(CANCER RESEARCH 49. 770-779. Februar} 15. 1989]

Review

Magnetic Resonance Spectroscopy of Tumors and Potential in VivoClinicalApplications: A ReviewPeter F. Daly1 and Jack S. Cohen2

Clinical Pharmacology ¡J.S. C.J and Pediatrie Oncology fP. F. D.¡Branches, National Cancer Institute, NIH, Bethesda, Maryland 20892

Abstract

The development of nuclear magnetic resonance (NMR) spectroscopyas an established research tool for noninvasive studies of cancer cells andfor in vivo studies of tumors in animals and humans has led to thepossibility that this technique may be used in the future for clinicalresearch studies and monitoring of therapy in cancer patients in combination with magnetic resonance imaging. This article provides a briefqualitative explanation of NMR spectroscopy and then reviews the celland animal studies detailing which biochemicals can be observed in vivoby "I", "( , and 'II NMR. The human studies done to date and their

potential for diagnosis and monitoring of therapy are then discussed. Inaddition, "F NMR spectroscopic studies of fluorinated drugs and 'II and"P NMR studies relating to drug resistance are mentioned. The current

technical limitations and developing improvements are indicated also.

Introduction

A growing body of research concerning magnetic resonancespectroscopy in cancer has developed over the past 10 yearsbecause of its potential for noninvasive localized in vivo measurements of concentrations of specific water soluble metabolites. As with other radiological methods the biological/medicalutility of the results (the spectra) are more important to theclinician than details of how they were obtained. Therefore, thisreview will use as a focal point the currently published humanin vivo "P spectra of tumors and discuss both the basic biochemical and clinical implications. 'H, "F, and I3C MRS3 will

also be discussed. Qualitative explanations of the key termsused in MRS will be given, but an extensive detailed explanationis beyond the scope and purpose of this review. More detailedaccounts of the methods of spectroscopy and imaging writtenfor radiologists have been published (1,2).

The suggested clinical applications of this technique to oncology include /// vim human diagnosis (3-11), in vivo humanmonitoring of chemo- and radiation therapy (3-5, 8-10, 12,13), prediction of drug resistance (14-16), prediction of met-astatic potential (17, 18), and prediction of hormonal responseof breast cancer (19, 20). Many of these potential applicationsare currently beyond the capabilities of human spectrometersdue to problems of obtaining spectra only from the volume oranatomic structure of interest and the low signal:noise ratio for

Received 3/17/88; revised 8/8/88, 10/31/88; accepted 11/10/88.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 inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

' Present address: Pittsburgh NMR Institute. 3260 Fifth Avenue, Pittsburgh,

PA 15213.2To whom requests for reprints should be addressed, at Clinical Pharmacology

Branch, Building 10, Room 6N119, NIH, Bethesda, MD 20892.' The abbreviations used are: MRS, magnetic resonance spectroscopy; DPDE,

diphosphodiester; GPC, glycerophosphorylcholine; GPE, glycerophosphoryl-ethanolamine; PC, phosphocholine; PCr, phosphocreatine; PDE, phosphodiester;PE, phosphoethanolamine; PME, phosphomonoester; NMR, nuclear magneticresonance; FUra, 5-fluorouracil; FdUMP, 5'-fluoro-2'-uridine monophosphate;FUTP, S-fluorouridine triphosphate; FUH:, 5.6-dihydrofluorouracil; FUPA, o-fluoro-iJ-ureidopropionic acid; FBAL, a-fluoro-tf-alanine; FdUDP, 5-fluorodeox-yuridine diphosphate; FdUTP, S-fluorodeoxyuridine triphosphate; FdUrd, 5'-deoxy-5-fluorouridine.

in vivo 31PNMR. For these reasons, tumors in vivo in animals

(21-25), human biopsy material (26, 27), and acid extracts oftumors (14, 28-30) have provided biochemically detailed cor

relating data not easily obtained by human in vivo studies.However, biochemical interpretations of differences in the spectra between normal and malignant tissue, or the pathophysiol-ogy producing changes in the spectra following radiation andchemotherapy, have been hampered by the heterogeneity ofmalignant growths and lack of a complete understanding of thebiochemical source and pathways of some of the signals. Intact,metabolizing cells can be studied by embedding them in agarosegel threads (14, 31-34, 104) or growing them in basement

membrane gel threads (35). These model systems allow forhomogeneity, high resolution and separation of signal whichoverlap /// vivo, and biochemical manipulation of functionalmetabolizing intact cells in order to assign and understand thepathways of the observed metabolites (34, 36).

