(CANCER RESEARCH 58. 1825-1832. May I. 1998)

Correlation of High-Resolution Magic Angle Spinning Proton Magnetic ResonanceSpectroscopy with Histopathology of Intact Human Brain Tumor Specimens1

Leo Ling Cheng,2 I-Wen Chang, David N. Louis, and R. Gilberto Gonzalez

Deparlmenl of Pathology [L. L. C., D. N. L.¡.Nuclear Magnetic Resonance Center und Division of Neuroradiology, Department of Radiology ¡I.W.C., R. G. G.j. NeuroMirgiSen'ice ¡D.N. L.¡,Massachusetts General Hospital. Harvard Medical School, Boston. Massachusetts 02129

ABSTRACT

High-resolution magic angle spinning proton (HUMAS 'III magnetic

resonance spectroscopy produces well-resolved spectra of metabolites

from intact tissue specimens. Here we report the results of a preliminarystudy of 19 human brain tumors obtained by applying this method.Among these 19 cases were 2 low-grade astrocytomas, 1 anaplastic astro-

cytoma, 8 glioblastomas, 6 meningiomas, and 2 schwannomas. In addition,autopsy human brain tissues from two subjects without any known neurological diseases were used as normal controls. The HRMAS 'H magneticresonance spectroscopy measurements were performed at 2 ( ' on a 400-

MHz NMR spectrometer with a HRMAS speed of 2.5 kHz. From theseHRMAS 'H MR spectra, we measured the concentrations of 11 metabo

lites, the ratios of 15 metabolites (resonances) to creatine (at 3.03 ppm),and the spin-spin relaxation time for these metabolites (resonances). Our

results indicate that these parameters have the potential to characterizetumor types and grades with statistical significance as well as identifytumor biochemical characteristics. In particular, we found that comparedwith metabolite concentrations and metabolite spin-spin relaxation time,

the metabolic ratios presented the highest sensitivity in differentiatingnormal tissue from tumors, as well as in distinguishing between tumorgroups. Of 15 analyzed metabolic ratios, 12 showed statistical significancein differentiating normal tissue from low-grade and anaplastic astrocyto

mas, 13 showed statistical significance in differentiating normal tissuefrom glioblastomas, 14 showed statistical significance in differentiatingnormal tissue from schwannomas, and 9 showed statistical significance indifferentiating normal tissue from meningiomas. Moreover, our resultsstrongly indicate that the resonance ratio of inositol (at 4.05 ppm) tocreatine may help distinguish tumor type. Our results suggest that theHRMAS method for intact tissue measurement may function as an adjunct to histopathology and contribute to improved accuracy for braintumor diagnoses.

INTRODUCTION

Clinical data have shown that survival rates among brain tumorpatients vary considerably depending on the type and grade of neoplasm, and that accurate diagnosis can significantly alter therapeuticplanning, potentially improving the clinical outcome (1-3). At present, the diagnosis of brain tumors relies extensively on histopatholog-

ical evaluation of cellular morphological changes of tissue specimens.Despite worldwide efforts to standardize tumor evaluation criteria, themorphological classification of human brain tumors can yield somewhat varied interpretations. This uncertainty results from the inherentcomplexity of tumor cell biology, as well as from discrepancies thatstem from the descriptive nature of morphology-based histopathology.

It is now well known that in addition to exhibiting morphologicalchanges, neoplasms demonstrate an altered metabolism (4). Thesemetabolic changes are currently believed to precede histologicallyobservable cellular transformations (5). Thus, quantification of alter-

Received 9/10/97: accepted 2/23/98.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1Supported in part by an American Cancer Society Institutional Grant IRG-173H (to

L. L. C.), by USPHS Grants NS24279 (to D. N. L.) and NS34626 (to R. G. G.). and by theMassachusetts General Hospital Nuclear Magnetic Resonance Center.

~To whom requests for reprints should be addressed, at Pathology Research. Massachusetts General Hospital CNY-7, 149 13th Street, Charlestown. MA 02129.

ations in tumor metabolism could help to establish biochemical criteria crucial to a more detailed and precise classification of neoplasmsand could ultimately lead to improved diagnostic accuracy.

Over the past decade, despite unceasing effort by scientific andmedical researchers, few techniques that can accurately measure metabolic change within the context of clinical care have been forthcoming. Difficulties in technique and application have primarily beenoccasioned by the subtleties inherent in tumor metabolic alterationsand by limited success in the development of rapid methods requiredfor pathological evaluation. Among the candidate techniques, MRS3

has been considered to have the highest likelihood of success, due toits superior sensitivity in measuring changes within the chemicalenvironment. Thus far, a number of 'H MRS studies of human brain

tumors have been conducted either on intact tissues (6-9) or, mostcommonly, on solutions of tissue extracts (10-17). These studies,

which used conventional MRS techniques designed for the analysis ofaqueous homogeneous solutions, were attempts to correlate the characterization of neoplasms with 'H MRS-measurable alterations in

cellular metabolism.As a consequence of the fundamental physical differences between

tissues and aqueous solutions, the study of nonsolution tissues byusing solution methodologies results in broad-line spectra of low

resolution. These results are therefore of limited use for diseaseevaluation. Alternatively, the MRS analysis of solutions made fromtumor extracts has also been considered. Solutions of tissue extractscan produce high-resolution 'H MR spectra of metabolites that are

soluble in water. However, even if the length of extraction procedurescan be tolerated (a minimum of several hours) or reduced, the destructive nature of extraction may alter the corresponding spectrum toan unknown degree. For this reason, the correlation reached betweenextract spectroscopy results and disease states, and therefore thepractical utility of the extraction approach in a clinical laboratory,remains questionable (4).

