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Meta-Analyses of Studies on Bone MarrowMicrometastases: An Independent PrognosticImpact Remains to Be SubstantiatedBy Ilona Funke and Winfried Schraut

Purpose: In 1997, the immunocytologic detection ofisolated umor cells in bone mamow, termed micrometos-tasis, will be optionally included in the tumor-node-metastasis (TNM) classification indicated Ml,i). In thepresent meto-analyses, 20studies, which included 2,494patients, regarding the prognostic influence of a posi-tive bone marrow micrometastases lBMMl status on, ~- ~ - ~ -relapse-freeand/or overall survival were analyzed.Materials and Methods: The literature search in-cluded the Medline and Current Contents bibliographicdata bases from August 1980 o June 1997. The statisti-cal evaluation considered the prognostic nfluenceof theprevalence of micrometastatic cells in bone marrow onrelapse-free and/or overall survival. The comparableeffect estimate and its corresponding 95% confidenceinterval (Cl) were calculated with the Mantel-Haenszelmethod using the originally published data of the re-trieved studies.Results: The presence of epithelial cells in bone mar-row was detectable in all carcinoma types, with a

median prevalence of approximately 35%. Fourteen of20 studies found a positive correlation between positiveBMM status and reduced relapse-free survival by uni-variate analysis, but only five of 11 studies confirmedpositive BMM status as an independent predictor ofshort disease-free survival. Regarding overall sum'val,positive BMM status was identified univariately in fiveof 12 studies, but multivariately in only two studies, asan independent factor of poor survival. Despite theheterogeneity of the studies, calculation of the relativerisk (RR) for reduced relapse-freesurvival was possiblefor breast cancer, which -resulted n a Mantel-~aenszelRR (R bH) f 1.34 (95%CI, 1.27 to 1.42).Conclusion: In conclusion, the results suggest thatthe prognostic impactof epithelial cells in bone marrowremains to be substantiated by further studies usingstandardized methodic protocols before its entrance inthe TNM classification.J Clin Oncol 16:557-566. o 1998byAmerican Soci-etyof ClinkalOncology.

I VARIOUS TYPE S OF CARCINO MA, the prevalence data suggest the cautious interpretation of handling theof isolated tumor cells in bone marrow gains increasing BMM status as a prognostic indicator for a variety of tumoracceptance as an indication of disseminated malignant types and strengthen the necessity of defining a consensusdisease. In 1980, Sloane et al l were the first to use an on methodology and standards for the design of furtherirnrnuno cytochemical approach for the detection of disserni- studies.nated carcinoma cells in bone marrow aspirates of breastcancer patients and termed them micrometastatic cells. MATERIALS AND METHODS

Since this pioneering work, a number of studies have Literature Search and Selection Cr iteriafocused on the prognostic impact of the bone marrowmicrometastases (BMM) status in a variety of epithelialtumors and correlated the presence of imm unocytologicallydetected carcinoma cells in bone marrow with establishedclinicopathologic factors and/or follow-up parameter^.^-^^ Inaddition, evaluation of phenotypic characteristics of indi-vidual micrometastatic cells, as well as the experimentalapplication of other techniques such as polymerase chainreaction (PCR), fluorescence activated cell sorter, and en-zyme-linked irnrnunoadsorbent assay (ELIS A) analysis wereperformed.3148

The literature search included the Medline and Current Contents(program MacSPIRS, version 2.4, Silver Platter International SoftwareNV) ibliographic data bases fro m August 1980 to June 1997 using theterms: bone marrow, micrometastases, carcinoma, immunocytochemis-uy, and prognosis. Letters to the editor, abstracts, reviews, and articlespublished in a book or without an English abstract were excluded fromfurther consideration . From the original articles, those with less than 20patients or not enough data to ca lculate at least a relative risk (RR) as acomparable effect estimate for relapse-free andlor overall survival werealso excluded from the meta-analyses. Some studies were based on thesame patient series. To exclude multiple tested cohorts, the data publishedmost recently by these authors were included. To clanfy this important issue.