The magnetic resonance spectrometer is essentially a powerful magnet coupled to a highly sophisticated radiowave transmitter and receiver. The strength of the magnetic field of theearth is approximately 0.5 Gauss, and 10,000 Gauss are equalto 1 Tesla (T). Current human spectrometers operate from 0.5to 4.0 T. In general the quality (signal resolution and signal: noise ratio) of the spectra improves with the strength of themagnetic field. In biological MRS a brief (us) pulse of MHzradiofrequency energy is transmitted into the tissue or cells ofinterest which are situated within the strong magnetic field.This energy is absorbed and then released back to the surroundings at characteristic frequencies (the resonance frequencies)over ms, and the resonance signal is received (see Fig. 1, A andC) and computer analyzed to produce the spectrum (Fig. 1, Band D). Different nuclei within molecules absorb and releasethe energy at specific frequencies which are characteristic of thetype of chemical bond the nuclei are in (Fig. 2). Only moleculeswhich are free to tumble give a sharp enough peak to be seenabove the random noise in the baseline. This restricts the signalsobserved to water soluble metabolites and fats. For example,the phosphorus nuclei can be in a phosphomonoester bond, aphosphodiester bond, a diphosphodiester bond, P¡,etc., andthese bonds cause phosphorus to emit the absorbed energy atthe different and characteristic frequencies (Fig. 2). The resulting spectrum (Fig. 2) gives information as to what biochemicalsare present (the position of the peak along the horizontalfrequency axis termed the chemical shift), and how much ofeach (the area under each peak is proportional to the concentration). In Fig. 2 all the signals are clustered near 162 MHzand a distance of 162 Hz along the horizontal scale is definedas 1 ppm. The ppm scale is easier to use than expressing theabsolute MHz frequencies, although the 0 point is arbitrarilydefined and different laboratories have used different conventions as to where to place the origin and which direction ispositive.

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MAGNETIC RESONANCE SPECTROSCOPY OF TUMORS

TIME B FREQUENCY(MHz)

TIME D FREQUENCY(MHz)

Fig. 1. A. In a simple one pulse experiment of pure water a burst of radiofre-quency energy is projected into and absorbed by the sample. After a short pausethe radiofrequency emitted by the hydrogens of water is then recorded. B.Computerized analysis of the signal in A produces the spectrum B which is amapping of the intensity and the frequency(ies) of the signal. C and D. In a moreviscous solution the signal C from the hydrogens of water is composed of a widerdistribution of frequencies than in A. This distribution of frequencies results in amore rapid decay (relaxation) of the signal and the broader peak in D.

IN vivo

Fig. 2. A, 3IP spectrum at 9.4 T of MDA-MB-231 human breast tumor in anude mouse; B, 3'P spectrum at 9.4 T of MDA-MB-231 human breast cancer

cells proliferating in basement membrane gel threads. PE and PC are the dominant water soluble metabolites producing peaks (resonances) in the (PME) regionof "I* tumor spectra, and GPE and GPC are the dominant sources of signal in

the phosphodiester (PDE) region. The peak marked Pi is intracellular Pi at pH6.9; *, extracellular P¡at pH 7.35; peak with arrow may by PI in a pH 6.4compartment such as lysosomes (Ref. 3S, reprinted with permission).

Metabolites Observed in 31P, 13C,and 'H MRS

31P MRS of Tumors. 3IP MRS of tumors and malignant cellsconsist primarily of phosphomonoester, phosphodiester, phos-phocreatine, PI, nucleoside triphosphate, and DPDE peaks (28).One report of a Morris hepatoma observed significant amountsof /nyo-inositol l,2-(cyclic) phosphate (37). myo-Inositol hasalso been observed in an Adriamycin resistant cell line (38).The nucleoside triphosphate peaks are predominantly the threephosphorus nuclei of ATP with smaller contributions fromUTP, GTP, and CTP (28). The three phosphorus nuclei in thea, ß,and y positions of nucleoside triphosphate resonate atdifferent frequencies (Fig. 2). The P¡peak shifts position with

acid/base status allowing measurements of both intra- andextracellular pH to be obtained from 31P MRS (see Fig. 2).

However, at the present magnetic field strength used for humanin vivo studies (1.4 and 1.9 T) the P¡peak is broad and notresolved into its intra- and extracellular components makingthe pH measurement an average with a large standard error(10, 12). Despite this, significant pH differences have beenobserved in human in vivo studies between tumors and normaltissue (6, 10, 12).

Since many different biochemicals have the same type ofchemical bond, this can cause clustering and overlap of numerous different metabolites in the same region of the spectrum,making peak identification in vivo or in intact cells difficult(39). Assignments can be made more rigorously using acidextraction but this leads to questions of artifact caused by thisdestructive technique. During the past 10 years the PME peakshave variously been attributed to sugar phosphates (38, 40, 41),AMP (13), or PC plus PE (26,28, 30, 34,43,44). For example,glucose 6-phosphate, fructose 6-phosphate, AMP, ribose 5-

phosphate, PC, PE, and many other compounds are all PMEs(39). A 31P MRS study on intact metabolizing human breast

cancer cells, using substrates and inhibitors of the enzymes ofphospholipid synthesis and degradation (Fig. 3), identified thesepeaks as PE and PC produced by the synthetic enzymes cholinekinase and ethanolamine kinase (34), and not by phospholipaseC as has been suggested (28, 29, 45, 46). This assignment tothe synthetic pathways is in agreement with the work of enzy-mologists who have found millimolar concentrations of PC inHeLa and L6 myoblast cells produced by choline kinase (47).Similarly, the PDE peaks derive from glycerophosphorylcho-line, and glycerophosphorylethanolamine, which are membranebreakdown products (34). The DPDE peaks have been identified as UDP-glucose, NADP(H) (4, 14, 28, 43, 48), and theglycoprotein/glycolipid precursors, UDP-/V-acetylglucosamineplus UDP-jV-acetylgalactosamine (24,49). Therefore, 31PMRS

gives information concerning phospholipid synthesis (PE andPC) and degradation (GPE and GPC), cell energetics (PCr/

f-ATP

•¿�V-

Fig. 3. MDA-MB-231 human breast cancer cells embedded in agarose andperfused with 2 mm ethanolamine. The enhancement of the PE peak results fromactivity of ethanolamine kinase, the diminution of the PC peak is caused by thecompetitive inhibition of choline kinase by ethanolamine, and the appearance ofGPE and GPC results from inhibition by ethanolamine of GPE and GPCphosphodiesterase (from Ref. 34, reprinted with permission).

