proton spectroscopy provides accurate pathology on biopsy and...

Review Articles

Proton Spectroscopy Provides Accurate Pathologyon Biopsy and In Vivo

Carolyn Mountford, DPhil (Oxon),1–3* Cynthia Lean, PhD,1–3 Peter Malycha, FRACS,4

and Peter Russell MD, FRCPA1–3

In the last 25 years, MR spectroscopy (MRS) has movedfrom being a basic research tool into routine clinical use.The spectroscopy method reports on those chemicals thatare mobile on the MR time scale. Many of these chemicalsreflect specific pathological processes but are complicatedby the fact that many chemicals change at one time. Thereare currently two clinical applications for spectroscopy.The first is in the pathology laboratory, where it can be anadjunct to, and in some cases replacement, for difficultpathologies like Barrett’s esophagus and follicular ade-noma of the thyroid. The spectroscopy method on a breastbiopsy can also report on prognostic indicators, includingthe potential for spread, from information present in theprimary tumor alone. The second application for spectros-copy is in vivo to provide a preoperative diagnosis and thisis now achievable for several organs including the prostate.The development of spectroscopy for clinical purposes hasrelied heavily on the serially-sectioned histopathology toconfirm the high accuracy of the method. The combinationof in vivo MRI, in vivo MRS, and ex vivo MRS on biopsysamples offers a modality of very high accuracy for preop-erative diagnosis and provision of prognostic informationfor human cancers.

Key Words: spectroscopy; body; pathology; biopsy; in vivoJ. Magn. Reson. Imaging 2006;24:459–477.© 2006 Wiley-Liss, Inc.

PROTON (1H) MAGNETIC RESONANCE SPECTROS-COPY (MRS) is now providing an adjunct to and in somecases an alternative to pathology for the diagnosis ofhuman diseases, etiology of pain, and subsequently,pain management. The expectation that MRS would

develop at the same rate as MRI produced disappoint-ment and fueled the thought in many that it was aninferior technology.

The reason for the slow emergence of spectroscopy asa diagnostic modality will surprise many. Spectroscopyis highly accurate, providing pathological and in somecases prognostic data with a hitherto unprecedentedaccuracy (1). So why was the gestation period so long?Spectroscopy was so sensitive it required correlationwith very detailed histopathology and patient outcometo prove its accuracy. Unlike MRI, which slotted neatlyinto the armamentaria of the radiology community,spectroscopy evolved from the collaboration betweenscientists, surgeons, and pathologists. Only when thegroundwork had been done with large and detailed clin-ical databases on biopsy material did the radiologycommunity become involved.

We have undertaken correlative pathology with spec-troscopy on biopsy for 25 years chemically mapping thehuman body. For the first 15 years, the goal was to de-velop the technology for analysis of biopsy specimens.Now, with high-field body spectroscopy, some but not allorgans can be examined by spectroscopy at 1.5, 3, or 4 T.As with the biopsy program, there is no fast track. If thecorrelative pathology is not undertaken with appropriatecare and precision the outcome will do justice to the sen-sitivity of the spectroscopy method. A good example of thiswas an 11% error rate in routine hospital examination ofprostate tissue solely due to sampling errors. The errorswere identified by MRS and confirmed in a blinded studyof serial sectioning of all tissues examined (2).

Neurospectroscopy will not be covered in this reviewas this was the subject of a recent review by Ross andDanielson (3). This review will also not cover magicangle spinning (MAS) since many of the chemical shiftscan be altered due to tissue degradation.

THE EARLY HISTORY OF PROTON MRS

The first report of a high-resolution 1H MRS from intactviable cancer cells was made in the United States byBlock (4) in 1973, who suggested the method might leadto pathologically relevant information. In 1978, Danielset al (5) in Oxford produced the first 1H spectra from ratadrenal glands. A series of experiments undertaken inSydney in 1978 indicated that MRS could identify the

1Institute for Magnetic Resonance Research, University of Sydney, Syd-ney, New South Wales, Australia.2Department of Magnetic Resonance in Medicine, University of Sydney,Sydney, New South Wales, Australia.3Department of Pathology, University of Sydney, Sydney, New SouthWales, Australia.4Department of Surgery, University of Adelaide, Royal Adelaide Hospi-tal, Adelaide, South Australia, Australia.*Address reprint requests to: C.E.M., Institute for Magnetic ResonanceResearch, Royal North Shore Hospital, St Leonards, NSW 2065, Aus-tralia. E-mail: [email protected] June 5, 2005; Accepted May 16, 2006.DOI 10.1002/jmri.20668Published online 8 August 2006 in Wiley InterScience (www.interscience.wiley.com).

JOURNAL OF MAGNETIC RESONANCE IMAGING 24:459–477 (2006)

© 2006 Wiley-Liss, Inc. 459

sequential or stepwise alterations in cells prior to themmanifesting frank malignancy, by light microscopy. Theexamination, by MRS, of excised intact thymus from aninbred strain of mice (6) indicated that the chemicalcomposition of the thymus cells changed prior to suchchanges being identifiable cytologically or histologi-cally. Thus MRS measured a continuum of changestaking place in the thymus as the final preleukemicperiod ended and neoplasia occurred (7,8).

Using another mouse model (9), it was shown that 1HMRS distinguished between tumor cells with a capacityto metastasize and those that produced only locallyinvasive tumors. Assignment of the one-dimensional(1D) MR spectra indicated multiple differences. How-ever, of particular interest was a resonance with a longT2 relaxation rate, at the chemical shift of 1.3 parts permillion (ppm), which clearly identified those primarytumors with capacity to metastasize (10–12). The as-signment of the potentially diagnostic and prognosticresonances was made possible by two-dimensional (2D)MR methods (13–15). The majority of the resonanceassignments were made by 2D MRS and many corre-lated with biological or clinical criteria.

It is now known that these initial observations re-corded on animal models could be extrapolated to cells,biopsies, and tissues in vivo (1). Importantly the capac-ity of MRS to determine both neoplastic status andbiological potential from human biopsies with histolog-ical correlation approaching 100% has now been dem-onstrated (16).

HISTOPATHOLOGY

Histopathology has been the gold standard of the 20thcentury but it is also well recognized that correlationwith clinical outcome is one of statistical probabilityrather than a medical certainty. In the first instanceMRS was tested for its capacity to discriminate betweenwell documented and highly reproducible stages ofpathological process such as cancer and precancer ofthe uterine cervix (17). Later, MRS was used to deter-mine if there were useful spectral differences betweenhistological variants within a neoplastic range that cor-related closely with predicted clinical outcome. Thequestion being addressed was “could MRS do what pa-thologists found difficult or not possible.” Two goodexamples are follicular neoplasms of the thyroid (18)and dysplasia in Barrett’s esophagus (19).

Much of the earlier difficulties stemmed from extremesensitivity of the MRS method and the need to examinewith great care the histological features of the test sam-ple taken from a lesion, the nature of which varied fromarea to area. It was not possible to simply to use thecrude “global” pathological diagnosis of the patient’ssalient disease as a measure of the expected MR find-ings and hope that the correlation would be other thanmediocre. For the MRS method to achieve its full poten-tial, detailed step-sectioning of the specimen for his-topathological analysis of the precise piece of tissueexamined was needed. The exact percentage of eachtype of tissue type or cellular composition needed to berecorded. For example, for the prostate percentages ofglandular, stromal, prostate intraepithelial neoplasia

(PIN), and inflammatory disease need to be comparedwith the amount of malignant tissue present in eachslide. These meant large volumes of pathology, clinical,and spectral information were compiled for evaluation.This principle applies today to examination of the re-sected organ following in vivo spectroscopy.

THE BASIS OF THE MRS METHOD

MRS reports on pools of chemicals in cells and tissuesas they alter (many at the same time) with aging and thedevelopment of disease processes (17,20–40). The mol-ecules in question may be metabolites or originate fromareas on the cell that also allow molecular motion suchas cell-surface markers or mobile lipid pools. A sum-mary of assignments made for both 1D and 2D spec-troscopy of cell and tissues are listed in Tables 1 and 2.