Recently, we developed a new method to measure 'H MR spectra

of intact human and animal tissue with high spectral resolution. Byapplying MAS, a technique originally developed for the study ofsolid-state NMR, we demonstrated (18, 19) and other researchers

corroborated (20) that it is possible: (a) to greatly improve the spectralresolution of intact tissue by using MAS; and (b) to successfullycorrelate disease states with the spectroscopic results obtained byusing MAS on tissue specimens.

In this report, we present a preliminary study of human braintumors conducted by applying our newly developed method, HRMAS'H MRS (19). Strong correlations between HRMAS 'H MRS results

obtained from intact tumor specimens and neuropathological evaluation are observed and discussed. In addition, we present a discussionand comparison between spectroscopic results obtained with HRMASapplied on tissue specimens, on the one hand, and results obtained byusing solution measurement on chemical extracts for the same specimens, on the other.

' The abbreviations used are: MRS. magnetic resonance spectroscopy; MR. magnetic

resonance: MAS. magic angle spinning: HRMAS. high-resolution MAS: GBM. glioblastomas: PCA. perchloric acid: ID. one-dimensional: Lac. láclate:Pch. phosphorylcholine,Choi, choline; Cr, creatine; NAA. W-acetylaspartate; t2. spin-spin relaxation time.

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HRMAS 'H MRS OF HUMAN BRAIN TUMORS

MATERIALS AND METHODS

Collection of Tissue Specimens. Tumor and normal brain tissue specimens were obtained from the Department of Pathology at MassachusettsGeneral Hospital. Brain tumor specimens from 19 subjects were characterizedaccording to the revised 1993 WHO criteria for brain tumors (21). Thesesamples, each weighing between 20 and 60 mg, included two low-grade

astrocytomas (WHO grade II), one anaplastic astrocytoma (WHO grade III),eight GBMs (WHO grade IV), six meningiomas, and two schwannomas.Autopsy human brain tissues (including gray, white, and deep-gray matters)

from two subjects without any known neurological diseases were used asnormal controls. All samples were stored at —70°Cuntil ready for MR

spectroscopic analysis.Chemical Extraction. A PCA extraction procedure was used to process

human meningioma specimens. Tissues from three subjects (100, 300. and 300mg) were pooled to create a significant volume of the extract solution. The

extraction was conducted according to a literature procedure (22). In brief, thefrozen tissues were pulverized to a powder in a mortar containing a medium ofliquid nitrogen. A solution of 10% PCA (1 ml/g tissue) was added dropwise tothe sample while pulverizing continuously until a powder of the mixture wasformed. This mixture was then centrifuged at 25,000 rpm at 4°Cfor 15 min in

a Beckman TJ6 refrigerated centrifuge. The supernatant was removed and kept

cool in ice, and the pH was adjusted to 9.0 by KOH. The resultant saltprecipitate was removed by a second 10-min centrifugation. The supernatant

was frozen, lyophilized. dissolved in deuterium oxide, lyophilized again, and

kept for future spectroscopic analysis.'H MRS. The spectroscopic procedures used in the HRMAS study of the

tissue specimens, as well as that applied in the study of the extract solution,have already been described (19).

In brief, HRMAS experiments were performed at 2°Con a MSL400 NMR

spectrometer (proton frequency at 400.13 MHz) by using a BD-MAS probe

(Bruker Instruments, Inc.. Billerica, MA). Temperature was controlled by aVT-1000 unit, in combination with a MAS-DB pneumatic unit (Bruker Instru

ments, Inc.). Sample spinning rate was stabilized at 2.500 ±0.001 KHz. Arotor-synchronized Carr-Purcell-Meibom-Gill pulse sequence [90-(T-180-T)n-

acquisition] was used as a 12 filter. t2 filters were applied in order to further

reduce the interference of tissue water and macromolecules, such as lipids. onspectral resolution. The interpulse delay (T = lirlw, = 400 /is) was synchro

nized with the rotor rotation (where ^ is the sample spinning speed in timeunits, and w/27r represents the MAS speed in kHz.) The value for n was 750(2nr = 600 ms). The 90-degree pulse length was adjusted individually for each

sample and varied from 7.1 to 10.6 /MS.The number of transients was 512. with

an acquisition time of 819 ms. A repetition time of 3.0 s and a spectral widthof 8 KHz (20 ppm) were used. t2 was measured by using the same Carr-Purcell-Meibom-Gill sequence with 64 transients and by varying n from 200 to900 (160 to 720 ms). 'H MR spectra of the solution of meningioma extractswere acquired at 20°Con the above-mentioned NMR spectrometer by using a

5-mm probe (Bruker Instruments. Inc.). Spectral acquisition parameters included a one-pulse excitation, a repetition time of 15 s, a spectral width of 4

KHz, 32K complex points, and an average of 64 transients.Before Fourier transformation and phase adjustment, all free induction

decays were subjected to 1-Hz apodization. TMS at 0.00 ppm was used as an

external chemical shift reference, from which the internal reference of the Lacdoublet was determined to be 1.32 and 1.34 ppm (4, 23).