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MAGNETIC RESONANCE SPECTROSCOPY OF TUMORS

NTP/Pi), pH (P¡position), and glycoprotein/glycolipid synthesis (DPDE).

'H MRS of Tumors. The 'H MR spectra of tumor extractsconsist of many more resonances than the 31P spectra. Unfor

tunately many of these peaks overlap and are difficult to resolvefrom one another even in vitro. In acid extracts these resonanceshave been assigned to various amino acids plus láclate,choline,PC, PE, creatine, PCr, and glucose (28). It should be noted thatsignal resolution is superior in extracts compared to ;// vivobecause of the increased motion of metabolites in nonviscoussolution. Significantly, recent developments in the ability toobtain localized in vivo 'H spectra of metabolites other than

water and fat show that in vivo human spectra from normalbrain exhibit a prominent /V-acetylaspartate peak which is usually markedly diminished or absent in gliomas (50). In vitro 'H

MRS of intact tumor cells and biopsies reveals a large peakwhich under high resolution conditions can be resolved intofour components resulting from méthylènegroups of neutrallipids and the methyl group of a fucolipid (Fig. 6). As discussedbelow, changes in these peaks have been reported to correlatewith malignancy, metastatic potential, and drug resistance (16,51).

13C MRS of Tumors. 12C constitutes 98.9% of naturally

abundant carbon and is not visible by MRS methods. Theresonances of I3C (1.1% natural abundance) are visible in extracts and have been assigned to láclate,creatine, phosphocrea-line, PE, PC, and various amino acids (28). However, in vivo(25), mosl of ihese resonances are noi resolved and many areonly small shoulders on ihe large melhylene peaks of lipids,which is noi presenl in ihe waler soluble extracls. One potentialresearch use is Ihe injeclion of I3C labeled melabolites such as

glucose in order lo sludy metabolism in vitro and in vivo. Thishas been done successfully in studies of lumor cell glycolysis(25, 34, 40, 52).

Potential Clinical Uses of MRS3IP MRS

Published speclra of human lumors (see Table 1) oblained invivo (3-10, 12) or from biopsy material (26-27) from morelhan 70 palienls consislenlly show 2- lo 4-fold or more increased levels of PMEs (PE and PC) (Figs. 2 and 4) whencompared lo Ihe lissue of origin for breasl (26), lung (27), liver(6), muscle (3, 12), melanoma (7, 12), squamous cell (7, 12),and bone (5, 8-9, 12) cancers. In vivo human sludies show lhalmelaslalic neuroblasloma has a markedly higher PME peaklhan ihe surrounding liver (4), and brain lumors in vivo ire-einemK show an increased PME peak (10), decreased PCr (10),and increased pH (6). PE and PC are known lo have highconcentrations in colon cancer cells (48) and in in vivo speclraof human colon lumors in nude mice (25). Speclra have noi yelbeen published comparing /// vivo human cancers of ihe colon,pancreas, ovaries, testes, adrenal gland, and lymph nodes to thenormal tissue of origin. Work on human biopsy material (26)has shown thai the PE and PC peaks were 2- to 4-fold higherin malignant breast lumors when compared lo benign fibroad-enomas of ihe breasl (Fig. 4). As shown in Table 1, whencompared lo Ihe normal lissue of origin, many prevalenl solidlumors in adults (lung and breast), adolescents, and children(neuroblastoma, rhabdomyosarcoma, osteogenic sarcoma,brain lumors) show a characlerislically high PME concenlra-lion and also abnormal pH, increased P¡,increased GPE andGPC, and ease of signal acquisilion (3, 9, 26) due to ihe highcell density of many tumors (Fig. 4).

Table 1 Phosphomonoesters in cancer: published human studies

Tumor PMECA/PMENMU

BrainAstrocytoma (6)Oligodendroglioma (6)Meningioma (6)I'm latti ni»nia(10)

Metastatic lymphoma (10)Astrocytoma (10)

Lung (27)

Breast(26)Fibroadenoma (26)

Liver (6)

Liver métastasesNeuroblastoma (4)Endometrial carcinoma (6)

Kidney (13)

Muscle (3, 12)

BoneEwing's sarcoma (9, 12)

Histiocytoma (5)Osteosarcoma (8, 12)Lymphoma/bone (9)

SkinMelanoma (7, 12)Squamous cell carcinoma (7, 12)

2-4+2-4+

3-4+

23-4

2-32-32-32-3

3-4+3-4+

°PMECA/PMENML,ratio of PME peak of tumor to that of normal tissue of

origin unless ratio specified under tumor column.