Many of the biochemical processes that are amenableto being monitored by MRS occur in parallel, but thechemical manifestations do not necessarily alter in thesame direction; i.e., some can be commencing whileothers are shutting down. In a 1D spectrum there arehundreds of resonances forming composites with dif-ferent T1 and T2 relaxation values. With time it can bedetermined which of the chemical criteria for each or-gan and disease state are important and which have thecapacity to interfere with a definitive diagnosis.

SHIMMING

Optimization of magnetic field homogeneity (shimming)is crucially important for spectroscopy. The first exper-iments undertaken on cells and tissues preceded auto-matic computer based shimming (41,42). The latestgenerations of vertical spectrometers have pulse fieldgradients available allowing gradient shimming. Whatused to be considered an “art” was transformed into anautomated operation.

Body spectroscopy in vivo still suffers from lack ofadequate automated shimming particularly at thehigher field strengths of 3 T and above. It is particularlyimportant for in vivo spectroscopy to have the higherorder shims available. The intricacies of shimming arewell covered in a two part series by Dr. W. Hull (41,42).For body spectroscopy to reach its full potential this isan area in need of attention. However the problem is noworse than trying to shim on biopsy specimens in thelate 1970s!

SPECTRAL EDITING

The MRS method is amenable to two types of spectralediting, during data acquisition and postacquisition(15). The advantage to postacquisition processing isthat, in principle, no resonances are lost due to filteringand are available for a series of different processingprotocols. The many assignments can be made by 1Dand 2D spectroscopy (43). By comparing resonance in-tensity and, in some cases, line width at half height (adangerous option in the view of these authors) correla-tions with clinical criteria can be made.

460 Mountford et al.

EFFECTIVE CONTRAST MATERIAL ONSPECTROSCOPY

Whether or not contrast material may be administeredbefore clinical spectroscopy appears to be organ-depen-dent and warrants consideration (44).

STATISTICAL CLASSIFICATIONSTRATEGY—MANAGING LARGE VOLUMES OFDATA

The interpretation and management of complex data-bases including large volumes of biomedical data in-cluding spectroscopy, pathology and patient statistics,requires a special methodology. Pattern recognitionmethods are now in common use for many biophysicalapplications including proteomics and metabolomics(45).

A multistage statistical classification strategy (SCS)was developed specifically to address these issues. Themethod has allowed automated analysis of both ex vivoand in vivo spectroscopic data using highly accurateand reliable mathematical classifiers for a variety ofclinical applications. For reviews see Lean et al (16) andSomorjai et al (45). The application of SCS methodologyprovides classifiers that distinguish, for example, be-nign from malignant specimens, with accuracies ap-proaching 100% when a small proportion of samplesthat are unable to be accurately classified are excluded

(often due to lack of signal-to-noise ratio [SNR]). Whenthese samples are included, accuracy percentages inthe mid-90s can still be achieved.

Unlike most other mathematical methods of data anal-ysis the SCS method identifies the spectral regions fromwhich the diagnostic and prognostic information is taken.This allows the chemical resonating in these regions to beconsidered and the biochemical pathways involved in thedisease process analyzed. However, it must be remem-bered that with such a method the results are only asgood as the pathology included in the database and thatadequate numbers of samples must be included in thetraining sets to ensure that the data is not overclassified.

BREAST

Breast cancer is the most common cancer to affectwomen in Western countries, with an incidence ofabout 80–100 newly diagnosed patients per 100,000inhabitants per year (46). Presurgical assessment of thebreast lesion with accurate sizing spatial location andan accurate assessment of extent of disease has thepotential to radically improve patient management.

Comparison of Breast Diseases on Fine-NeedleBiopsy at 8.5 T

The 1H MR spectra obtained from breast fine-needleaspiration samples that were taken from more than

Table 1Assignment of Resonances in 1H MR One-Dimensional and COSY Spectra

Assignment of resonances in 1H MR one-dimensional

Molecules Abbreviation Moiety Chemical shift (ppm)

AminesCholine, phosphocholine,

glycerophosphocholineChol -N(CH3)3 3.2

Spermine, spermidine, polyamines PA -NCH2- 3.1Creatine, phosphocreatine Cr -NCH3 3.0

LipidsTriaclglycerol (fatty acyl chain) Lip CH3-CH2-CH2- 0.90

Lip CH3-CH2-CH2- 1.33Lip COO-CH2-CH2- 1.6-1.7Lip CH3-CH2-CH2- 2.02Lip CH�CH-CH2- 2.08Lip COO-CH2-CH2- 2.3Lip CH2-CH�CH 5.38

Amino acidsAlanine Ala CH3-CH- 1.49Glutamate, glutamine Glu, Gln -CH-CH2-CH2-COO-/NH3� 2.2Glutamate, glutamine Glu, Gln -CH-CH2-CH2-COO-/NH3� 2.6Isoleucine Ile -CH3 0.97Leucine Leu -CH3 0.97Lysine Lys H3N�-CH2-CH2-CH2- 1.7Lysine Lys H3N�-CH2-CH2-CH2- 3.03Threonine Thr CH3-CHOH- 1.33Threonine Thr CH3-CHOH- 4.25Valine Val -CH3 1.03

OtherLactate Lac CH3-CH- 1.33Citrate Cit COO-CHAHB-C(OH)- 2.4-2.7Taurine Tau H3N�-CH2-CH2-SO3

- 3.25Taurine Tau H3N�-CH2-CH2-SO3

- 3.43Inositol Ins -CH�CH- 5.32

Proton Spectroscopy on Biopsy and In Vivo 461

1000 breast lesions during surgery or through skinpreoperatively (Fig. 1) showed a clear distinction be-tween frankly malignant, ductal carcinoma in situ(DCIS) with microinvasion, DCIS without microinva-sion, and benign tissue (47,48). The correlative histo-pathology (shown on the right in Fig. 1) was obtained bythe surgeon intraoperatively by taking a tissue samplefrom the end of the needle track to ensure the spectrawere accurately compared with the correct diagnosis. Itis clearly seen in Fig. 1 that an increase in cellularmetabolites that are monitored by MRS occurs duringtumor development. Of particular importance is theappearance of a large number of chemically active spe-cies that are present in the spectrum from DCIS withmicroinvasion and not apparent in the spectrum from aspecimen of DCIS without microinvasion. It is furtherstressed that special techniques such as immunohisto-chemistry may be needed to discriminate betweenclosely related stages in a neoplastic spectra such asthe presence or absence of microinvasion in a cancer-ized lobule associated with DCIS (Fig. 1b and c). How-ever, on visual inspection of the MRS data, fibroade-noma remained a false positive (47).

It can also be seen in Fig. 1 that there is a cleardistinction between the spectra from frankly malignanttissue and DCIS with microinvasion and those frombenign and DCIS without microinvasion. Differencesinclude increased choline to creatine ratios and in-creased in the amounts of chemicals in 2.0–2.4 ppmspectral regions with the capacity for invasion.

These spectral differences are consistent with the lit-erature reports showing that choline metabolites alterwith aging and cancer in the breast. MRS of breast celllines showed that the total choline-containing phos-pholipids metabolite levels increase with progressionfrom normal to immortalized, to oncogene-transformed,to tumor-derived cells (35). Aboagye and Bhujwalla (35)demonstrated a glycerophosphocholine (GPC) to phos-phocholine (PC) switch with cell immortalization in on-cogene-transformed and breast tumor cell. They alsoreported an increase in PC levels and total choline-containing phospholipid metabolite levels. Sitter et al(49) used MAS MRS to analyze breast tissues and per-chloric acid extracts and assigned more than 30 metab-olites. However, the chemical shifts recorded for MASand for cell extracts are not necessarily the same as

Table 2Assignment of Resonances in 1H MR COSY

Molecules Abbreviation MoietyChemical shift (ppm)

F2 F1

LipidsTriglyceride A (CH2)n-CH2-CH3 0.90 1.33

B �CH-CH2-CH2- 1.33 2.08C -CH�CH-CH2- 2.02 5.38D -CH2-CH�CH- 2.84 5.38E -CH2-CH2- 1.33 1.62F O�C-CH2-CH2- 1.60 2.30G -O-CH2-CH-O 4.12 5.26

4.26 5.26G’ -O-CH2 4.09 4.29

AminesCholine Chol N-CH2-CH2 3.50 4.07Phosphoryl-choline PC N-CH2-CH2-OP 3.61 4.25GlyceroPC GPC N-CH2-CH2-OPO 3.69 4.38

Amino acidsAlanine Ala -CH-CH3 1.49 3.79Histidine His -CH-CH2-ring 3.22 3.95Leucine Leu -CH-CH3- 0.97 1.78Lysineb Lys -CH2-CH2- 1.72 3.05Threonine Thr HOCH-CH3 1.33 4.27Valine Val -CH-CH3 1.03 2.34

OtherTaurine Tau H2N-CH2-CH2-OS 3.28 3.50Lactate anion Lac -CH-CH3 1.33 4.12Fucose Fucl -CH-CH3 1.33 4.27

Unassigned 0.90 1.501.20 3.651.30 4.651.50 1.951.95 3.802.10 2.402.20 2.402.65 2.853.15 3.353.60 3.90


those recorded in intact viable cells and tissues andcare must be taken.