Tissue water was used as an internal standard to estimate the concentrationof brain metabolites. The resonance of tissue water (varying from 4.83 to 5.01ppm depending on individual samples) was measured without presaturation.The concentrations of metabolites in tissue specimens were estimated by usinga previously discussed formula (19). This estimation is based on the integration(with Lorentzian and Gaussian curve-fitting) of resonance intensities of metabolites and tissue water from /2-filtered (600 ms) spectra on the t2 values

measured for metabolites and water with each specimen, and on an approxi

mation that brain tissue contains 80% of water relative to its weight, i.e., 44mmol/g (11, 19).

Unless otherwise specified, a two-tailed and unpaired Student's t test was

used to analyze the statistical significance of using metabolic alterations and I2variations to differentiate tumors from normal tissue, as well as betweenvarious tumors.

RESULTS

HRMAS 'H MRS of Brain Neoplasms. InFig. 1, the HRMAS 'H

MR spectra obtained from human tissue specimens of normal brainand various tumors are represented. These spectra were acquired at2°Cwith a Carr-Purcell-Meibom-Gill pulse sequence and a /, filtering

time of 600 ms. By examining the spectra, visible differences inmetabolic resonances between different pathological specimens canbe recognized.

Measurement of Metabolic <2.Most of the resonances shown inFig. 1 can be assigned to various metabolites found in the normalhuman brain. Selected resonances and their corresponding metabolitesare listed in Table 1, together with the means and SEs of their /2,measured for various pathological specimens. Because of the smallnumber of specimens available for this preliminary study, "Normal"

in the table represents the mean of individual measurements of normalgray matter (n = 3), white matter (n = 2), and deep-gray matter(n = 2). Similarly, the low grade/anaplasticastrocytoma group (LG &AA) includes both low-grade astrocytomas (n = 2) and anaplasticastrocytomas (n = 1).

Resonance Ratio and Metabolic Concentration. The alterationsin tumor metabolism reflected in HRMAS 'H MR spectra of tissue

specimens, as shown in Fig. 1, can be quantified either by an assessment of changes of resonance ratios or by an estimation of metabolite

AnaplasticAstrocytoma

Glioblastoma Normal

4.00 3.00 2.00 1.00 4.00 3.00 2.00

Chemical Shift (ppm)1.00

Fig. I. HRMAS 'H MR spectra of intact tissue specimens from various types of human

brain tumors and normal brain tissue. *. possible chemical contamination during pathological processing (44).

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HRMAS 'H MRS OP HUMAN BRAIN TUMORS

Table l Chemien/ shift, mean, and SE of I, for brain metabolites

Normal LG&AA'' GBM Schwannoma

" CS, chemical shift in ppm.* Mean, mean of metabolite I2 in ms.' SE, SE of metabolite t-, in ms.' LG&AA, low-grade/anaplastic astrocytomas.

Meningioma

MetaboliteLacInositotCrGlycinePchChoiGlutaminaGlutamateNAAAcétaleAlaLipidses"4.121.334.053.933.033.553.223.202.432.352.011.921.480.95Meanfr242.6247.7316.8364.6516.3336.4341.3442.5326.1290.0311.9520.5355.3312.2SE'10.819.917.417.724.223.412.730.228.420.88.747.528.724.9Mean220.0226.0207.0211.3269.7277.0306.0346.3329.0154.0291.0354.5210.0250.0SE15.442.914.024.821.725.17.120.589.5Mean387.9459.0353.7319.5424.1398.3329.9321.0342.7324.4362.0479.5325.1346.1SE28.452.952.531.333.290.420.345.034.641.823.048.039.728.5Mean250.5381.0262.5291.5326.0314.0404.0285.0250.0372.0308.0SE39.537.018.538.530.018.040.056.023.0Mean283.7323.0334.535

1.0283.6340.8337.0419.6333.3305.4206.0266.0348.8363.5SE29.337.594.544.749.827.449.471.847.117.026.231.349.4

concentrations. Fig. 2 presents the ratios of various metabolites overthe Cr resonance at 3.03 ppm. The resonance intensities (peak height)used to generate this chart were measured directly from ^-.-filtered(600 ms) HRMAS 'H MR spectra, thus minimizing any possible error

of resonance peak integration. Peak height can be used in suchestimations, because the examined resonances of different metabolitesin an observed spectrum are reasonably well separated and haveapproximately similar linewidths. Depending on the individual spec-

troscopy measurement evaluated, the SE of the measured resonancelinewidths in any given spectrum represents only 5.8-14.3% of the

mean measured resonance linewidth.A total of five different groups of specimens are shown in Fig. 2:

(a) normal controls; (b) low-grade/anaplastic astrocytomas; (c)

GBMs; (d) schwannomas; and (/) meningiomas. Again, the normalcontrol category includes gray, white, and deep-gray matter, whereasthe low-grade and anaplastic astrocytoma category contains two casesof low-grade astrocytomas (WHO, grade II) and one case of anaplastic

astrocytoma (WHO, grade III). Resonances of known origins, i.e.,those that have been assigned to their corresponding metabolites (4,19, 23), are represented by the names of the corresponding metabolites as well as by resonance frequencies (in ppm); e.g.. Lac (Lac,4.12), inositol (¡nos,4.05), glycine (G/v, 3.55), Pch (Pch, 3.22), Choi(Choi, 3.20), glutamine (Gin, 2.43), glutamate (Glu, 2.35), NAA(NAA), alanine (Ala, 1.48), and lipids (Lipids. 0.95). As reportedpreviously (19), in this chart, NAA includes both NAA at 2.01 ppmand its degradation product, acetate, at 1.92 ppm. Several distinctiveresonances that have not yet been positively assigned (3.98, 3.93, and3.27 ppm) are also included in this figure.