NOBUAL MUSCLE

y-ATP a-ATP ß.„f

BREASTCAHCNOMAa-ÄTP

y-ATP

J3-ATP

RHABDOMYOSARCGMA

Fig. 4. A and B, comparison of biopsies of a 1.2-g human mammary fibroadenoma and a 0.58-g human breast carcinoma (from Ref. 26, reprinted withpermission). Note the two prominent PME peaks (PE and PC) in the carcinomawhich are not present in the benign mass. In addition, the malignancy gave moreintense signals despite its smaller size. The biopsy samples were bathed in mediawith a 10 HIMI', concentration resulting in extremely large extraneous P, peakswhich have been truncated for clarity. C and D, in vivo human spectra from thesame patient comparing normal muscle to a rhabdomyosarcoma (from Ref. 3,reprinted with permission). The prominant PME peak and greater signal intensityof the tumor are evident here also. UDPG, UDP-glucose.

Choline kinase and elhanolamine kinase, which produce PCand PE, are examples of "key enzymes" (53-55). Key enzymes

are involved in Ihe regulation of Ihe rale and direction ofsynthelic or degradalive pathways and confer a growlh advantage to Ihe malignanl cell (53-55). An example includes Iheincreased activity of the synthetic enzyme thymidine kinasecombined with decreased activily of Ihe calabolic enzyme thymidine phosphorylase (53). Choline and elhanolamine kinaseshave Ihe following characlerislics of key synlhetic enzymes: (a)they regulate rate and direclion of a pathway, although noinecessarily Ihe rale limiting step; (b) Ihey control one wayreaclions; (c) Ihey are frequenlly ihe firsl enzyme in Ihe palh-way; (d) isoenzymes exist; and (e) Ihey are regulated in multiple

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MAGNETIC RESONANCE SPECTROSCOPY OF TUMORS

ways (47). An increase in choline kinase activity is known torequire increases in the amount and synthesis of the enzyme(47), and choline kinase has been shown to have its activitydoubled in nonmalignant cells by growth factors (56, 57), witha corresponding doubling in the concentration of PC and synthesis of phosphatidylcholine. The increased PE and PC observed in the spectra of tumors can be viewed within this widerperspective of key enzymes with a change in activity from thenormal cell that reflects differences in gene expression (53, 54).Cells at log phase, the growth of which is inhibited by tumorgrowth factor ß,produce 31P spectra with PC and PE peaksonly one-half the intensity of the same cell line grown withouttumor growth factor ß(58). Further work may reveal thatspecific oncogenes result in the production of growth factorswhich cause increased production of choline and ethanolaminekinase.

High levels of GPC and GPE have also been observed intumors but not as consistently as with PE and PC (13, 25, 34,59). These high levels may be the result of decreased activity ofthe enzyme GPC phosphodiesterase (34). This enzyme also hascharacteristics of a key degradative enzyme: (a) it regulates therate of a pathway (60); (b) it is frequently a common enzymefor two or more pathways (61); (c) it is the last step in a pathway(61); and (d) it is the target of feedback regulation (62). It isinteresting to note that in one human lymphoma cell linestimulated by vasopressin (63) an increase in phospholipidsynthesis was the result of decreased flux down the catabolicpathway without any increase in the baseline level of the anabolic pathway.

Diagnostic Potential. Although there is a change in the enzy-

mology of many cancer cells there is no one pattern for allcancers (55,64), and the differences in activities of key enzymesis greater from normal when there is a larger difference ingrowth rate between the malignancy and the normal tissue oforigin (53, 54, 65). This may be why brain tumors which arefrequently slow growing (65) do not consistently demonstrateas large a change from normal in their "P spectra (6, 10). Also,

certain organs such as brain (10), pancreas (66), kidneys (12),and luteinized ovaries (67) have high levels of PE and PC evenbefore malignant transformation. This may be due to a highlevel of phospholipid synthesis even without cell division orgrowth, as in normal brain which needs to maintain the extensive myelin sheaths of nerve cells (68, 69).

For these reasons, "P MRS will not be pathognomonic for

cancer but still has the potential, if the limitations of the currenttechnology are overcome, to be a useful piece of diagnosticevidence in the initial evaluation and staging of a patient inconjunction with biopsy. The spectrum of a mass would not beinterpreted in isolation, but within the context of the history,physical examination, and appearance of the mass on imaging.Many masses are benign and are composed primarily of fibroustissue (fibrosis, fibroadenomas, fibromas, neurofibromas), fattissue (lipomas), cysts, benign polyps, hamartomas, heman-giomas, leiomyomas, etc. Breast, thyroid, ovarian, asymptomatic mediastinal, and uterine masses in adults; persistentadenopathy, cervical, soft tissue, and mediastinal masses inchildren; and abdominal masses in infants are frequently benign(70, 71). Aspiration cytology or thin needle biopsies can givefalse negative results (72), because tumors can contain significant proportions of inflammatory, reparative, necrotic, and/orfibrotic tissue; and occasionally tumors may be difficult orimpossible to biopsy due to the location of the mass (e.g.,brainstem), or the poor surgical risk of the patient. Because 31P

MRS is noninvasive and can sample the entire mass, the com

bined pattern of a magnetic resonance image of a mass in anabnormal anatomical location with a 3IP spectrum showing

high concentrations of PMEs, P¡,GPE, GPC, and ATP, plusan abnormal pH, would rule out many of the fibrous and otherbenign lesions and could be useful in guiding which portions ofa tumor or tumors are most appropriate for surgical biopsy.