Clinical Study of Fine-Needle Aspiration BiopsySpecimens at 8.5 T

One of the most revealing studies into the diagnosticpower of 1H MRS analyzed by the SCS method is therecent study of fine-needle aspirate biopsies (FNAB).The quality of spectra obtained from these specimenscan be seen in Fig. 1 (47) and assignments in Table 1.Visual inspection of these spectra where the choline tocreatine ratio was measured gave an accuracy of 96%for determining malignant status. Using the SCSmethod the distinction between benign and malignantbiopsy specimens gave a sensitivity of 95% and speci-ficity of 96% with an overall accuracy of 96% (Table3). Interestingly, fibroadenoma ceased to be a falsepositive when the SCS method was implemented to

analyze the MRS data. The SCS method did not im-prove on malignant vs. benign but the rest of theinformation available is not obtainable by anothersingle method to date.

From the same spectra obtained from tissue in theprimary tumor and using the SCS method, lymph nodeinvolvement could also be predicted with an overallaccuracy of 95%. Similarly, from the same spectrumfrom the primary tumor, vascular invasion in the defin-itive surgical specimen was predicted with an overallaccuracy of 94% (47) (Table 3). Estrogen and progester-one receptor status was correctly predicted with 87%and 91% accuracy, respectively (31), and low grade(Grades 1 and 2) distinguished from high grade (Grade3) tumors with an accuracy of 94.7%. This is the firstclinical report of information on the invasive, meta-static, and receptor status of a tumor being available byinspection of a cell sample of the primary tumor.

Figure 1. Breast biopsies at 8.5 T. 1H MR spectra ofbreast fine-needle aspiration biopsies (FNAB) with SNR�10. a: Malignant. b: DCIS with microinvasion. c: DCISwithout microinvasion. d: Benign. Shown on the leftwith the comparative histology slide on the right. sweepwidth (SW) of 3597 Hz, 8192 data points, 256 accumu-lations, and a relaxation delay of two seconds. (Re-printed, in part, from Mountford CE, Somorjai RL, Ma-lycha P, Gluch L, Lean C, Russell P, Barraclough B,Gillett D, Himmelreich U, Dolenko B, Nikulin AE, SmithIC. Diagnosis and prognosis of breast cancer by mag-netic resonance spectroscopy of fine-needle aspiratesanalyzed using a statistical classification strategy. Brit-ish Journal of Surgery 2001;88:1234–40, with permis-sion from John Wiley & Sons Ltd., on behalf of theBritish Journal of Surgery Society Ltd. Also reprinted inpart from Lean CL, Russell P, Mountford CE. Magneticresonance spectroscopy: a new gold standard for medi-cal diagnosis. Journal of Women’s Imaging 2000;2:19–30 with permission from Lippincott Williams &Wilkins.)


The MRS method is dependent upon there being suf-ficient cells for an adequate SNR in the spectrum, i.e.,5 � 106. A routine audit of FNAB cytology specimensshowed there was a 15% rate of inadequate cellularityin the specimens (50). Also, it should be noted that theMRS method is not currently robust when applied tocore biopsies due to the high content of fat. The effect ofhigh levels of fat on an MR spectrum is described below.

Thus with adequate sampling the application of MRSto the examination of FNAB from the breast has thepotential to revolutionize breast cancer management byproviding both diagnosis and staging parameters priorto surgery. The number of patients examined so far isjust over 1000. It is anticipated that a further 1000 willbe needed for robust classifiers to be available. Themethod is currently undergoing clinical acceptancetesting.

Spectroscopy of The Breast In Vivo at 1.5 T andHigher Field Strengths

There are now a series of reports on the application ofMRS to breast in vivo. A range of breast coils and pulsesequences have been utilized. It was initially, and con-tinued to be, proposed that the broad composite reso-nance from choline and choline containing compoundsat 3.2 ppm was a marker for malignant disease (51).This observation has been contentious for many years,bringing into doubt whether MRS could be diagnosticfor breast cancer in vivo (51–58). At the higher fre-quency of 4 T, the intensity of the choline resonancerelative to other resonances was also considered as apossible diagnostic method (59).

A flaw in these studies (except for Kvistad et al (54))was the absence of a cohort of apparently healthy vol-unteers and lactating mothers. The term “broad com-posite resonance” gives license to a range of chemicalshifts and with such a rudimentary means of visualinspection of the data some of the volunteers, and all ofthe lactating mothers, had a broad composite reso-nance in roughly the 3.2 ppm spectral region.

Stanwell et al (60) demonstrated that, with carefulreferencing, the spectra from women with invasive ma-lignant disease had a resonance at 3.23 ppm, whereasin the spectra obtained from those women that hadhealthy breast tissue or lactating mothers (Fig. 2), thechemical shift was different, at 3.28 ppm. With this

discrepancy corrected the sensitivity and specificity ofthe method was 84% and 100%, respectively, leaving a16% false-negative of concern. This can be explained bythe basic chemistry of the breast.

As was initially described by Roebuck et al (53) thevariable and intense contribution to the MR spectrafrom lipid and fat in the breast poses a problem for 1Dspectroscopy. In the Stanwell et al (60) study, it would

Table 3SCS-Based Classification for Pathology, Nodal Involvement, and Vascular Invasion, Tumor Grade, ER Status,and PgR Status From Fine-Needle Aspiration of Primary Breast*

Classification Spectral regions (ppm) Accuracy

Benign vs. malignant 1: 0.87-0.92; 1.20-1.25; 1.63-1.67; 1.79-1.82; 1.95-1.98; 2.85-2.87; 2.95-2.97; 3.19-3.33 96.1%2: 1.19-1.23; 1.32-1.38; 1.79-1.82; 1.96-2.00; 2.13-2.15; 2.70-2.76; 2.92-2.94; 2.97-2.99

Lymph node involvement (yes/no) 1: 0.43-0.51; 0.64-0.77; 1.10-1.20; 1.56-1.59 95.0%2: 0.44-0.51; 0.67-0.71; 1.13-1.19; 1.56-1.60; 1.86-1.96

Vascular invasion (yes/no) 1: 0.47-0.55; 0.57-0.62; 0.86-0.92; 1.00-1.03; 1.69-1.71; 1.99-2.05; 2.55-2.56; 2.63-2.72 91.9%2: 0.75-0.81; 0.90-0.94; 1.03-1.12; 1.21-1.24; 1.59-1.63; 2.00-2.04; 2.24-2.27; 2.70-2.74

Grade 1 and 2 vs. grade 3 0.85-0.95; 0.98-1.01; 1.95-2.00; 3.50-3.58; 3.67-3.70 94.7%ER-positive vs. ER-negative 1.57-1.61; 1.85-1.90; 2.58-2.64; 3.50-3.53; 3.62-3.71 90.6%PgR-positive vs. PgR- negative 0.48-0.52; 1.59-1.62; 1.86-1.90; 2.57-2.63; 3.50-3.53; 3.91-3.96 86.7%

*See Refs. 47 and 102. Accuracy for the test was obtained using the crisp data, i.e., when specimens classified as fuzzy were excluded. Aclass assignment was fuzzy if the class probability was less than 75%. ER � estrogen receptor, PgR � progesterone receptor.