Today, BMM status is almost accepted as a usefulparameter to define precisely the tumor stage of the indi- From the Deparhnent of Surgery, Universio of Munich, Munich,vidual cancer patient and will be inc luded in the tumor-node- Gennany.metastasis (TNM) classification as a facultative prognostic Submitted March 1,1997 ; accepted ~e ~ te rn b e r,1997.factor indicated Mlo).4 9 Address reprint requests to Ilona Funke, MD, Department of Surgery,Klinikurn Grophadern, Marchioninistrape 15, 81377 Munchen, Ger-

In the present meta-analyses, a critical evaluation of the ,ny; Emailfrcnke~gch,meduni-rnuenchen,de,published data concerning the prognostic relevance of 0 998 b y American socieo of clinical oncology.micrometastatic cells in bone marrow was performed. These 0732-183x/98/1M)2-0021$3.00/0

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8 FUNKE AND SCHRALJTthree studies were contacted and the informationtwo cases. Neverthe-bsequent publication bias by not publishing nonsignificant

by the authors using a standardized summary form. Theere compared , and in case of discrepancy, the report wasachieved. Scanning the references ofrelevant studies.

ical M ethodsstatistica l evaluation considered the prognostic influence of the

95% confidence interval (CI) were calculated with the(RRm) method using the originally published data of

in a log-linear scaleThe RR estimates the quan titativet of the risk of patients with a positive BMM status in comparisontus. The 95% CI illustrates theRR within a 95% CI. n breast cancer, an overall RR was

orRR was calculated using the formula RR(Mv,= eP; C1959i(RR)=

X SE), where SE is the standard error, if this was noty l calcula-407 software byary, NC.

del to Estimate the Level of Sensitivity Dependinghe Number ofAnalyzed Bone Marrow-Derived Cells

assumption of a 100% sensitivity of the antibody used tomarrow sample, the

binomial distribution and according to the large number of analyzedbone marrow-derived cells that contain a relatively small number ofmicrometastatic cells by a Po isson distribution. The resulting probabil-ity to detect at least one epithelia l cell in n bone marrow-derived cellscan be approximated by the following formula: w = 1 - 1 - )O = 1 -e-o P. This formula was used in Table 5 .

RESULTSThe literature search identified a total of 112 articles.nKenty studies, which included a total of 2,494 patients,fulfilled the selection criteria and were included in theanalysis of the influence of the BMM tatus on relapse-freeandlor overall survival.

Patient Ch aracteristics and Prevalenceof Micrometastatic Cells

All studies included in the present meta-analyses were ofthe cohort type. The majority of the data was collected inbreast cancer (Table 1). The size of the cohorts varied from25 to 727 patients and included series that differed in theTN M classification and the residual tumor after surgery (Rclassification).The prevalence of a positive BMM tatus inbreast cancer ranged from 2% to 48%, with a medianprevalence of 35%. Similar to breast cancer in other tumortypes such as carcinomas of the gastrointestinal tract, lung,and head and neck, study cohorts and clinical characteriza-tion of the investigated patients were heterogeneous. Themedian prevalence of 38.5% (range, 26.9% to 59.7%) wasnot significantly higher than in breast cancer.

bbk 1. PatientCdkaiveJ and Prwaknce of BMMFinlA h r , Year TumorTm No. ofMenh Pnnrrlence of BMM I%) TNMSbges: R Clossilication

1988 Breast 285 27.0 T1 -4NO-3MO;nsp1990 Breast 25 48.0 TO-2NO-1MO;nsp1990 Breast 121 16.5 T I -2NO-1MO;nsp1991 Breast 49 36.7 T1 -3NO-3MO;nsp1991 Breast 39 33.3 T1 -4NO-3MO;nsp991 Breast 350 25.4 TO-4NO-3MO;nsp1991 Breast 71 38.0 TI -4NO -3Ml;R21992 Breast 50 2.0 T1 -3NO-3MO;nsp1994 Breast 100 38.0 T1 -4NO-3MO;nsp1996 Breast 727 43 .3 T1 -4NO-2MO;nsp1997 Breast 109 31 .1 T1 -2N0,MO; RO-R21990 ' Colon/rectum 156' 26.9 T1 -4NO-3MO-1;nsp991 S h c h 97' 35.0 T1 -4NO-340-1;nsp1992 Cdon/rectum 88 31.8 T 1e4NO-3MO; RO1994 Head/neck 73 37.0 T I -4NO-3MO-1;nsp1995 Lung 43 40.0 T I 4NO-3MO;nsp