3IP Monitoring of Therapy. 3IP MRS also has the potential

to be helpful for diagnostic dilemmas related to treatment ofknown malignancies. Fibrous masses frequently develop following radiation therapy of tumors (e.g., Hodgkins lymphoma).Unfortunately, these masses can also be recurrences or persistence of the original tumor, but cutting through previouslyirradiated skin has an increased risk of poor wound healing.Treatment of tumors can also result in fibrosis or necrosis ofthe mass without a change in size which sometimes makes serialmeasurements on radiological images a very crude method withwhich to evaluate response. The ability to noninvasively observeATP, the basic biochemical signature of living tissue, could bea much more refined method for evaluation of response tochemotherapy or radiation therapy (Fig. 5).

Table 2 shows the spectral changes which have been reportedfollowing various treatments in tumors grown in animals(Groups 1 to 3), and from in vivo human studies (Group 4).The number of pluses and minuses is meant to indicate roughlythe extent of change. Group 1 shows the effects of the biologicalresponse modifiers interferon and tumor necrosis factor. Theinitial changes seen in the tumors studied were 2-fold alterationsin the level of PE and PC precursors and breakdown products(GPE and GPC). These changes appeared days prior to theappearance of necrotic areas within the tumor. However, sinceonly the static levels were measured rather than flux, it is notpossible to say what effect interferon and tumor necrosis factorhad on these pathways. A decrease in PC could be alternately

Prt -Tr»*tm»ni

3 hr Post-Treatment

-20 10 20

Fig. 5. Complete loss of ATP (Peaks I, II, and III) and an increase in P¡(PeakIli are easily seen in the spectrum of a human glioblastoma growing in a nudemouse following 50% lethal dose of vincristine. These changes did not occur inthe normal tissues of the mouse (from Ref. 22, reprinted with permission).

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Table 2 Effects of therapy on "P spectra

TumorGroup

1Friend leukemia (73)Friend leukemia(29)Group

2Breast cancer (23)Myeloma (23)RIF-1 (23)RIF-1(23)Group

3Neuroblastoma (22)Glioma (22)Glioma (22)Lung cancer(22)Group

4Ewing's sarcoma (5)*

Osteosarcoma (5)Lymphoma (10)*Neuroblastoma (4)*TreatmentInterferon

Tumor necrosisfactorAdriamycin

CytoxanCytoxanRadiationCytoxan

CytoxanVincristineAdriamycinRadiation

ChemotherapyRadiationRadiation and Cyt/

Ver/Dtic'Spectral

changes"Dose

PME P, pH PDE PCrATP—

—¿�+

+200 mg/kg i.p. - ++150 mg/kg i.p. —¿� ++-,+300

mg/kg i.v. ++200 mg/kg i.v. +

2 mg/kg i.v. ++5 mg/kg i.p.++Unstated

-,+Unstated - - +2420 rad —¿� +

900 radUnstated

°+, increase; —¿�,decrease; —¿�,+,decrease followed by increase.* In vivo human study.' Cyt/Ver/Dtic, Cytoxan/vincristine/5-(dimethyltriazeno)imidazole-5-carboxamide.

explained as either decreased activity of choline kinase (EC2.7.1.32), indicating decreased flux, or increased activity ofcholine phosphate cytidylyltransferase (EC 2.7.7.15), indicatingincreased flux and utilization of enyzme precursor. Studies onhuman breast tumor biopsies have indicated that estrogenreceptor positive tumors have low levels of GPE and GPC andestrogen receptor negative tumors have high levels of thesecompounds (19). These results suggest that the visibility of thephospholipid pathways which are intrinsically connected withthe growth of the cell may be a probe of the sensitivity of tumorsto biological response modifiers.

Groups 2 and 3 demonstrate two patterns observed in vivowhen protruding s.c. tumors in animals were studied in highfield strength magnets. One advantage of this model is that theanatomic source of the signal is more reliably identified (23,25), as compared to the present methods of spatial localizationfor in vivo human spectra of deep seated masses. A somewhatparadoxical effect of therapeutic doses of single agents orradiation therapy is demonstrated by Group 2. In this groupthe tumors demonstrated decreased P¡and increased PCr, before any noticeable decrease in the tumor diameter. Since theseeffects were observed prior to the subsequent decrease in thetumor size and loss of ATP signals and are seen in 3IP spectra

of normal tissue following reversal of ischemia, a plausibleexplanation is that a percentage of the tumor cells are killedand removed, which results in decompression of the tumor andincreased perfusion before a change in size. A combined //; vivo" 1*NMR and histológica! study of tumors in animals support

this hypothesis (75). In Group 3, mice were given 10% lethaland 50% lethal doses of the chemotherapeutic drugs listed (Fig.5). This was accompanied within hours by the almost totaldisappearance of ATP and the rapid increase of P¡(Fig. 5).These two changes indicate rapid cell death caused by thesevery high overdoses of single agents, and histological sectionsof the tumors revealed predominantly necrotic areas.