Figure 2. In vivo breast at 1.5 T. Proton single-voxel spectra(TR � 135 msec/TE � 2000 msec, 256 signal averages). a:From a patient with histologically confirmed invasive ductalcarcinoma. b: Lactating volunteer. c: Spectrum recorded inthree out of 40 nonlactating volunteers. The spectra are pro-cessed by zero-filling the 2048 data points to 8192 data points,applying a Gaussian apodization function of 1.5 Hz with a 15%echo offset before fast Fourier transformation. In the spectrumfrom the cancer bearing patient, resonances can be assignedto creatine-containing compounds (3.04 ppm), phosphocho-line (3.22 ppm), and glycerophosphocholine/taurine/myoi-nositol (3.28 ppm). In the spectra from the lactating volunteerand the false-positive nonlactating volunteer the dominantresonance is centered at 3.28 ppm, consistent with glycero-phosphocholine/taurine/myoinositol. The spectra were refer-enced to the methylene resonance of lipid at 1.33 ppm andwater at 4.74 ppm. (Reprinted from Stanwell P, Gluch L, ClarkD, et al. Specificity of choline metabolites for in vivo diagnosisof breast cancer using 1H MRS at 1.5T. European Radiology2005. 15:1037–1043, with permission from Springer.)


appear that in approximately 84% of patients with ma-lignant disease the choline resonance at 3.23 ppm canbe seen despite the signals from fat. In the remaining16% the fat is superimposed over choline-containingcompounds, thus masking the diagnostic markers.

The effect of increasing levels of lipid, short T2 relax-ation value, on smaller amounts of chemicals (choline-containing metabolites, with a long T2) is shown in Fig.3. The T2 relaxation value and hence the slope does notalter, but with increasing amounts of lipid the reso-nances with a long T2 are masked. Lipid suppressiontechniques cannot be guaranteed to overcome thisproblem; hence, if choline containing metabolites arenot observed, it cannot be assumed that they are notthere. The potential for false negatives can only be over-come by the implementation of 2D correlated spectros-copy (14,61), which will separate the lipid from themetabolites in a second dimension. This technology hasnow being extended to magnetic resonance spectro-scopic imaging (MRSI), providing the ability to monitormultiple lesions in the breast (62).

Now and in the immediate future the combination ofcontrast enhanced MRI and in vivo MRS to identify thelesion(s) and an image guided biopsy that is subse-quently examined by MRS at 8.5 T offers a diagnosticprocedure likely to result in an extremely high level ofdiagnostic accuracy.

PROSTATE

Prostate carcinoma is the most common cancer affect-ing men in the United States (63) and Australia (64).Histological examination remains the standard for di-agnosis and classification of prostate neoplasms (65),yet correlation between histological features and clini-cal outcome is modest at best. Prostate cancers dem-onstrate a range of biological potential. The optionsfacing the patient are clinical observation, hormone de-privation therapy, surgical procedures, and radiation or

cryosurgical therapies. Disease in the prostate is heter-ogeneous and, therefore, spectroscopic mapping of theprostate offers an opportune means of accurately iden-tifying spatial location and diagnosis.

In contrast to all other organs, except perhaps thebrain, the first example of spectroscopy of the prostatewas undertaken in vivo in the late 1980s using anendorectal coil (66–68). Subsequently the UniversityCollege, San Francisco (UCSF) group, led by JohnKurhanewicz, S.J. Nelson, and colleagues, has devel-oped in vivo spectroscopy of the prostate such that it isnow implemented on a routine basis in approximately40 hospitals in the United States. In Europe, Heerschapet al (69) and van der Graff et al (70) have developed asimilar method, albeit with software developed for adifferent manufacturer. Both use an endorectal coil,allowing relatively high-resolution anatomical imagesas well as 3D chemical shift imaging (CSI) (71).

The in vivo diagnostic method is based on high levelsof citrate that are present in healthy tissue. In contrast,malignant tissue is characterized by low levels of citrateand increased levels of compounds involved in phos-phatidyl choline and phosphatidylethanolamine synthe-sis and hydrolysis (choline, phosphocholine, glycerophos-phocholine, ethanolamine, and phosphoethanolamine)contributing to the spectral region known to contain cho-line.

The development of in vivo MRSI (see Kurhanewicz etal (72)) was a major advancement providing a 3D map ofcontiguous volumes of 0.24–0.34 cm3 of voxels thatmap the entire prostate (Fig. 4). The MRS and MRSI areacquirable within the same examination and alignmentis possible. This can be seen in Fig. 4d. The citrateresonance is absent but there is a strong choline reso-nance apparent. In contrast, in Fig. 4e there is a strongcitrate resonance but choline and creatine are alsopresent at significantly smaller than a citrate reso-nance. The in vivo method involves suppressing thelipid both inside the voxel of interest and outside thevoxel of interest.

Initial results of endorectal 3 T 1H MR spectroscopicimaging showed spatial, temporal, and spectral resolu-tion advantages at the higher field strength (73). Thecomparison of the 1.5 and 3 T data is seen in Fig. 5. Thespectroscopic data collected at 3 T show the relevantmetabolites to be separated from each other and fromwater and lipid, improving the diagnostic capability.However, this study, like most at 3 T, indicates thatshimming at 3 T is considerably more difficult.

It was almost a decade after the first in vivo study ofthe prostate that the first comprehensive study on pros-tate biopsies was published by the National ResearchCouncil of Canada at the higher frequency of 8.5 T (40).Using the SCS method, the sensitivity and specificityachieved in this study were 100% and 95% for distin-guishing benign prostatic hyperplasia (BPH) and can-cer, respectively. Examination of the patient databaseshowed that routine hospital histopathological diagno-sis had been used to determine study and controlgroups. No PIN or proportion volumes of each type ofdisease state and each tissue volume were reported.Since the SCS method is dependent upon correct his-topathological data the possibility that pathological en-

Figure 3. Schematic representation of the effect of increasinglevels of lipid (short T2) on those resonances with a long T2

relaxation value, e.g., choline (C.E. Mountford, unpublishedresults).


Figure 4. In vivo prostate MRI and MRSI at 1.5 T. A representative reception-profile corrected T2-weighted FSE axial imagetaken from a volume data set demonstrating a large tumor in the right midgland to base. The selected volume for hypointenselesion in right midgland (a) and overlay of spectral grid (b). c: Corresponding spectral array from b. Spectra in regions of cancerdemonstrate dramatically elevated choline (d, red box) and a reduction or absence of citrate and polyamines relative to regionsof healthy peripheral zone tissue (e, green box). The strength of the combined MRI/MRSI exam is demonstrated when changesin all three metabolic markers (choline, polyamines, and citrate) and imaging findings are concordant for cancer. (Reprinted fromKurhanewicz J. Swanson MG. Nelson SJ. Vigneron DB. Combined magnetic resonance imaging and spectroscopic imagingapproach to molecular imaging of prostate cancer. Journal of Magnetic Resonance Imaging. 16:451–63, 2002, with permissionfrom Wiley-Liss, Inc, a subsidiary of John Wiley & Sons, Inc.)

Figure 5. Comparison of prostate spectroscopy at 1.5 and 3 T. 1H-MR spectra from voxels in the peripheral zone of a prostateat 1.5 and 3 T are shown. The position of the voxel for which spectrum at 1.5 T (a) and at 3 T (b) originate is indicated (c). Spectraat 1.5 T (e) and at 3 T (f) correspond to the voxel (d) located in a slice in the base of the prostate 8 mm above the slice in (c). Thecitrate resonance is centered at 2.60 ppm. Notice the high spectral resolution at 3 T, e.g., in separating the lines of the citrateresonances and separation of choline and creatine peaks. (Reprinted from Futterer J, Scheenen T, Huisman H, Klomp D, vanDorsten F, Hulsbergen–van de Kaa C, Witjes A, Heerschap A, J. B. Initial experience of 3 Tesla endorectal coil magneticresonance imaging and 1H-spectroscopic imaging of the prostate. Investigative Radiology; 39:671–680, 2004, with permissionfrom Lippincott Williams and Wilkins.)


tities or variants may have been included in one clas-sifier was considered. Furthermore, with the verycomplex zonal anatomy of the prostate the various mix-tures of glandular and stromal tissues needed to betaken into account.