1996 Lung 139' 59.7 T I -4NO-3MO;RO1996 Stomach 180 ' 53.0 TI-4NO-2MO-1 O-R2996 Pancreas 42' 57.1 T1 -4NO-2MO-1; RO-R21996 Esophagus 90' 41.1 T1 -4NO-3MO-1;RO-R2

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META-ANALYSES ON PROGNOSTIC IMPACT OF MICROMETASTASES 559Methodology BMM Status and Classical C linicopathologic Factors

Methodologic procedures may have a remarkable influ-ence on sensitivity and specificity of tumor-cell detectionrates in bone m arrow. The 20 studies differ in the number ofinvestigated bone marrow -derived cells, antibody selection,staining procedure, and control series of healthy donors(Table 2). Both smears and cytospin preparations of mono-nuclear cells isolated f ~ o mone marrow samples have beenused. The number of c ells analyzed was given in only ninestudies and ranged from 1.5 X lo5 to 5.0 X lo6 cells perpatient. Twenty-four different monoclonal or polyclonalantibodies or antibody cocktails directed against differentantigens were used for immunocytochemical identificationof isolated tumor cells in bone marrow. The greatestexperience is available with the antibodies EMA, directedagainst a n epithelial cell-surface antigen,53 and CK2, di-rected against the cytokeratin component no. 18.54,55 peci-ficity controls were reported in 10 studies that used bonemarrow samples from patients without malignant diseaseand the same imrnunocytochemical approach performed inbone marrow aspirates from c ancer patients. In the largestcontrol group, six of 215 false-positive results were de-scribed, which corresponds to a specificity of 97.2%.

The presence of epithelial cells in bone marrow wascorrelated with established clinicopathologic parameters in13 of 20 studies (Table 3). In breast cancer, four of sixstudies described a positive correlation (P < .05) betweenthe presence of BMM and a positive axillary lymph nodes t a t u ~ . ~ ~ ~ , ~ ~ ' ~n addition, in some studies, positive BMMstatus was found to correlate with further parameters ofadvanced cancer disease, such as pT stage, enlarged tumorsize, and vessel invasion,2g7 although contrary results wereobtained. With the exception of the study reported by M ansiet al,' who demonstrated an inverse correlation betweenBMM and estrogen receptor (ER) status, most of the studiesfound no association between these two variables. In othertumor types, with the exception of two studies in gastrointes-tinal cancer^,'^.]^ no correlation between BMM status andclassical parameters of tumor progression was detectable.Impact of BMM Status on Relapse-Free Survival

According to the selection criteria of the present meta-analyses, all studies considered the impact of BMM statuson relapse-free survival. Seven analyses focused on asubpopulation, ie, curatively resected patients (R0),'4.19-2

Table 2. -K C&a in StudiesomBMMFirstA u k No,ofCells Anabze.4 Antibodies U d h4whod ControlPotisnh'

Berner2 nsp EM4 (pdyclonal)t AP Noneirk; 'lSP UCR.LON.M8.4+ AP NoneSolvadori4 nsP Mbr 15 IF NoneCoJ nsP C26; TI 6; AE- 1l IF NoneDcarnalqd 'lSP EM4t AP NoneMansi7 "SP EM At AP NoneSingleto+ "sP AE- 15; AE-35; MAK 65; 1 13Flt; 260F9 t; 31 7G St AP NoneCwrtemanche9 nsP UCR.LON.MB+ AB-POX NoneH o r t - ~ k ' ~ nsP EM4t; 12H121; anti-CK8,18,195 AP NoneDie('' nsP 2 E l l l AB-AP 0/21 I(Molinol2 "SP Mbr 1,$M br 8,s MO V 85 MOV16,s MLuCI5 IF NoneSchlimokl3 1.5 x 105 CK25 APAAP 0/75Schlirnok" 1.5 x l o 5 CK25 APAAP 0/10211Lindemann'5 3.0 x 105 CK25 A?AAP 0/ 102 1Wollen berg1& 1.0 X 106 anti-CK19t APAAP 0/18CoteI7 5.0 x 106 CK25; W 4 5 . 2 5 APAAP 0/65PandlB A X 14-1.6 x 106" CK25 APAAP 6/2 1 5 11Jauchl9 1.0 x 106 CK25 APAAP 1/A6Thorbana 5.0 x 105 CK25; KLlS; W - B /B 3 APAAP 0/25Thortanll 4.0 x 105 CK25; KL l5; A45-B/B35 APAAP 0/30