Fig. 3 shows estimations of metabolite concentrations for selectedresonances, which were obtained by using tissue water as an internalreference (see "Materials and Methods"). Metabolite concentrations

for various pathological specimens are also classified into five groupssimilar to those in Fig. 2. However, unlike Fig. 2, only resonanceswith assigned metabolite origins, including Cr at 3.03 ppm, arecalculated for their concentration and presented in the chart.

HRMAS 'H MRS versus Tissue Extracts. To compare the similarities and differences between HRMAS 'H MR results and tissue

extraction results and to further assess the completeness of the commonly used PCA extraction procedure, human meningioma specimenswere subjected to both 'H MRS analytical procedures. Fig. 4a shows

a spectrum of meningiomas obtained from tissue specimens withHRMAS, whereas Fig. 4, b and c, shows the spectra of extracts fromthe same specimens. Fig. 4a represents a weight-normalized sum of

three HRMAS spectra obtained independently from three individual

meningioma specimens included in the tissue extraction; the resultingextract was subsequently used to obtain the results shown in Fig. 4, band c. To obtain Fig. 4a, the three HRMAS spectra were addedaccording to the ratio of tissue weight (1:3:3) used in the extraction(see "Materials and Methods"). Examining Fig. 4, we noticed remark

able differences between the HRMAS and the extract spectra obtainedfrom the same tissue specimens. One such difference is labeled withan arrow in this figure for the Choi-containing metabolites. We willfurther elaborate the implications of these differences in the "Discussion" section.

DISCUSSION

HRMAS 'H MR Spectra of Brain Neoplasms. Encouraged by

advancements in the in vivo brain MRS during the past decade, manyex vivo 'H MRS studies have been undertaken to classify brain

neoplasms, to more accurately interpret in vivo observations, and tobetter understand the cell biology of brain neoplasms. However, theseex vivo efforts have been largely hampered by the traditional techniques of conventional solution NMR. Whereas water solutions ofchemical extracts cannot truthfully reflect the in vivo condition oftumor metabolism in tissue, poor spectral resolution of intact tissuespecimens cannot identify a number of important tumor-related me

tabolites. For example, it is impossible to separate the resonance ofChoi (3.20 ppm) from that of Pch (3.22 ppm) in tissue spectraobtained by means of conventional solution NMR methods (6-9).

The low 'H MR spectral resolution of tissue specimens is due partly

to the nonsolution characteristics of tissue and partly to the inhomo-

geneity of the magnetic field experienced by the specimen (magneticsusceptibility.) To overcome the effects of magnetic susceptibility, acapillary sample handling procedure was introduced that has provedrelatively effective in narrowing spectral linewidth (24). However,even with this procedure, the resulting spectral resolution was toobroad to allow accurate assignments of resonances (7, 8).

Two-dimensional correlation spectroscopy has been suggested to

improve spectral resolution, i.e., to separate individual resonancesspectroscopically (4, 6, 9, 25), and more than 50 tumor-related bio

chemical species have been identified by using this method (4, 26).Unfortunately, each two-dimensional correlation spectroscopy meas

urement requires at least several hours to complete, a time scale thatprecludes its routine application and adoption in many importantclinical settings, such as intraoperative consultation. Investigationscarried out by using conventional ID 'H MRS on intact tissue were

focused on estimating ratios of different spectral regions and/or using

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HRMAS 'H MRS OF HUMAN BRAIN TUMORS

op

tiII£.iiU•;T

:,JDo~^nnnHnnJ

Lac4.12

Ino* Crl?) Gly Peti Chol Gin Glu

Metabolites ([M]/[Cr,3.03] %)

Lipidi

Fig. 2. The means and SEs of selected ratios (percentages) of resonances over Cr (3.03ppm) measured from I2-filtered (600 ms) HRMAS 'H MR spectra of intact tissue

specimens of the normal human brain and various tumors. Normal represents the mean ofindividual measurements of normal gray matter (n = 3), white matter (n = 2), anddeep-gray matter («= 2). Similarly, Love-Grade and Anaplastic Astrocylomas representsthe mean of low-grade astrocytomas (n = 2) and anaplastic astrocytomas (n = 1).