Group 4 shows the human studies done to date. Ensuringthat the magnetic resonance spectrum is derived only from awell defined volume or anatomic structure on the magneticresonance image is known as spatial localization. Spatial localization of deep seated masses is improving and is an active area

of research (76), although still not perfected, and the sig-nalrnoise ratio in a few of these human studies was low (5, 7-8). Interpretation of these limited number of results is thereforetentative. A combined MRI/MRS study of an intracraniallymphoma (10) showed a change of the spectra back to that ofnormal brain seen as a decrease in PME and P, with an increaseof PCr, and this corresponded to a marked decrease in the sizeof the mass on the image. A decrease in the PME resonancewas seen in the human neuroblastoma and osteosarcoma studiesalso, most likely representing a loss of signal from cancer cellsand a corresponding increase in signal from normal tissue. Theeffect of radiation therapy on Ewing's sarcoma (5) is similar to

that of the RIF-1 tumor of mice interpreted as decompressionwith improved perfusion.

MRS studies have also confirmed that tumors are moresensitive to hyperthermia than normal tissue (41, 78). Onestudy (78) of human tumors in nude mice used the spectrometerboth to create heat within the tumor by greatly increasing theamount of projected radiofrequency energy, and then to observethe loss of ATP as the tumor died from this treatment.

31PSpectral Correlations with Drug Resistance. Development

of drug resistance is one of the major clinical problems inoncology, and changes in the NMR spectra between sensitivecells and a corresponding resistant subline have been reported.3IP spectra of Adriamycin resistant MCF-7 breast cancer cells

grown in culture and suspended in agarose threads demonstrated a 50% decrease in PC and PE, an 80% decrease in GPCand GPE, a 500% increase in PCr, and a 90% decrease inDPDE when compared to Adriamycin sensitive MCF-7 cells(14). A similar decrease in phospholipid metabolites was observed in vivo in Adriamycin resistant versus Adriamycin sensitive mammary tumors of mice with 30 to 60% reduction inPC, PE, and GPC, although significant changes in the levels ofPCr and DPDE were not observed (15). A rationale for reductions in the phospholipid precursor and degradation productsrelating to drug resistance is not clear, although studies haveclaimed that drug resistant cells have decreased membranefluidity related to alterations in phosphatidylcholine and phos-phatidylethanolamine (80, 81). Other reports claim that theamount and activity of choline kinase are altered in relation to

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MAGNETIC RESONANCE SPECTROSCOPY OF TUMORS

drug detoxification (82, 83) and that resistant leukemia cellshave increased amounts of phospholipids and cholesterol esterwith a 50% decrease in the triglyceride/phospholipid ratio (16).If further studies show consistent changes in the levels ofphospholipid metabolites as cells become resistant, this wouldbe a useful observation for clinical application as well as astarting point for determining the mechanism relating drugresistance and phospholipid turnover.

'// MRS Relaxation Studies

Another MRS parameter providing useful information is theamount of time required for the absorbed radiofrequency energyto be released back by the nuclei, which can vary from milliseconds to seconds depending on the nucleus and the chemicalbond. The overall rate of the signal decay (Fig. 1, A and C) isdetermined by two simultaneous mechanisms measured by theexponential time constants, Tl and T2 (see Refs. 1 and 2 fordetails). In a low viscosity water solution (Fig. 1, A and B), allthe radiofrequency energy of a particular nucleus, such as thehydrogens of water, would be released over a narrow range offrequencies. This signal decreases exponentially over time (Fig.1/4) and is called the free induction decay. This narrow rangeof frequencies would give a narrow peak in the spectrum (Fig.1B). In a more viscous solution the hydrogens of water give offtheir energy over a wider range of frequencies producing abroader peak (Fig. 2D). The net effect of this wider range offrequencies is to cause a faster signal decay in the free inductiondecay, associated with a shorter T2 (Fig. 1C). A mathematicalanalysis shows that T2 is inversely proportional to the peakwidth at half-height, i.e., narrow peaks imply a longer T2 thanbroad peaks. Narrow peaks (long T2s) frequently indicate a lessviscous, more liquid environment where molecules can tumblewith less constraint. Biological MRS, then, can give information not only as to what major water soluble biochemicals andfats are present and in approximately what quantities but alsohow viscous or liquid is the molecular environment of a particular metabolite.

A number of intriguing in vitro 'H MRS studies of T2

relaxation times relating to cancer have been published andappear to have a biochemical correlate. The méthylènepeaktends to be very prominent in cancer tissue in vivo (25) and invitro (16). This "méthylène"peak of cancer cells, biopsy mate

rial, and serum lipoproteins has been studied at high resolution(Fig. 6), and this work (16, 84, 86) has demonstrated that theméthylèneregion can, by present methods, be resolved into atleast four overlapping peaks. In the cancer cells studied andserum from cancer patients, one of these resolved peaks isstated to be a resonance from the fucose moiety of a fucolipid(16) located on the surface of newly described proteolipids (17,86). These proteolipids are found in the serum of cancer patientsand are also associated with the membrane of cancer cells, butare not present in normal controls. Although methyl and méthylènepeaks are normally (but not always) well resolved fromeach other, this fucolipid peak within the "méthylène"region

derives from the methyl moiety of the fucose. Prior to thediscovery of these proteolipids MRS measurements (87) hadshown that in cancer cells, this peak had a markedly prolongedT2 (greater than 350 ms). Furthermore, when cancer cells aretreated with trypsin (87) or a fucosidase (16) there is a markeddecrease in the number of métastasesresulting from cells injected into nude mice associated with a decrease of this peak(see Fig. 6), and the remaining peak has a T2 of only 150 ms.This implies that the resonance from the malignancy associated