Abbreviations: AP, alkaline phosphotose; IF, immunofluoreuence;AB-POX, avidin-biotinparoxidaw; AB-AP, avidin-biotinalkaline phosphahw; APAAP, alkalinphosphahswantialkalinephosphotow.'Positive number to tested number.tht i-e pih eli al cell-surfocsantigm.+Antimilk fot

5hti-c ytokero tin components.(AntCpolyrnorphic epithelial rnucin./mereconcrd groups represent ex ~ s io n sf previous studies and are not independent.**Diffirent numbersof bone marrow-derived cells were ono bzed within the same patient cohort. .

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FUNKE AND SCHRAUTk3. C a m h i o n ofBMMond Established dinicopalhdogicPammekKs

Clinicopdhdogic PornmetenFintA& Significant bnakfions* Nonsignificantbnolafiont

pN, V-inv Histdogic type, ER, tumorsize

Nat tested Not teshdNot tested Not testedNot tested Not testedpN Tumor size, ER

nsi7 pT, pN , tumor size, ER, V-inv Age, menopausal status&ry9 Na ttarted Not tested

Nat tested Not tested- pT, pN , ER, PR, manopouxrl

s w s , grading, histologicmid 1 pT, pN , grading, meno- ER, PR, Sphase fractionpausal status

- Tumor size, pN, menopausalstabs, ER, proliferativeindexDuke's stage, loccllizaiion -

Histologictype, pN ,Mstage -- pT, pN , age,a,rading,

localization- pT, pNO, gradingNot tested Not tested- pT, pN , grading; histulogic

'Ypehl9t B o r n a n - r n pT, UlCC stage, pN, grad-ing; histologic type,lymphangiosis

Not tested Not tested- Sex, age, UICC-stage, R

classificationAbbreviations: V-inv, vcrscular invasion; ER, eshogen receptor stubs; PR,

eskron receptor stutvs; UICC, Internationalunion Against Cancer.* P< 05 .tCorrelations were given only b r h e RO subgroup.

at least 12 months13 (Table 4). Different statisti-such as the X2, Fisher's exact probability,

between BMM status and relapse-free survival.p duration, ranged from 118 to 114 monthse6Four-

status than in patientsbone marrow using nonmulti-

ree s u r v i ~ a l . ' ~ ~ ~ ~ ~ ' ~ ~ ' ~ . ~ ~n contrast, five breast cancer stud-anBMM tatus as an independent predictort relapse-free survival d~ ra ti on .~ ~5 .~ ~8 J9

Additionally, in five breast cancer studies, the localizationof tumor recurrence was considered. Three studies showedthat patients without distant metastases but a positive BMMstatus at the time of surgery subsequently developed metas-tases in the skeleton, which i s a preferential secondary site inthis tumor type.2,6,7However, two other studies could notconfirm a preferential osseous manifestationImpact of BMM Status on Overall Survival

The impact of BMM status on overall survival wasevaluated with nonmultivariate procedures in 12 studies(Table 4). Five analyses showed a positive correlationbetween the presence of isolated tumor cells in bone m arrowat the time of diagnosis and a shortened overall survivalduration. However, in only two studiesI0,l1was the positiveBMM status identified as an independent predictor of poorsurvival.Calculation of the RR for Relapse-Free Survival