Resonances with identified metabolites are shown in terms of both the names of thecorresponding metabolites and the chemical shift values (in ppm), whereas, for theunassigned resonances, resonance ratios are presented by their chemical shift values alone.NAA represents the sum of NAA at 2.01 ppm and its degradation product of acetate at 1.92ppm. The chart for Normal is vertically scaled differently (20X) from those for tumors topresent normal metabolism with better clarity.

pattern recognition tools of mathematics (4, 8, 27-30). These ID

measurements may have an advantage over classical histopathology,because a relatively large volume of samples can be analyzed withless human power involved. Results have shown that the detection ofcancer cells with ratios of spectral regions obtained by means of ID'H MRS might be more sensitive than regular histopathology, thanks

to the reduction of sampling errors inherent in sample preparation forhistopathological evaluation (5). Nevertheless, poor tissue ID spectralresolution, which prevents both accurate measurement of individualmetabolite and effective correlation between metabolic changes andtumor biology, makes the usefulness of these methodologies somewhat limited.

Fig. 1 shows that superior spectral resolution is attainable with

HRMAS on intact brain specimens of normal tissue and varioustumors. The results reported in this figure strongly confirm our previous conclusions regarding the utility of HRMAS for high-resolution

analysis of ex vivo brain tissue (19). The HRMAS technique can notonly reduce the resonance line-broadening caused by tissue nonsolu-

tion characteristics, but it can also minimize the influence of magneticsusceptibility effects on spectral linewidth. Such high spectral resolution of HRMAS has been observed only in homogeneous solutionsand has never been reported in intact brain tissue. Only by applyingHRMAS can individual resonances be monitored and measured accurately. The close association of tissue HRMAS measurements within vivo physiology further allows us to assess important biologicalcharacteristics of tumor metabolism, such as metabolic concentrationand r2, for a number of brain metabolites. However, here, it is worthnoting that for both normal brain and tumor specimens, the high exvivo concentration of Lac, a final metabolite of glucose anaerobic

I*g£<«T

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60

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30

20

10

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»2010

arJJl

Ala Lac Lipidi1.48 1.33 0.95

Metabolites ([m M])

Fig. 3. The means and SEs of selected metabolite concentrations (/Amol/g wet tissue ormM) calculated from (2-filtered HRMAS 'H MR spectra of intact tissue specimens for the

normal human brain and various tumors. Metabolite concentrations presented werecalculated by using tissue water as an internal reference and corrected for t2 effects forindividual metabolites. Metabolites are identified by both their names and their chemicalshift values (in ppm). NAA represents the sum of NAA at 2.01 ppm and its degradationproduct of acetate at 1.92 ppm.

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HRMAS 'H MRS OF HUMAN BRAIN TUMORS

HRMAS Intact Tumor tissue

Extract Solution Portion

LC.

4.00 3.00 2.00

Chemical Shift (ppm)1.00

Fig. 4. The 'H MR spectra of meningiomas obtained from intact tissue by HRMAS (a).

from a water-soluble PCA extract by conventional solution NMR procedure (b). and from

lipid precipitates from extraction by HRMAS (r). Lipid portion of sample is usuallydiscarded without further analysis. Choi and its derivative metabolites are labeled by anarrow above spectrum (a).

processes (31), is likely to result for two principal reasons. It is wellknown that even at rest and under well-oxygenated conditions, brain

tissue produces a very small amount of Lac (<0.5 HIM; Ref. 32),which is continuously removed by the venous blood. Under conditionsof poor oxygénation,however, such as ischemia, the level of Lac canbe as high as 40 mM (33). Such an anaerobic production of Lac bybrain tissue would explain our ex vivo observation of ~38 mM Lac in

normal brain tissue, shown in this figure. Also, tumors are likely tocontain even less glucose than normal brain tissue and thus are likelyto produce smaller amounts of Lac under anaerobic conditions (forinstance, low-grade and anaplastic astrocytomas). However, tumor

necrosis also results in a perceptible accumulation of Lac, as shown inthe case of GBMs.

The metabolic concentrations quantified from the HRMAS spectraof this study are either higher than or similar to literature data ontissue extracts of normal and various tumor tissues (11, 13, 16).Among the metabolites we analyzed, Cr is the one most often considered to be primarily associated with cytoplasm and thus lessaffected by restricted tissue molecular motion. Given its close association with cytoplasm, concentrations of Cr determined by means ofHRMAS are more likely to resemble results obtained by use ofextraction. On the other hand, the calculated HRMAS concentrationsof less-mobile metabolites, such as NAA, are likely to be significantly

different from those measured by means of extraction. This can beseen in Fig. 5 by comparing our calculated HRMAS concentrations ofCr and NAA to analogous findings reported in PCA extraction literature.

/2, an important physical parameter of tissue metabolites, is directlyrelevant to the study of in vivo MRS and imaging. Ex vivo measurement of t2 with tissue specimens can have an immediate impact on thedesign of metabolite-selective in vivo diagnostic technology. Metab

olite i2 values of normal specimens measured in this study, shown inTable 1, are in close agreement with our previously reported resultsfor the control regions from a study of a Pick disease case (19). /2variations for all measured metabolites (Lac, inositol, Cr, Pch, glutamate, NAA, acetate, and alanine) are between —9.2and 10.5% of the

previously reported values, with the exception of the metabolites Cr

and acetate (19). For Cr and acetate, the observed variations are 54and 31%, respectively. This agreement establishes the reliability ofmeasurements of tissue metabolic t2 that can be obtained by using theHRMAS methodology. Here, we observed that for the group oflow-grade and anaplastic astrocytomas, the mean of metabolite /2s

decreased 24.1% from that of normal brain, whereas the mean forGBMs increased by 10.1% when compared to normal controls. Thisstatistically significant (P < 0.0009) t2 variation may result frommicroscopic changes in the physical states of tumor metabolites.Longer /2s indicate that the metabolites exist in a more liquid-like

environment, which may contribute to the observed t2 increases inGBMs. On the other hand, the decreases in t-, observed in low-grade

and anaplastic astrocytomas suggest a more solid appearance for thesetumors. The significance of these observations and their possiblecorrelation with tumor biology require further investigation.