5432

PPM

14 12 14 12

PPM

Fig. 6. 'H MRS of a suspension of rat mammary adenocarcinoma cells. Note

the prominent méthylènepeak at 1.3 ppm which is resolved in A into separatepeaks. In B the same cells have been treated with trypsin with a marked decreasein one of the peaks. This peak is proposed to be the fucose moiety of a surfacefucolipid involved in métastases(from réf.87, reprinted with permission).

fucolipid is superimposed on a normal lipid peak with a shorterT2, but the superimposed fucolipid resonance can be identifiedby its prolonged T2.

Studies of one rat model consisting of a malignant metastaticmammary adenocarcinoma and a derived nonmetastatic clonedemonstrated that only the metastatic line had a peak in theméthylèneregion with a T2 value of greater than 350 ms. Dataon human ovarian and colon cancer biopsy material demonstrate that the vast majority of these have a long T2 value whichis consistent with their known natural history to metastasize ifnot resected early. However, proof that this correlation willhold in general requires large numbers of human samplesshowing that a T2 less than 350 ms is rarely associated withmétastases,and/or an explanation in terms of the biochemicalmechanism of metastasis. These 'H MRS observations have

their correlation in biochemical studies using techniques otherthan MRS where numerous reports have appeared since 1971showing that malignant cells have increased levels of fucolipids(88-92). Other in vitro 'H MRS studies show increased

amounts of glycoproteins (18, 93) which have also been observed by more classical techniques (94).

Much publicity surrounded the release in 1986 of a studywhich measured the average of the widths at half-height (acrude T2 measurement) of the methyl and méthylènepeak inthe serum of different patients (95). These peaks arise predom-

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inantly from the mobile components of fats which are free totumble in the liquid center of proteolipids. This study claimedthat the average width at half-height of the serum methyl andméthylènepeaks was narrower for cancer patients than fornormal controls and most benign neoplasia, although there wassignificant overlap when compared to the serum of pregnantwomen or men with benign prostatic hypertrophy. This couldimply a paraneoplastic phenomenon where the serum proteolipids of cancer patients have a more liquid environment thanthose of the normal controls, although a precise biochemicalexplanation of this effect is not yet known. Many others attempted to reproduce these results, and although some observeda stastistical difference between normals and cancer patientsthe degree of overlap was 20 to 30% (96, 97).

Since both of the above-described "H MRS studies concen

trate on the méthylèneregion of the spectrum and both note along T2 value (narrow peak) in this region, it may seem thatthey are both observing the same biochemical phenomenon.However, this is not necessarily the case. The high resolutionwork on cells, tumor biopsies, and proteolipids has focused onindividual peaks, whereas the peak widths of serum methyl andméthylèneresonances represent an average of many unresolvedpeaks.

"FMRS

Although fluorine does not occur naturally in the body it is arelatively easily detected nucleus by MRS and studies of fluor-inated chemotherapeutics and anesthetics have been performedboth in vitro and in vivo (98-103). FUra can be metabolized toits active products, FdUMP and FUTP, or it can be detoxifiedvia a catabolic pathway from FUra to FUH2, then to FUPA,and finally to FBAL (102). In an in vivo study of FUra metabolism of Lewis lung carcinomas implanted in mice it wasobserved that the liver used predominantly the detoxificationpathway with visible peaks arising from FUra and FBAL,whereas spectra from the tumor revealed FUra and FdUMPwith no visible FBAL ( 102). The FdUMP peak may also containcontributions from FdUDP and FdUTP. In vivo human spectrafollowing therapeutic doses of FUra have been reported (100)and reveal the relative amounts over time of FUra and its finalcatabolite, FBAL, in the liver during detoxification. The metabolite FUPA was observed in rodents but not in humans, indicating a difference in catabolism between species.

The fluorine nucleus gives well separated MRS peaks andcan detect fluorine compounds in body fluids in vitro if theirconcentration is 10 ¿¿Mor more (98) and has the advantage thatno chemical extraction of the fluids or special labeling of thedrug is required. A study of human urine during a 6-day FUra

schedule (99) revealed that FBAL was 79% of the excretedmetabolites followed by FUra (11%), FUPA (8%), F~ (2%), and

FUH2 (trace). The majority of the metabolites were excreted inthe first 6 h and FBAL appeared in significant quantities withinl h after infusion. A similar study on human blood samples(98) followed the catabolism of FdUrd during treatment andshowed that its major catabolites were FBAL, FUH2, andFUPA. Since these are metabolites of FUra this implies thatFUra is an intermediary in the catabolism of FdUrd.