The RR was calculated for each study using the Mantel-Haenszel procedure (RRm) and is presented in a log-linearscale in Fig 1A. The RRMH f three breast cancer ~ t u d i e s ~is lower than 1, which indicates that the presence ofepithelial cells in bone marrow is not associated with anincreased risk for a short relapse-free survival duration. Inaddition, three further breast cancer s t u ~ l i e s ~ . ~ ~ith anRRm slightly greater than 1, but a 95% CI less than 1, alsodid not show a significant impact on relapse-free survival. Incontras t , in f ive breas t cancer ~tudies? ,~,~, '~ .~~he RRMHvaried between 1.3 and 4.5, which indicates that thepresence of BMM was significantly correlated with areduced relapse-free survival. The overall RR MH for allbreast cancer studies was 1.34 (95% CI, 1.27 to 1.42).However, the Breslow-Day test for homogeneity (P = ,001)demonstrated that a pooled analyses of these studies has tobe interpreted with caution because of the too large range ofthe individual results. In other cancer types, with oneexception,19 all RRM values, including their 95% CIS,reached significant levels (Fig 1A). Similar to the resultsobtained in breast cancer, the 95% CIS varied less in stud ieswith large patient numbers than in small patient cohorts.

In 11 studies, a multivariate analysis was performed totest the independent influence of BMM status on relapse-free survival (Fig 1B). Six studies4,5p8.12-16p19ould not beconsidered because insufficient data were given in theoriginal articles for calculation of the RR(,, an d the 95%CI, respectively. In breast cancer, the RR(,,, demonstra testwo s t ~ d i e s ' ~ J ~ith significant results of an RR(,,) of 4.10(95% CI, 1.8 to 9.1)1 and 3.06 (95% CI, 1.91 to 4.90),re~ pec tiv ely .'~ wo studies in colorectal and lung cancer

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META-ANALYSES ON PROGNOSTIC IMPACT OF MICROMETASTASES 56 ITab4. Prognolc hnpoctof BMMonR e k p F r e aand OverallSurvivd

Prognostic SigniQmncet nd Sbti~hmi ralwtionUsedRelapse-Free Ovemll SUNIVO~

F~rrt ulhor No of Pahenb' Fo ll wU p hmnth) Non-mv mv Nal-mr mvBerger2 285 29+ +I I Not tested + Not testedKirk3 25 34+ - Not tested - Not testedSolvadori' 121 48s - - Not tested Not testedCotes 49 3% + -1 Not tested NOI testedDearna1q.d 39 114s + Not tested + Not testedMonsi7 350 76s + - + -Singleto$ 71 11s - -.. - ..Courtemanche9 50 2 1.5s - Not tested - Not testedHarbeck'o 100 34+ + + - +Diel" 727 369 +II +II + +Mdinolz 109 369 - - - -Schlimok'3 38 26.4+ + Not tested Not ested Not estedSchlimokl4 85 25.A+ + Not tested Not ested Not estedLindemonn15 88 3 5 . 6 + + Nat tested Not testedWollenberg'6 73 25+ + +** Not ested Not testedCoteI7 43 13.65 + Not tested - Not testedPantd'e 66 395 +tt +tt Not ested Not testedJouch19 109H 30.6+ -11 - 1 -11 -11Thorbanm 2A++ 156s + Not tesd Not tested Not testedTharban2' 42++ 15.5+ + Not esd + Not tested

Abbreviations. +, s~~nificuntorrelation between a positive B M M status and short relapse-hee/ovemll survival; -, nonsignificantcorrelation between a positiveBMM status and short relapsefree/werall survival; non-mv, nonmulhvariate esting was performed; rnv, multivariate esting was performed

'Number of potienh used for stat~stico~alwtion of the prognostic impact. This may be less than the original collectiw as described n Table 1tAs determined by the authors of the original articles.+Median follow-up.Mean follow-up.g k a l elapse not considered.I F epresence of micrometostatic cells had no significant prognostic impact, while tumor burden reached signih'conce."No RRvalues given by the authars ofthe orig~nol rticles.t ton ly pNO patients reached a significant difference++Only ROpatients cons~dered.