Classification of Brain Neoplasms. In this work, we have analyzed the potential of the HRMAS method with respect to the diagnosis of human brain neoplasms. Our analytic results are summarizedin Tables 2-4, respectively, for metabolite concentrations, metabolic

ratios, and the t2 values of metabolites. The matrices of these tableslist metabolites that can help to differentiate one histopathologicalgroup of tissue specimens from another. The degree of statisticalsignificance is indicated by the number of asterisks after the metabolite name. The greater the number of asterisks, the higher the level ofsignificance.

Table 2 shows that even with the small number of specimens availableduring this pilot investigation, the estimated HRMAS concentrations ofmany metabolites make statistically significant distinctions between normal tissue and tumor tissues, as well as between different types of tumors.For instance, the increase in Choi resonance at 3.20 ppm is sensitiveenough to allow the discrimination of normal controls from GBMs(P < 0.020) as well as from schwannomas (P < 0.013) and meningiomas(P < 0.034); it also allows us to distinguish GBMs from low-grade/

anaplastic astrocytomas (P < 0.030). As an even more sensitive measure,

1 2

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NAA

Normal LG-AA GBM Normal LG-AA GBM

Fig. 5. Comparison of metabolite concentrations of Cr and NAA measured by usingHRMAS in this study with literature values obtained by extraction procedures. The Crconcentration obtained in this study is similar or higher than those reported with extracts,whereas the NAA concentration measured by HRMAS is much higher than those measured by extracts. Slight differences exist in tumor grouping between this study and thepublished results. LG-AA of Y. K. is the average of values of aslrocytomas and anaplasticastrocytomas found in Ref. 13 from Y. Kinoshita et al.; LG-AA of J. P. is the value ofbenign astrocytomas adopted from J. Peeling and G. Sutherland in Ref. 16; LG-AA ofJ-P. R. U. is the value for astrocytomas grade 1-2 found in Ref. 11., Usenius et al.; GBM

of J. P. is the value for malignant astrocytomas found in Ref. 16, whereas GBM ofJ-P. R. U. is listed as astrocytomas grade 3-4 in Ref. 11.

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HRMAS 'H MRS OF HUMAN BRAIN TUMORS

Table 2 Matrix of selected brain metabolite concentrations measured with HRMAS HMRS for differentiation between different pathological .specimens

NAA, in the table, includes both measured resonances of NAA at 2.01 ppin and acetateat 1.92 ppm: see the text for details. Numbers in parentheses represent resonance chemicalshift in ppm. The resonance at 3.93 is tentatively assigned to the Cr metabolite. As anexample of the use of this matrix, the Choi resonance can be used to differentiatelow-grade/anaplastic astrocytomas from GBMs with a significance of P < 0.05. Similarly,the glycine resonance (Gly) can be used to distinguish GBMs from Schwannomas with aP < 0.005.

NormalLG&AA"Normal

NAA*Lac

(1.33)'LG&AAGBMsSchwannomasMeningiomasGBMsCr

(?,3.93)'Gly

(3.55)''Chol (3.20)*Cr(3.03)*Lac

(1.33)*Chol

(3.20)'SchwannomasChol

(3.20)*NAA*

Lac(1.33)''Gly

(3.55)''MeningiomasCr

(?,3.93)*Chol

(3.20)'Glu (2.35)''Ala(1.48)*Glu

(2.35)*Gly

(3.55)*

" LG&AA. low-grade/anaplastic aslrocyloma.* P < 0.05. two-tailed Student's f test.'' P < 0.0005, two-tailed Student's t test.dP < 0.005. two-tailed Student's / test.' P < 0.05. calculated according to one-tailed Student's I test, based on the hypothesis

that chol increases in tumors.' P < 0.00005, two-tailed Student's l test.

Table 3 Matrix of .selected brain metabolic ratios measured with HRMAS H MRS fordifferentiation between different pathological specimens

NAA. in the table, includes both measured resonances of NAA at 2.01 ppm and acetateat 1.92 ppm; see the text for details. Numbers in parentheses represent chemical shift inppm. The resonance at 3.93 is tentatively assigned to the Cr metabolite.