Using biopsy specimens, van der Graaf et al (39) iden-tified decreased levels of spermine at 3.0 and 3.3 ppmas a marker of malignancy in the prostate, but found invivo at 1.5 T that spermine was obscured in the 1Dspectra by choline and creatine at 3.2 and 3.0 ppm,respectively.

The second study of prostate biopsies, also at 8.5 T(2), addressed the issue of specimen sampling for cor-relation with MRS data. The spectra were correlatedwith serially sectioned tissues, identifying an error rateof 8% in routine hospital procedures due to incompletesampling of the tissue. No lipid suppression methodswere used and typical ex vivo MRS spectra (8.5 T) fromglandular BPH, stromal BPH, PIN, and invasive carci-noma are shown in Fig. 6. Also seen in Fig. 6 is acomparison of the spectra from excised tissues withdifferent proportions of adenocarcinoma present (5%and 50%). There is a clear distinction on visual inspec-tion between adenocarcinoma (50%), PIN, epithelial/glandular BPH, and glandular BPH. The presence ofcitrate in glandular BPH is consistent with the in vivoMRS of the prostate. However, no such citrate pattern isrecorded for stromal BPH. Citrate can still be seen inthe spectrum from PIN, as can the emergence of reso-nances consistent with choline and creatine. On visualinspection, the spectral separation of tissue with 5%adenocarcinoma from tissue containing only PIN wasnot easy. Resonances from lipid, amino acids, citrate,choline, and creatine are seen in the spectra from bi-opsy specimens containing small levels of adenocarci-noma and PIN, albeit at different levels. Once again, itcan be seen that there is a gradation in the spectra frombenign, stromal, and glandular BPH, through PIN, tofrankly malignant tissue with increasing volumes oftumor.

The Swindle et al (2) study also divided the biopsyspecimens from BPH into patients with and withoutcancer elsewhere in the prostate and compared thesewith adenocarcinoma specimens. When serial sec-tioned histopathology was used for correlation withMRS, a sensitivity of 100% and specificity of 94% wasachieved. Depleted citrate and elevated choline mea-sures alone were not accurate markers of malignancysince citrate levels remained high when only a smallamount of malignant tissue was present (Fig. 6). A re-cently developed mathematical regression method (74)identifies small volumes of carcinoma (less than 50%)with accuracies approaching 100%. Thus the combina-tion of different cell types (e.g., stromal, glandular, andzonal differences) can be documented and (combinedwith accurate histopathology) can offer a highly accu-rate diagnosis of malignancy in biopsy examinations at8.5 T.

It would therefore appear that while histology re-mains the routine standard, spectroscopy could pro-vide important biochemical information to improve ourunderstanding of tumor development and progressionin the prostate since, clearly, there are different signa-

Figure 6. Spectroscopy on prostate biopsies. 1H MR (8.5 T)spectra of prostate biopsy specimens, 256 accumulations, sweepwidth 3597 Hz, and a pulse repetition time of 2.14 seconds. Thewater peak was suppressed by selective gated irradiation. a:Adenocarcinoma (50% of the tissue was made up of malignanttissue). b: Adenocarcinoma (5% of the tissue was made up ofmalignant tissue). c: Prostatic intraepithelial neoplasia (PIN). d:Stromal BPH (95% stromal, 5% glandular). e: Glandular BPH(85% glandular, 15% stromal) are compared. (Reprinted in partfrom Swindle P, McCredie S, Russell P, Himmelreich U, KhadraM, Lean C, L., Mountford CE. Pathologic characterization of hu-man prostate tissue with proton magnetic resonance spectros-copy. Radiology 2003;228:144–151, with permission from theRadiological Society of North America.)


tures for glandular and stromal BPH, PIN, and adeno-carcinoma.

There remain contentious issues with the in vivo ex-perimental protocols for prostate. Inner voxel lipid sup-pression clearly impacts on the spectral profile of theprostate and comparison of non-lipid-suppressed spec-tra from the region of interest is warranted. The newmathematical regression methods developed by Somor-jai appear to offer well-needed assistance in the inter-pretation of the chemical composition of potential mul-tifocal and heterogeneous prostate disease. Thechemical information allowing definitive and detailedpathology now needs to be collected and interpreted ina manner that will allow all pathology subtypes to beidentified with high accuracy.

In vivo prostate spectroscopy is currently operationalin many centers around the world, albeit with differentprotocols. With the significantly improved signal tonoise ratio at 3 T it is now a matter of time before thereare robust classifiers for automated pathological diag-nosis and an accurate identification of the extent of thedisease and the effect of the disease on the neurovas-cular bundles.

CERVIX

Cancer of the uterine cervix was chosen by the pathol-ogist (P.R.) for the first 1H MRS study on clinical biop-sies (26) since the tissue is readily biopsied and thepreneoplastic and neoplastic spectra are very well es-tablished with good correlation with clinical outcomes.

Punch Biopsy

The 8.5-T spectra obtained from adenocarcinoma of thecervix is typical of adenocarcinoma tissue from anyorgan (17). The main spectral difference between be-nign and malignant was the presence of strong lipidsignals in addition to choline and creatine in the spec-trum from the malignant biopsies. The MRS methodrecorded an accuracy of 99% in distinguishing preinva-sive from invasive cervical cancer.

The next outcome from this study is equally interest-ing. Current taxonomy divides preinvasive cervical dys-plasia or cervical intraepithelial neoplasia (CIN) intothree degrees of severity. It is debatable whether theseconstitute a linear progressive development or differentdisease entities. Certainly, there is often a mixture ofCIN lesions in each piece of tissue. Neither visual in-spection of the MRS data nor the application of SCS tospectra from preinvasive cervical tissues, CIN 1, CIN 2,and CIN 3 (I.C.P. Smith and Ray Somorjai, unpublishedresults), allows any clear delineation, which probablyreflects the influence of mixed infections and high-riskand low-risk human papillomavirus infections in thesepatients.

Cervix In Vivo

The London (England) group (75) compared preinvasiveand invasive cervical lesions in vivo at 1.5 T, using anendovaginal coil, and confirmed the earlier report oncervical biopsies that lipid levels are more than double

in cervical tissue containing cancer cells compared tonormal control tissue (17). The 1H MR spectra withcorresponding MRI from a patient with cervical canceris shown in Fig. 7. The study reported on 51 women:nine were volunteers with benign uterine disease; 10with CIN; and 32 with invasive malignant disease (28with squamous cell carcinoma and four with adenocar-cinoma). It would appear from this study that elevatedlipid levels detected by MRS in vivo are independent oftumor load in the volume of tissue sampled. However,resonance peaks were all phased to choline and theirchemical shifts were assigned relative to choline at 3.2ppm. They reported that the sensitivity for detection oflipids in vivo using the single-voxel method was limitedby technical and patient-related factors, including im-perfect slice-selection profiles.

Potential developments in the preoperative diagnosisinclude use of ex vivo MRS on biopsy, material fromproven cervical cancers to predict the presence of nodalspread, and use of in vivo spectroscopic imaging topredict the size and spread of the primary tumor.

THYROID

Thyroid nodules are very common, and the vast major-ity is biologically benign. The difficulty facing the clini-cian is selecting the few genuinely malignant thyroidnodules for surgery (76), when a correct diagnosis canonly be made on excised material examined histologi-cally. Fine-needle aspiration biopsy, although accuratefor distinguishing papillary, medullary, and anaplasticcarcinoma, is unable to distinguish benign from malig-nant follicular neoplasms. The criteria for malignancyare capsular or vascular invasion at the periphery of theneoplasm. This requires surgical removal of the entiretumor and extensive laboratory examination. Thus, thediagnosis of malignancy depends very much on sam-pling and is, at times, pure chance. This dilemma waseditorialized by Ernest L. Mazzefarri (76).

Intraoperative Biopsies

In the first MRS study, tissue was obtained intraoper-atively from 53 consecutive patients undergoing partialor total thyroidectomy for solitary thyroid nodules (28).Typical 1D 1H MR spectra (8.5 T) of follicular adenomaand follicular carcinoma are compared in Fig. 8.