dem onstrated a RR(,,, of 2.98 (95% CI, 1.45 to 6. 12)15 and2.4 (95% CI, 1.1 to 5.5),18 respectively.Calcula tion of the RR for Overall Survival

In breast cancer, a significant impact of BMM status onoverall survival was demonstrated in four of eight studies,with RRs of 3.0,6 1.4,' and 1.21 (Fig 2A). The largeststudy" shows an RRMHof 1.19 (95% CI, 1.13 to 1.24).Although the results of a multivariate analysis were givenfor five breast cancer studies by the authors of the originalart icles (see Table 4). only the data from three s t u d i e ~ ~ . I ~ - ~ ~were suitable for calculation of the RR(,,) for overallsurvival (Fig 2B). One studylo with 100 patients found asignificant Rq,) of 3.7 (95% CI, 1.3 to 10.5). Anotherstudy, which included the largest patient series investigatedthus far, found an RR[,,, of 2.14 (95% CI, 1.08 to 4.22).Hitherto, only these two studies demonstrated a significantRR of a positive BMM status for overall survival in any typeof cancer. In contrast, the other study7 with 350 patientsdemonstrated an Rh,,, of 1.62, but the 95% CI (0.78 to

3.34) did not reach significance. In other tumor types, therewas not a significant correlation between positive BMMstatus and sho rt overall survival time by either nonmultivari-ate or multivariate analyses (Fig 2).

DISCUSSIONIdentification of individual cancer patients at high risk fo r

developing tumor relapse despite complete surgical resec-tion (RO resection) is one of the main goals in clinicaloncology. Detection of a single dissem inated epithelial cellin bone mimow as a subclinica l indicator of tumor dissemi-nation was pioneered by Neville's group in breast can-cer1,2.25.56-58nd was confirmed for various tumor types byseveral laboratories within the last 17 y e a r ~ . ~ ~ - ~ On additionto studies focusing on meth~dology,"~~n some cancerstudies, positive BMM status has been identified as anindicator of poor prognosis.2.5-7.10.11~13318~20.21ecently, BMMstatus has gained increasing clinical interest. In breast cancerand lung cancer, the detection of epithelial cells in bonemarrow was used to select patients for bone marrow purging

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FUNKE AND SCHRAUTA Fa

K i t 3

F i i l . ~ c d c u b ~ ( m m h r a w ~ i n ( ~ ) b n o ~ m n a r a n d ( @ )& ancers with 95%CIS(lymmdric inh ogarilhmic scab)as e s t im drid dthe pmvaiencedBMM fadapse. (8) In d a mu lhda t aevalvariar,h&fankp#isalroJ#wn.Inccuedattdha&ad ~ & a g i r m h f o r h & , b ~ ~ ~ a s i g n h n t o rnansign'ht pamnek, h a ed s f i n d i d by braces) ware seta&han'ly left or rightd he line hat i n d i i n RRd . (*) RR,., bmastcancer; ( 0 )&,other c a w .

and to m onitor chemotherapeutic responsewB7; a random -ized multicenter study was initiated to single out high-risknode-negative patients for conventional adjuvant therapy.88Moreover, immunocytochemical identification of isolatedtumor cells in bone marrow was suggested for monitoringthe efficacy of treatment protocols.B9Today, BMM status isproposed as an entry in the TNM classification as afacultative prognostic factor indicated Mla1.49

The present meta-analyses included all studies publishedbetween 1980 and June 1997 that dealt with the prognosticinfluence of BMM status on relapse-free andlor overallsurvival and was performed to prove the prognostic value ofa positive BM M status according to the recomm endations ofinternational cancer c~rnmittees.~"'Most of the prognosticstudies were performed in breast cancer, and showed that thepresence of micrometastatic cells in bone marrow wasdetectable in 35% (median) of cancer patients investigated.