NormalLG&AA"Normal

Lac(4.12)*Inositol(4.05)*(3.98)'Cr

(?,3.93)''Gly(3.55)*(3.27)''Pch

(3.22)*Choi(3.20)'Gin(2.43)*NAACLac

(1.33)rLipid(0.95)*GBMsLac

(4.12)c(3.98)'Cr

(?,3.93)''Gly

(3.55)*(3.27)''Pch

(3.22)'Choi(3.20)'Gin

<2.43rGlu(2.35)'NAA''Ala

(1.48)'Lac(l.33)cLipid

(0.95)*SchwannomasLac

(4.12)''Inositol(4.05)''(3.98)''Cr

(?,3.93)'Gly(3.55)'(3.27)''Pch

(3.22)''Choi(3.20)*Gin(2.43)'Glu(2.35)*NAA'Ala

(1.48)*Lac(1.33)'Lipid

(0.95)'MeningiomasLac

(4.12)*(3.98)*Cr

(?,3.93)'Gly(3.55)*(3.27)'Gin

(2.43)*Glu(2.35)*Ala(1.48)*Lac

(1.33f

LG&AA Inositol (4.05)' Inositol (4.05)' Ala (1.48)'(3.98)'

Inositol (4.05)'' Gly (3.55)'

GBMsSchwannomasMeningiomas(3.98)'

Gly (3.55)*Choi(3.20)*Glu

(2.35)'Ala(1.48)*Lipid

(0.95)'Glu

(2.35)'Ala(1.48)*Inositol

(4.05)'NAA'

" LG&AA. low-grade/anaplastic astrocytoma.b P < 0.005. two-tailed Student's / test.' P < 0.05. two-tailed Student's ( test.a P < 0.00005. two-tailed Student's I test.' P < 0.0005. two-tailed Student's I test.

Metabolic ratios can be measured directly from spectra and haveimportant utilities for in vivo diagnosis because of their visualsimplicity; in fact, most of the I'Mvivo brain MRS studies are

reported in terms of metabolic ratios or metabolic profiles, ratherthan absolute metabolite concentrations. In Table 3, we analyze thediagnostic significance of metabolic ratios shown in Fig. 2. Comparing Table 3 with Table 2, it is clear that the metabolic ratiosseem to be more sensitive than the estimated concentrations for thepurpose of differentiating one group of pathological specimensfrom the other. This difference in sensitivity may be attributed topossible errors and uncertainties introduced during the process ofconcentration calculation. For example, resonance integration and?2 estimation procedures can be susceptible to different sources oferrors.

We have further examined our findings by comparing them with I'M

vivo observations reported by other researchers in a recently publishedMRS study of 91 brain tumor cases (34). In their report, metaboliteprofiles were constructed with six resonance peaks (Chols, Cr, NAA,Ala, Lac, and lipid) from individual I'Mvivo spectra classified accord

ing to tumor types (WHO grade II. Ill, and IV astrocytomas; menin-

giomas; métastases;and nontumorous brains). Based on the analysesof these metabolite profiles, the success rate in tumor classificationwith in vivo MRS was determined to be 99%. Here, we constructHRMAS metabolite profiles according to the metabolite concentrations (in Fig. 3) of Pch + Choi (equivalent to the I'Mvivo Choi

intensities), Cr, NAA, Ala, and lipid. Despite the value of the ex vivoLac levels as a metabolic marker suggested by both Tables 2 and 3,Lac was excluded from our profiles, because ex vivo levels of Lacfollow different mechanisms from that of I'Mvivo levels. The metab

olite profiles we obtained agreed in many respects with the reportedprofiles. For instance: (a) the higher the grade of astrocytoma, thelarger the Chol:Cr ratio, due to increased cell proliferation; (h) thehigher the grade of astrocytoma. the lower the NAA due to decreasednumbers of neurons, and the higher the lipid intensities associatedwith the development of necrosis; and (f) increased Ala levels wereobserved with meningiomas. Nevertheless, the following differencesbetween our study and the I'Mvivo investigation were observed: (a) the

relative intensity of Pch + Choi to other metabolites measured withHRMAS seems higher than that of Chols reported I'Mvi'vo for all types

of tissues, including the normal controls. This difference may indicatethat these metabolites possess certain solid-like characteristics that aremore difficult to detect with I'Mvivo procedures; and (b) the relative

Table 4 Matrix of selected brain metabolic t: measured with HRMAS '// MRS for

differentiation bet\\'een different palholttgical specimens

Numbers in parentheses represent chemical shift in ppm. The resonance at 3.93 istentatively assigned to the Cr metabolite.

NormalLG&AANormal

LG&AA"Inositol

(4.05)*Cr(?,3.93)*Cr(3.03)''GBMsLac

(4.12)*Choi(3.20)'Cr(3.03)'NAA

(2.02)''Lac

(1.33)*Lac(4.12)'Cr

(3.03)'Lac(1.33)'SchwannomasCr

(3.03)*Acetate(1.92)'Lac

(1.33)'MeningiomasCr

(3.03)*NAA(2.02)''Acetate

(1.92)*

NAA can separate normal tissue from low-grade/anaplastic astrocytomas

(P < 0.0042) as well as from GBMs (P < 0.0000015). We have alsonoticed that total Choi concentrations (Choi at 3.20 ppm + Pch at 3.22ppm) can significantly impair our ability to use the apparent sensitivity ofChoi increases at 3.20 ppm for tumor differentiation. This observationreinforces our view that by resolving and measuring individual resonance, HRMAS is crucial for the purpose of better understanding tumormetabolism.

GBMs

Schwannomas

Meningiomas" LG&AA. low-grade/anaplastic astrocytoma.h P < 0.005, two-tailed Student's t test.'' P < 0.05, two-tailed Student's t test.

JP< 0.0005. two-tailed Student's I test.