Visual inspection of the data and measuring the ratioof resonances at 1.7 and 0.9 ppm, MRS distinguishednormal tissue from carcinomas (proven clinically or his-tologically) with an accuracy of 100% (28). When thisresonance ratio was calculated for all follicular adeno-mas, some specimens grouped with the benign lesionsand others with the carcinomas. One possible explana-tion is that adenomas with a malignant spectral patternare in the process of malignant transformation. Whenthe SCS method was applied to this thyroid data anaccuracy of 100% was reported for normal thyroid vs.carcinoma (77).

It is important to recall that the specimens in thisparticular study were obtained intraoperatively, i.e., af-ter ligation of blood supply to the thyroid. In order forthis biopsy method to be of use it had to be extended to


FNAB obtained preoperatively. However, the thyroid isa very vascular organ and the FNAB specimens con-tained significant amounts of blood, which masked thediagnostic chemical signature (W. Mackinnon, C. Lean,P. Russell, L. Delbridge, P. Malycha, and C. Mountford,unpublished results).

Thyroid Spectroscopy In Vivo

There are now three reports of in vivo spectroscopy ofthe thyroid at 1.5 T (78) and 3 T (1). The group in HongKong (78) used a standard volume neck coil to show thefeasibility of in vivo MRS at 1.5 T by examining eightcarcinomas (including follicular, anaplastic, and papil-lary) and five apparently normal thyroid glands. A spec-trum obtained from an anaplastic carcinoma (TE � 136msec) is shown in Fig. 9. The analysis was centered onthe presence or absence of choline to creatine ratios.

A special purpose designed multiring surface coil wasbuilt for undertaking in vivo MRS of the thyroid at 3 T(79). A typical 3-T spectrum from a solitary thyroid

nodule is shown in Fig. 10. The T1-weighted axial imageof the thyroid lesion is compared with a spectrum of athyroid biopsy of the same pathology but recorded at8.5 T. The lesion is a benign adenoma as determined byMRS on the biopsy and confirmed histologically.

It can be seen that the in vivo spectra obtained at both1.5 T and 3 T are directly comparable to those recordedat the higher field strength of 8.5 T and on biopsymaterial from patients with the same diagnosis. Thedevelopment of the MRS method for determining accu-rate diagnosis would remove the necessity for thyroid-ectomy for many women. However, while this methodshows potential, a large study involving serially section-ing the entire thyroid now needs to be undertaken andclassifiers need to be developed.

OVARY

Ovarian cancers occur in one out of 57 women. Thedisease is commonly asymptomatic until it has metas-

Figure 7. In vivo spectroscopy of the cervix. 1H MR spectra with corresponding MR image and biopsy in cervical cancer: in vivopoint resolved spatial selection (PRESS) (TR � 1600 msec, TE � 135 msec) (a), ex vivo Carr Purcell Meiboom Gill (CPMG) at TE �135 msec (b), and ex vivo pulse-collect (c) spectra from a patient with cancer. a: Triglyceride –CH2 and –CH3 signals, Cho, creatineplus phosphocreatine (Cr), and a peak at 2 ppm are seen. Assignments of (b) and (c) are 1 � triglyceride –CH3, 2 � triglyceride–(CH2)n–, 3 � triglyceride –COCH2CH2–, 4 � triglyceride –CH2CH2CO, 5 � triglyceride –HC_CHCH2–, 6 � triglyceride –COCH2,7 � triglyceride –HC_CHCH2HC_CH, 8 � amino acid –CH3, 9 � lactate –CH3, 10 � alanine –CH3, 11 � acetate –CH, 12 �glutamate –CH2, 13 � creatine –CH3/lysine –CH2, 14 � choline–N (CH3)3, 15 � phosphocholine–N (CH3)/glycerophosphocholine–N–CH2, 16 � phosphatidylcholine –N (CH3)/taurine –(CH2–NH), 17 � unidentified resonance, 18 � taurine –(CH2–SO3), 19 �glycine–_CH; 20, creatine –CH2, and 21 � lactate –CH. In (a), triglycerides are in-phase relative to choline. In (b) and (c),triglyceride –CH2 and –CH3, Cho, and Cr are increased. d: A T2-weighted fast spin-echo image (TR � 4000 msec, effective TE �88 msec) using an endovaginal coil 37 mm in diameter (arrowheads) shows an irregular mass of tumor centrally within the cervix(arrows) filling 70% of the voxel of interest (marked). A � anterior, P � posterior. e: Section from the hematoxylin andeosin–stained biopsy (approximately 400�) analyzed by ex vivo MRS shows malignant cells with hyperchromatic nuclei anddense eosinophilic cytoplasm (arrows) occupying approximately 40% of the biopsy. (Reprinted from Mahon M, Cox IJ, Dina R,Soutter PW, McIndoe M, Williams A, deSouza NM. 1H magnetic resonance spectroscopy of preinvasive and invasive cervicalcancer: in vivo-ex vivo profiles and effect of tumor load. The Journal of Magnetic Resonance Imaging 2004;19:356–364, Withpermission from Wiley-Liss, Inc, a subsidiary of John Wiley & Sons, Inc.)


tasized with 70% to 75% of women presenting withadvanced stage disease; the five-year survival for thesewomen is only 20%. This poor overall outcome hascreated a bias towards aggressive therapy for all womenwith ovarian disease. Quality of life is important indeciding the extent of surgical procedures for patients.For ovarian cancer patients, this includes preservingfertility potential and minimizing the negative fertilityeffects of cancer treatment in young women (80).

In particular, conservative surgical management ofearly-stage epithelial ovarian cancer (81) offers much tothe patient when the disease is staged and typed accu-rately. It would be important for those patients with

genuinely benign disease of those with so-called “lowmalignant potential” (LMP) tumors, which may or maynot be malignant, if their diagnosis could be establishedprior to surgery, thus allowing more confident triagingfor appropriate therapy.

The LMP tumors, which frequently occur in youngwomen, account for 14% of ovarian surface epithelial/stromal neoplasms. These commonly encountered“surface epithelial stromal tumors” (82) have notionallybenign and malignant counterparts that are separatedby a quite arbitrary histological criteria, which may notcorrelate with the clinical outcome for the patient.

Thus no guidelines currently exist, at the interfacebetween benign and malignant, which provide for areliable prediction of the biological potential in a givencase of ovarian epithelial neoplasia. Many women un-dergo unnecessary surgery and therapy for tumors thathave no malignant potential (82). The extra ovarian“implants” associated with such LMP neoplasms areoften thought of as metastases, leading to unnecessarytreatment in many cases. Recent molecular geneticstudies (83,84) have cast doubt on the premalignantpotential of the proliferating (LMP) serous tumors, mak-ing it more critical that they be accurately distinguishedfrom their frankly malignant counterparts.

Intraoperative Biopsies

In an initial study of approximately 70 cases, it wasshown that MR spectral differences distinguished be-tween subsets of ovarian epithelial/stromal tumors(23). Typical 1D MR spectra from histologically normal,benign, proliferating (LMP) and malignant ovarian tu-mors of serous and mucinous types were compared inthis report. In the malignant category, visual inspectionshowed that while the malignant serous and mucinoustumors had very similar 1D MR profiles, the benigncategory showed slight differences. In the LMP category,mucinous tumors had a far more active chemical profilethan serous LMP tumors, with the mucin being clearlyvisible at around 3.8 ppm. An explanation is that somemucinous LMP tumors are already exhibiting MR evi-

Figure 8. 1H MR (8.5 T; 37°C) spectra from follicular carci-noma (a); benign follicular adenoma (b). Spectra were acquiredwith 256 accumulations, residual water was suppressed usinggated irradiation. Diagnostic resonances at 0.9 ppm (CH3,lipid) and 1.7 ppm (CH2, lysine) are denoted. �N(CH3)3 � N-trimethyl from choline containing metabolites, HOD � resid-ual water. (Reprinted from Mountford CE, Doran ST, Lean CL,Russell P. Cancer pathology in the year 2000. BiophysicalChemistry, 1997; 68:127–135, with permission from Elsevier.)

