho, low NAA, high lac FLAIR C.E. FSE T1 ADC TRACE PRESS TE 270ms

29. DWI TRACE ADC TRACE DWI TRACE ADC TRACE After chemotherapy At diagnosis b = 800 DWI ADC

30. 6 years old girl: glioma of the midbrain (WHO I) Mild reduction of NAA/Cr and mI/Cr PRESS TE 40ms

31. Boy, 4 years old: Astrocytoma WHO II-III Important reduction of NAA/Cr and moderate increase of Cho/Cr

32. b VOXEL 8 STEAM TE 20 ms 6 years old boy: medulloblastoma CSI PRESS TE 270 ms CSI PRESS TE 270ms: high choline peak (Cho/Cr 4.09), low NAA intensity signal. Small amount of lactate. STEAM TE 20ms: evident lipids peak. a

33. a DWI TRACE ADC TRACE Mean ADC value 1.2 10-3 mm2 /sec Medulloblastoma. 750 m.v. Nr. of cell: 0.083mm2

34. PRESS TE 135ms b STEAM TE 20ms 6 years old girl.: PNET-MB .. STEAM 20: evident mI, lipids and Glx peaks PRESS TE 135ms: high choline peak (Cho/Cr 12.4) and low NAA intensity signal. a

35. STEAM TE 20ms PRESS TE 135ms 11 months old girl: pinealoblastoma. PRESS 135: high choline peak (Cho/Cr 2.6), low NAA signal intensity. Presence of lactate. STEAM 20: evident lipids and mI peaks.

36. Mean ADC value 0.4 x 10-3 mm2 /sec DWI TRACE ADC TRACE Pinealoblastoma 900 m.v. Nr. of cell: 0.083mm2

37. 6 years old girl: choroid plexus carcinoma. All metabolites, included Creatine, but Choline are reduced Cho/Cr 31.5 Mean ADC value 1.0 10-3 mm2 /sec PRESS TE 135ms reducedCr neoplasm do not produce NAA

38. 5 years old girl: germinoma. PRESS TE 135ms high lipids signal. Mean ADC value 0.57 10-3 mm2 /sec Nr. of cell: 0.083mm2 320 m.v.

39. Cho/Cr 6.1 Lac/Cr 6.8 NAA/Cr CSI PRESS TE 135ms PRESS TE 270ms Girl, 23 months metastasis from rabdomyosarcoma

40. Cortical Dysplasia TAYLOR TYPE Increased mI/Cr ratio STEAM TE 20ms

41. STEAM TE 20ms PRESS TE 40ms Boy, 20 months: Tuberous sclerosis Increased mI/Cr ratio

42. ADC vs.Cells number correlation Germinoma • more highly cellular > • smaller intercellular spaces > lower ADC JDGG Pinealoblastoma PNET MB-PNET Pylocitic Astrocytoma (solid portion)

43. BRAIN ABSCESS vs. TUMOR a b DWI ADC DWI is able to discriminate between abscesses and tumors: a) brain abscess; b) supratentorial ependymoma

44. Boy, 6 years old: brain abscess Most of the metabolites are reduced. Lipids and lactate are present

45. Conclusions Brain tumors in children are highly heterogeneous for histology, prognosis and therapeutic response. Diagnosis and therapy of those, most of which are low grade, can be complicated because of their frequent adjacent location to crucial structures that limits biopsy. The utility of combining data from biologically important intracellular molecules, obtained with proton MR spectroscopy and from water mobility, obtained with diffusion imaging, is clearly addressed to increase the diagnostic accuracy in determining the clinical grade of pediatric brain tumors.

46. DWI enable to better differenciate between low- grade and high-grade tumor: high-grade gliomas have lower ADC values than low-grade gliomas. ADC maps are easily generated from routine fast diffusion-weighted imaging by use of software available on many MR systems. ADC may be a more direct indicator of changes in the brain than are other physical parameters. The degree of diffusion is strongly affected by microscopic biological structures such as the number, type and spatial arrangement of cells. These structures create barriers to the free diffusion of water so changes in diffusion may more directly reflect changes occurring within and between cells.

47. It still debating if ADC measurement can be used to determine the extent of tumor infiltration and to differentiate infiltration from peritumoral edema. It has been suggested that tumor infiltration is characterized by lower ADC values than edema. In our experience we found that quite often this is true but we always prefer to be cautious adding spectroscopy data when is possible to perform CSI. We also utilize high-b-value DWI that increase the anisotropy so is more accurate in the assessment of infiltration. Since infiltration occurs within and along white matter tracts, diffusion tensor imaging may yield more useful information.

48. Proton MR Spectroscopy enables the measurements of multiple chemical metabolites in normal and abnormal brain parenchyma. Cho/Cr or Cho/NAA in a lesion correlate with higher cellular proliferation rate and reflect the presence of a more malignant and rapidly growing tumor. It is necessary to correlate MRI data since pilocytic astrocytoma has high Choline and lactate despite it is considered benign: usually Cho/Cr ratio is less than 3. NAA is considered a neuronal marker, which decreases with replacement of neurons by tumor (or other non-neuronal tissue, including necrosis). In choroid plexus carcinoma a very low NAA/Cr is characteristic since the tumor does not produce NAA.

49. MRS can be used to follow tumors over time, since patients can serve as their own control after obtaining a baseline scan. CSI may be useful in monitoring the surgical scar: elevation of Cho/Cr ratio is index of a relapse. We have found this index reliable mostly in anaplastic ependymoma. It has been suggested that children with higher total creatine levels are more responsive to radiation or chemotherapy. In our experience children with low-grade gliomas that have significantly higher baseline Cho/Cr ratio have more chance to have tumor that progress over 2 years than those that have stable tumors. Lactate is no more considered an indicator of malignancy since it is found in benign tumors such as pilocytic astrocytoma.

50. Lipids may be detected in enhancing and non enhancing tumor regions. For a reliable detection of lipid peak it would be better to use acquisition protocols with a TE 65ms that null lactate peak. Lipids represent microscopic tumor cell necrosis or membrane breakdown that may precede necrosis. Lipids may be present in viable tumor, presumably because of poor perfusion and hypoxia and they undergo major intensity changes during apoptosis. They are found also in radiation necrosis. Because glial tumors are grade according to their cellularity, proliferative activity and degree of necrosis, Cho mapping (increased cellularity and proliferative activity) may added value to MRI in childrens with brain tumors, especially when it is combined with lipid mapping (necrosis and/or apoptosis).