The position of the 19Fpeak from the fluorinated anesthetic

halothane varies depending on whether it is in water or fat, andtumor tissue appears to have a more lipid environment possiblydue to increases in the amount of phospholipids (103). An invivo study in tumor bearing rats following the fate of difluoro-

methylornithine, an inhibitor of ornithine decarboxylase, ob

served that difluoromethylornithine was not catabolized andwas 3 times more accumulated in tumors than in nonmalignanttissue (101).

In Vitro Systems

The MRS studies on the effects of radiation, chemotherapy,and biological response modifiers reviewed here illustrate thatalthough changes in the spectra are seen, a firmer understandingof the biochemical, histologie, and vascular perfusion mechanisms associated with tumors and their spectral responses totreatment is needed. To this end many perfusion systems supporting intact actively metabolizing cells have been devised (35,104-111). We have developed two in vitro perfusion systemsconsisting of cells trapped in agarose gel threads (104) orproliferating in basement membrane gel threads under standardcell culture conditions (35). The major advantages of the invitro perfusion systems are that a homogeneous population ofcells can be studied for hours or days at a high field strengthwhile simultaneously controlling the extracellular environment.Agarose can be used for anchorage independent cells such asleukemia cells (107), and the basement membrane gel has beendemonstrated to support the three dimensional growth of cellswhich can attach, such as mammary tumor cells or normal livercells (35, 112).

One of the results of these in vitro methods which had eludedin vivo techniques for 10 years was the assignment of the PMEpeaks in the human mammary carcinoma cell line MDA-MB-231 not only to the metabolites PE and PC as had beendemonstrated in other cell lines and tumors (26, 28, 30,43, 44)but also to the specific enzymes and hence the pathways producing them (34) (see Fig. 3). PE and PC have been observedto accumulate in large hypoxic tumors (28) which might suggestthat these metabolite pools are, at least partially, degradationproducts. However, hypoxia also leads to a more rapid decreaseof CTP than of ATP (113). Since the PE and PC substrates inthe synthetic phospholipid pathways require CTP as a cofactorin order to be further metabolized, this could explain the greateraccumulation of these phospholipid precursors in hypoxic tumors. Other studies which have been performed using in vitrotechniques include studies on bioenergetics (39), hyperthermia(31), glucose metabolism in drug resistant versus sensitive cellsby I3C MRS (39), levels of phosphate metabolites in sensitive

vs resistant cells (14), PCr levels in lung cancers of differenthistology (32), and changes in metabolite levels upon differentiation (114). The basement membrane gel thread technique(35) also allows cells to grow and survive under sterile standardcell culture conditions as well as enabling one to alter the pOz,pH, perfusion rate, temperature, and nutrient or drug concentrations. This model will now allow in vitro experiments ongrowing and proliferating cells which were previously not possible. Since nude mice models of human tumors and in vivoprobes (25) for observing I3C, 31P, and 'H in these mice can be

built, combined in vivo/in vitro studies on the same cell linehave the potential to provide information not possible by eithermodality alone.

Summary

Presently human in vivo spectra can be obtained at 1.5 T withresolution of the PMEs, PDEs, and P¡by use of surface coilswhich localize the signal to a volume immediately below thecoil (76). Research spectrometers exist with 4.0-T whole bodymagnets, and this should lead to better signal:noise ratio and

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resolution, although the spectra from the currently available1.5 T imagers/spectrometers may prove adequate for clinical 13purposes. Additionally, methods for assuring that spectra areobtained from a well defined volume of interest (spatial local- ¡,ization) are expected to improve (76). Currently the technologylimits obtaining 31P spectra from 10-cm3 volumes near thesurface or from 10- to 30-cm3deeper structures. 15

Spectroscopic studies of human tumors in vivoare still in thevery early stages of development and improved methods ofspatial localization are needed before MRS can be applied 16routinely for clinical purposes. Much of the in vitro and in vivowork is still preliminary and requires further work to determine !

the generality and the biochemical significance of the effectsseen. At the present time the most consistently reported in vivoobservation of 3IP MRS of tumors is the presence of a large

PME peak in malignant tissue due to the high levels of thephospholipid precursors PC and PE. In addition the presence 19of the P¡peak as a measure of pH, the high ATP levels (pergram) of many tumors, and decreases in PCr levels in sometumors versus normal tissue make 3IP an attractive method tobe developed for in vivo noninvasive diagnosis of malignantversusbenign growths as an adjunct to biopsy and for monitoring the effects of chemotherapy in conjunction with measuring 2ithe diameter of a mass on an image. Also, well resolved 'Hhuman in vivospectra of 14-cm3volumes from the center of thehead (115) have been obtained recently. PC, PCr, ./V-acetylas-partate, and lactate were easily seen. Since 'H NMR gives morethan 100 times the signal:noise ratio for PC (116) than 3IP 23NMR, 'H NMR could prove very useful in basic studies and

clinical applications to oncology. The use of shielded stronggradients" makes proton-localized spectra a much more prac- 24

tical proposition. The combination of MRS with magneticresonance imaging should provide complementary informationnot available by either modality alone. 25

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1989;49:770-779. Cancer Res   Peter F. Daly and Jack S. Cohen 

Clinical Applications: A ReviewVivoinMagnetic Resonance Spectroscopy of Tumors and Potential

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