Lac (4.12)'Cr (3.03)'NAA (2.02)'Acetate (1.92)'

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HRMAS 'H MRS OF HUMAN BRAIN TUMORS

intensity of NAA in our profiles seems less than its in vivo measurement for various astrocytomas. We attribute the higher in vivo NAAlevel to tumor infiltration, resulting in the contamination of normaltissue due to the volume-averaging effect of the large in vivo region of

interest (1 ml). This possibility was minimized by the HRMAStechnique, which used only 20-60 /M!of the specimens examined

individually by neuropathology.Finally, Table 4 illustrates the significance of /2 variations in

relation to various pathological conditions of specimens. Significantlydifferent t2 values can be used to develop insensitive techniques thatmay assist with in vivo discrimination among different neoplasms. Forinstance, Cr resonance at 3.03 ppm may be used to differentiateGBMs from low-grade and anaplastic astrocytomas by using a r2-

sensitive chemical shift image technique.The Advantages of HRMAS 'H MRS. Although chemical ex

traction of tumor specimens has been studied by MRS over the past 10years, the relationship between metabolite concentrations derivedfrom extracts and actual in vivo concentrations is difficult to assess.Metabolites that are either closely associated with cell membranes orhave low water solubility, such as lipids and phospholipids, are likelyto be poorly extracted. It is unfortunate insofar as recent studies haveindicated that many of these metabolites might be closely related totumor development and progression (35, 36).

A major difference between a HRMAS spectrum (Fig. 4a) and anextract solution spectrum (Fig. 4£>)concerns the intensities of metabolitesat —3.2ppm (arrow), which are normally referred as Choi-containingmetabolites in the in vivo literature (10, 34, 37-42). Compared with the

extract solution spectrum (Fig. 4b), the resonances in this region in theHRMAS spectrum (Fig. 4a) are substantially more intense relative to theintensity of the Cr resonance (at 3.03 ppm). This difference remains aftercorrection for r2 effects in the HRMAS spectrum. This comparisonindicates that routinely used chemical procedures may not fully extractthe metabolites that form the peaks of this spectral region. As a result, itis very likely that previous ex vivo extract studies have concluded incorrect concentrations for these metabolites. We further tested this observation by means of a subsequent HRMAS spectrum (Fig. 4c) of lipidprecipitates obtained by extraction from the same meningioma samples.Although the lipid portion of extraction is usually discarded withoutfurther analysis, our spectrum in Fig. 4c clearly shows presence ofresonance intensities in this region. Our finding is important in light of thesignificance of the functions of Choi metabolites in tumor proliferation ingeneral (35) and in in vivo MR observations of intensive resonancesappearing in the spectral region in particular (34). It is important toinclude these intensities in an analysis to more accurately interpret in vivoobservation. Unfortunately, no known extraction method can fulfill thisrequirement.

In addition, unlike tissue extraction, which requires larger amountsof tissue (approximately 200-500 mg), only a small amount of

specimens (<50 mg or /xl) is needed for a HRMAS analysis. Suchtissue samples can be selected from sites that are adjacent to the locusof histopathological examinations. In this way, correlations betweenHRMAS MRS and histopathology will be less affected by tumorheterogeneity (43). Consequently, the HRMAS method may permit acloser evaluation of metabolic alterations with respect to tumor severity, as determined by neuropathologists, for each individual regionwithin a particular tumor.

Finally, we wish to mention the existence of additional advantages ofthe HRMAS 'H MRS method: (a) this method is independent of theobserver's skill and experience, and the analysis can be completed in a

few minutes, unlike histopathology; (b) HRMAS preserves tissue structures intact during measurement and produces results of physiologicalrelevance, by contrast with chemical extraction; (c) HRMAS can measurevariable volume of tissue from microliters to milliliters and is therefore

suitable for measuring both small biopsy and large surgery specimens; (d)HRMAS can not only provide molecular markers as independent criteriareflecting certain aspects of tumor biology that may assist histopathologyto better classify brain neoplasms and improve diagnostic accuracy, but itcan also provide vital prognostic criteria that are inaccessible to theclassical histopathology; and (e) HRMAS is objective and allows for afull automation of the entire procedure, including digitization of analysesand computerization of reports, thus potentially reducing cost in certainpathological examinations.

Conclusion. The results of our study suggest that the HRMASmethod can produce well-resolved 'H MR spectra of intact tissue

specimens excised from a variety of brain tumors. We have shownthat metabolite concentrations, metabolic ratios, and even variationsin metabolic r-, can be used to distinguish normal tissue from tumors

and to differentiate between tumor types. These findings support thatthe HRMAS 'H MRS method can potentially serve as a valuable

adjunct to current neuropathological techniques and is likely to resultin the increasingly accurate diagnosis of human brain neoplasms.Tumor metabolism obtained from HRMAS 'H MRS measurements

may also have important implications for the understanding of tumorcell biology and for designing new diagnostic methodologies that usei/i vivo MRS and imaging.

ACKNOWLEDGMENTS

We thank Dr. M. J. Ma for collaboration in the specimen collection and J.Fordham for editorial assistance. L. L. C. dedicates his contribution to thiswork to the memory of his late sister, Wei Cheng (1962-1991), who died of

brain tumors.

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1998;58:1825-1832. Cancer Res   Leo Ling Cheng, I-Wen Chang, David N. Louis, et al.   Intact Human Brain Tumor SpecimensMagnetic Resonance Spectroscopy with Histopathology of Correlation of High-Resolution Magic Angle Spinning Proton

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