Figure 9. Anaplastic carcinoma of the thyroid in vivo. a: Proton MR spectrum acquired at TE � 136 msec from the primarytumor in a patient with an anaplastic carcinoma of the right lobe of the thyroid. The nominal voxel volume was 36 cm3.Prominent peaks detected were Cho (3.2 ppm), Cr (3.03 ppm), fatty acid (FA) (–CH2–) (1.30 ppm) and FA (–CH3) (0.90 ppm). b:Transverse T1-weighted MR image shows the position of the volume of interest for spectroscopy represented by the white squarebox placed within the primary tumor. (Reprinted from King AD, Yeung DKW, Ahuja AT, Tse GMK, Chan ABW, Lam SSL, vanHasselt AC. In vivo 1H MR spectroscopy of thyroid carcinoma. European Journal of Radiology, 2005; 54:112–117, withpermission from Elsevier.)


dence of an adenoma–carcinoma sequence as a presageto full malignant transformation, a process that is nolonger postulated for their serous counterparts (83,84).In the initial study, the 1D spectral analysis distin-guished carcinomas from benign tumors and normaltissue with a sensitivity and specificity of 88% and 97%,respectively (23). An independent study was under-taken by Wallace et al (85) in which the SCS methodwas used to analyze the data. Ovarian cancers weredelineated from benign tumors with an accuracy of98%. The method also distinguished untreated ovariancancer from recurrent ovarian cancer with an accuracyof 97%.

Adenoma–Carcinoma Sequence as Monitored by2D Correlated Spectroscopy

The 2D correlated spectroscopy (COSY) method wasdeveloped by Ernst (43) and first applied to cells in 1984(14). Typical 2D COSY spectra of normal ovarian tissueand serous carcinoma are shown in Fig. 11. The COSYspectrum of the poorly differentiated serous carcinoma(Fig. 11b) is typical of carcinoma from many organs(14,21,27,86). An increase in chemical activity can beseen in Fig. 11 from normal ovary to carcinoma of theovary, which is also consistent with the spectra re-corded for other organs exhibiting an adenoma–carci-noma sequence.

One particular part of the COSY spectrum that hasprovided a valuable insight into the presence of theadenoma–carcinoma sequence is indicated on Fig. 11b.This spectral region, F1 � 3.9–4.5 ppm and F2 � 1.0–1.6 ppm, contains the methyl-methine coupling fromlactate (1.33–4.12 ppm) and cross-peaks from cell-sur-face fucose (21).

It is this region of the spectrum that has identifiedalterations associated with loss of cellular differentia-tion and an increase in cell surface fucosylation (22,27)a phenomenon well documented by immunochemicalmethods (87–90). The increasing complexity of theCOSY fucosylation spectral pattern with tumor devel-opment and progression has been shown previously forcolon (22,27) and breast (1). The increasing complexityof the COSY fucosylation spectral pattern with ovarian

tumor development and progression is shown in Fig.12, where the expanded methyl-methine coupling re-gion from the COSY spectra are shown for normalovary, benign tumor, proliferating tumor, well-differen-tiated carcinoma, moderately differentiated carcinoma,and poorly differentiated carcinoma. Analysis of the 2DCOSY data showed that of FucII, IIb, or III were presentin spectra from carcinoma that were poorly differenti-ated.

Of the LMP ovarian tumors examined, three of nine(one serous and two mucinous) generated cross-peakFucIII in addition to FucI, FucII, and FucIIb and aretherefore spectroscopically consistent with being malig-nant. In stark contrast the remaining six LMP tumorsgenerated a single cross-peak at Thr/FucI spectral fre-quency as seen in Fig. 12a. Fibromas, benign epithelialtumors, and normal tissue also generated a singlecross-peak at the Thr/FucI spectral frequency as seenin Fig. 12a. Thus three of the LMP tumors were chem-ically akin to the malignant tumors, and six moreclosely resembled benign tumors.

In Vivo at 3 T

In vivo spectroscopy and imaging of the ovary at 3 T canprovide diagnostic information preoperatively on pa-tients with ovarian masses. The 3-T pelvic image of apatient with a suspected ovarian cancer is shown in Fig.13 (91). There is a mixed cystic and solid lesion mea-suring 4 cm in diameter. Two-dimensional CSI wasundertaken in the region identified by the box. Thespectral information obtained from the voxel placedover the solid lesion is shown to the left. This spectrumcontains choline (3.2 ppm), creatine (3.0 ppm), othermetabolites and at 0.9 (not shown on figures), 1.2 ppmassigned to the methyl and methylene of lipid and isconsistent with a malignant tumor. The voxel on theright is placed on the cystic component and the result-ant spectrum is to the right. This spectrum does show asmall contribution from choline, indicating the pres-ence of malignant cells. The ex vivo (8.5 T) (not shown)and in vivo (3 T) spectra are very similar. In the 2Dspectra (Fig. 11), the region 3.8–4.5 and 1.0–1.6 ppmshows cell surface fucosylation patterns consistent

Figure 10. Benign follicular adenoma of the thyroid in vivo at 3 T and on biopsy at 8.5 T. a: In vivo 3-T T1-weighted axial image.b: Corresponding in vivo 1H 3-T MR spectrum. (Undertaken at the National Research Council [NRC] of Canada, Winnipeg). Datawas obtained using a specially designed multiring surface coil). c: Ex vivo 1H, 8.5-T MR spectrum from a biopsy specimen of thesame pathology, spectrum was acquired with 256 accumulations, residual water was suppressed using gated irradiation.(Reprinted from Mountford C, Doran S, Lean C, Russell P. Proton MRS can determine the pathology of human cancers with ahigh level of accuracy. Chemical Reviews 2004;104:3677–3704, © 2004 American Chemical Society.)


with level of dedifferentiation (1,23). The independenthistopathological examination confirmed the spectraldiagnoses.

Ovarian tumors tend to present clinically when quitelarge (10 cm in diameter or larger). This makes the

implementation of single voxel relatively easy and thusamenable to routine clinical examination in vivo at 3 T.The accuracy of the spectroscopy method in vivo re-mains to be determined.

ESOPHAGUS

Endoscopic Biopsy

Adenocarcinoma of the lower esophagus in the Westernworld (92) is rising in incidence and accounts for 40% ofesophageal carcinomas in males (92,93).

Esophageal carcinoma is thought to be caused fromgastric reflux and a condition known as Barrett’sesophagus is considered to be a precursor (94), with a40- to 50-fold increased risk of developing malignancy.Histopathology distinguishes normal from invasive car-cinoma of the esophagus with a high level of accuracy.Histopathology is not, however, able to provide an ac-curate prediction of the potential of dysplastic Barrett’sepithelium (95,96).

In a study investigating 72 consecutive patients, 29non-cancer-bearing and 43 cancer-bearing, MRS ana-lyzed by SCS distinguished normal epithelium fromesophageal carcinoma and from Barrett’s esophagus.Typical 1D 1H MR spectra at 8.5 T are shown in Fig. 14.1H MRS combined with the SCS method distinguishednormal Barrett’s epithelium from carcinoma with anaccuracy of 100%, normal from Barrett’s with an accu-racy of 100%, and Barrett’s vs. carcinoma at 96% (19).The spectra from Barrett’s and adenocarcinoma con-tained a relatively intense choline resonance.

Of importance are the spectra from Barrett’s epithe-lium from a non-cancer-bearing patient and from acancer-bearing patient with the corresponding histopa-thology in Fig. 14. Histologically, these tissues are in-distinguishable, yet the MR spectra group the specimensfrom cancer bearing patients with adenocarcinoma.There is evidence supporting the existence of an adeno-ma–carcinoma sequence in the esophagus (97,98).These MRS data support the MRS method being able toidentify field change consistent with the presence of anadenoma–carcinoma sequence as reported for the thy-roid and colon. The MRS study on esophageal biopsieshas been in progress for over seven years. Patients areinformed of the research outcome of the MRS analysisand the number of those patients identified as havingtissue committed to malignancy are now presenting forsurgery (Falk G, Doran S, Phillips J, Lean C, Russell P,Mountford C, unpublished results). The test is ready forclinical acceptance testing to ascertain its ability topredict the biological potential of Barrett’s’ esophaguson biopsy.

To date there has been no recorded attempt to de-velop this technology for in vivo MRS diagnosis.

TRANSLATION OF SPECTROSCOPY INTO THECLINIC

For spectroscopy to be effectively transferred from anacademic setting into routine hospital use there needsto be a strong working collaboration between the mul-tidisciplinary team and a commercial entity. Education

Figure 11. Two-dimensional spectroscopy on ovarian biop-sies. a: Normal ovarian tissue. b: Poorly differentiated serouscarcinoma. 1H MR magnitude-mode COSY spectra; 64 accu-mulations, 200 experiments. Spectra processed using a Sinebell, Lorentzian-Gaussian (line broadening (Lb) � –40, Gauss-ian broadening (Gb) � 0.12), window function, in the t1 and t2

domain, respectively. A–F: from fatty acyl chains; G�: fromgerminal protons on the glycerol backbone of triglycerides;Chol: cholesterol; Lys: lysine; Lac: lactate. (Reprinted fromMackinnon WB, Russell P, May GL, Mountford CE. Charac-terisation of human ovarian epithelial tumours (ex vivo) byproton magnetic resonance spectroscopy, International Jour-nal of Gynaecological Cancer 1995;5:211–221, with permis-sion from Blackwell Publishing.)


of all participants is an important means of ensuringeffective translation into the clinic.

BIOPSY PROGRAM

Housekeeping issues, such as the plastic used for col-lection vials and syringes, and storage conditions forspecimens, are of paramount importance (99). As de-scribed earlier, biopsy techniques, particularly for theFNAB, are important as at least 5 � 106 cells are neededfor adequate SNRs to be obtained during sample test-ing. However, once these technical elements are undercontrol, the data will, in the future, be able to be ana-lyzed automatically by robust classifiers.

IN VIVO MRS

The technical difficulties have been well described forneurospectroscopy by Roland Kreis (100), and thesedifficulties apply equally well to other organs. The dif-ficulties initially experienced by the UCSF team in ex-tending their prostate program to other centers has nowbeen overcome due to a closer working relationshipwith a commercial partner and the significant time andeffort expended by Dr. Kurhanewicz and his colleaguesin assisting other sites. The same care and time willnow need to be given for breast spectroscopy to come ofage in vivo.

Technical variables such as the coils for each organ(whether they are transmit-receive or receive-only), are

of paramount importance for body spectroscopy. Cur-rently, there is a plethora of coils available for the breastand it remains to be determined which coils are suitablefor spectroscopic analysis in addition to MRI. Tradition-ally, coil manufacturers have set their sights on highquality images and ignored spectroscopy. This is nowchanging.

CONCLUSIONS

Proton MRS is now ready to be introduced into thepathology laboratory, initially for the breast, where itcan not only provide a diagnosis but also prognosticindicators. The technology is also ready to commenceacceptance testing to ascertain its ability to predict thebiological potential of Barrett’s esophagus lesions.

The combination of MRI and MRS in vivo with correl-ative MRS on biopsy specimens offers great accuracyfor the diagnosis and prognosis of some human can-cers. Currently, the only body organs for which this canbe undertaken routinely are the breast and the pros-tate.

In the case of prostate, multifocal disease is commonand the MRS method offers a method for mapping mul-tifocal disease. For breast, spectroscopy in vivo canidentify regions of abnormal pathology where ultra-sound and computed tomography (CT) can fail. Forboth breast and prostate the capacity to undertake im-age guided biopsy using 3D CSI will significantly im-prove the preoperative diagnostic accuracy. It remains

Figure 12. Two dimensional spectroscopy on ovarianbiopsies. Expanded methyl-methine coupling region(F1 � 3.9–4.5 ppm, F2 � 1.0–1.6 ppm) from the 2DCOSY spectra (Fig. 11) showing increasing complexity offucosylation pattern with histologic grade for serous tu-mors. Contour levels started at 6000 and increased ex-ponentially. a: Normal ovary. b: Benign tumor. c: Prolif-erating tumor. d: Well-differentiated carcinoma. e:Moderately differentiated carcinoma. f: Poorly differen-tiated carcinoma. Thr/Fuc I, Fuc III, II, IIb: resonancesattributable to bound fucose, with a contribution fromthreonine to Thr/Fuc I. (Reprinted from Mackinnon WB,Russell P, May GL, Mountford CE. Characterisation ofhuman ovarian epithelial tumours (ex vivo) by protonmagnetic resonance spectroscopy. International Jour-nal of Gynaecological Cancer 1995;5:211–221, with per-mission from Blackwell Publishing.)

Figure 13. MR of the ovary in vivo at 3T. Axial T2-weighted 3-T image showingcystic ovarian mass and from 2D CSI.Spectrum to the left is from malignantmass. Spectrum on right is from cysticcomponent. (Reprinted from MountfordCE, Stanwell P, Ferrier A, Bourne R, Do-ran S, Christie J, Chin R, and Russell P.In vivo spectroscopy and imaging ofthe ovary in vivo at 3 Tesla and spectros-copy on biopsy at 8.5 Tesla. Journal ofWomen’s Imaging 2005;7:71–76, withpermission from Lippincott Williams&Wilkins.)


to be clarified whether spectroscopy is more accurate atdetermining the extent of disease in these organs com-pared with MRI, as has been shown for the brain (101).

The proof that MRS can monitor disease as well aspredict tumor progression will be in the next five years,a mere 30 years after the initial observation. Time willprovide testament to the accuracy of MRS both on bi-opsy and in vivo in the cancer clinic. If correct, thespectroscopy technology will also apply to other areassuch as cardiac disease, and diagnosis of causation ofpain.

ACKNOWLEDGMENTS

We acknowledge the long-term contributions from theother clinical and scientific directors of the Institute forMagnetic Resonance Research (IMRR): Professors Mar-tin Tattersall and Tania Sorrell, Dr. Uwe Himmelreich,and Peter Stanwell, and more recent members, Profes-sors Brain Tress and Clive Harper. We gratefully ac-knowledge the mentoring of Dr. ICP Smith, and thecontributions made by his and our colleagues at theNational Research Council (NRC), Winnipeg, Dr. RaySomorjai, Dr. Brion Dolenko, Dr. Slavek Tomanek, Dr.Scott King, and Dr. S. Nikulin. We thank Dr. Herb

Kressel for his guidance in the field of clinical MRI andhis insight into how to implement MRS in the clinic. Wethank Xavier Bujwalla, Michael Garwood, Ingrid Gribbes-tadt, Mary Hochman, J. Jaganathan, John Kurhanewicz,and Bob Lenkinski for sharing their thoughts and goalswith us. We thank Drs. Roger Bourne and Phillip Kate-laris for their contribution to the prostate program, andDrs. David Gillette, David Clarke, and Lawrence Gluchfor their contributions to the breast program. We thankSinead Doran and Susan Dowd for their tireless datamanagement and present and past staff and studentswithin the Department of Magnetic Resonance of Med-icine. Thanks also to Dr. Andew Clouston for the esoph-ageal pathology, and to Associate Professors JeanettePhilips, Michael Bilous, and Robert Eckstein, and Drs.Stephen Fairy and Geoff Watson for sharing their ex-pertise in pathology. We thank Dr. Deborah Edwardand Sinead Doran for helping prepare this manuscript.We thank our colleagues and friends from around theworld for allowing us to describe their work and for theirmany interesting discussions and contributions to thisreview. We thank Professor Tom Reeve and his manysurgical colleagues in Australia and around the worldfor believing MRS could be the definitive diagnostictechnology and giving the technology a chance. Last,

Figure 14. Esophageal biopsies. 1H MR(8.5 T; 37°C) spectra of esophageal biop-sies from four different histopathologicalsubtypes. a: Normal esophagus. b: Bar-rett’s epithelium from non-cancer-bear-ing patients. c: Barrett’s epithelium fromcancer-bearing patients. d: Adenocarci-noma. On the right are the comparativehistology slides. Spectra were acquiredwith 256 accumulations, residual waterwas suppressed using selective gated ir-radiation, sweep width of 3600 Hz, 8000data points. (Reprinted from Doran S,Falk G, Somorjai R, Lean C, Himmel-reich U, Philips J, Russell P, Dolenko B,Nikulin A, Mountford C. Pathology ofBarrett’s oesophagus by proton mag-netic resonance spectroscopy and a sta-tistical classification strategy. AmericanJournal of Surgery 2003;185:232–238,with permission from Excerpta Medica,Inc.)


but not least, C.M. thanks Professor R.J.P. Williams ofWadham College, Oxford for the encouragement in1978 to investigate “[a] result that is very likely to beright but will be very difficult to prove.”

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