It is noteworthy that a similar prevalence was found in allother carcinoma types, and until now, no report has demon-strated a solid tumor type without immunocytochemicallydetectable epithelial cells in bone marrow. These datasuggest that the presence of epithelial cells in bone marrowis an indication of disseminated tumor in a l l cancer types,rather than an indicator for the future development of solidbone metastasis. This is supported by the fact that both themorphologic appearancez4 and the phenotypic characteris-t i~s~~-32-3~.~2993f the micrometastatic cells differ in varioustumor types, which suggests that the biologic features ofmicrometastatic cells are involved in the preferential local-ization of bone metastasis in breast and lung cancer, but notin gastrointestinal tumors.

Regarding the prognostic relevance of the presence ofmicrometastatic cells in bone m arrow, 14 of the 20 selectedstudies nonmultivariately showed a positive associationbetween positive BMM status and short relapse-free sur-vival. Combining the data of the breast cancer studiesdespite their heterogeneity, the RRMHor relapse-free survival was 1.34 (95% CI, 1.27 to 1.42). Analysis of theindependent prognostic significance, which is demanded foevery new prognostic factor,% was perform ed in only 11studies, although six g r o ~ p s ~ , ~ , ~ - ~ ~ , ~ ~ * ~ound a close associa-tion between the presence of micrometastatic cells in bonemarrow and conventional clinicopathologic parameters ofadvanced cancer. In five studies, positive BMM status was

A Ref a log R R l w 1 10 10Kmk 3 25 +-Dcumnlcy 6 39 -LC-C ~ m b50 4-S i l e uw S 71 -w t I0 100 tMOli.0 12109 I B -Mm i 7 150 +Dicl 1 1 727 +

17 43 -o--Jawh 19 109 -0-

Fg2. ( A J ~ w k u M ( m m h e m ~ m [ ~ ) b m a # c o n c a a notherca~enwilh95%~o&dm~eintervdr(#inh~ksori lhmicude )asa~ r i ddh~ l enodWMfaoma i run i va l . (Bm ~ d a m u h i v w i a t e b ~ ~ ~ M o n , h & f o r ~ l l w r v i v d ilncaroofa mu l l i vw i c l t e d d o n , & a g h luefahe RR,.,, hutesd asa * g n i i n tor nonrignhnt pammetar, these rsJlhlid4b ) m & o r b ' m ~ r i t y M ~ & h d h h r m M W s a n R R[*I R L , k t ancer; ( 0 ) RR" othercancas.

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META-ANALYSES ON PROGNOSTICMPACT OFMICROMETASTASES 563confirmed as an independent factor of reduced relapse-frees u ~ i v a l . ' ~ ~ ~ ~ , ~ ~ , ' ~ . ' ~n addition, five of 12 studies univari-ately showed a significant correlation between positiveBMM status and overall s ~ r v i v a l . ~ ~ ~ ~ ~ . ' ~ , ~ ~ultivariate analy-sis was performed by six investigators, but in only twobreast cancer studies was the presence of epithelial cellsidentified as an independent factor of reduced overalls ~ r v i v a l , ~ ~ . ' ~he study reported by Harbeck et a1 does notseem to be representative, since the CIS for all includedvariables are large and the covariable nodal status remainsnonsignificant by both univariate and multivariate analysis,while tumor grade gains an impressive value of 4.7 (95% CI,1.5 to 33.4).1

In the study reported by Die1 et al,Ii which included thelargest patient cohort, the prognostic impact of a positiveBMM status overcomes that of the nodal status, althoughprogesterone receptor status was identified as the mostimportant prognostic parameter. However, nodal status andpT stage, which are the most important classical risk factors,were arbitrary analyzed as polychotom ous variables, whichresulted in a severe underestimation of their prognosticimpact. In addition, although the applied antibody 2Ellcrossreacts with basophilic myelocytes and monocytes, thecontrol patient series is identical with that already publishedin the form er study." Due to these methodical objections,the suggestion that BMM status might replace axillarylymph node dissection remains to be substan tiated."

In addition, two studies, which failed to demonstrate aprognostic impact of BMM status per se, considered thequantitative tumor-cell burden in bone marrow and foundthat an increased number of epithelial cells, rather than thepresence of epithelial cells in bone marrow, was indepen-dently correlated with a reduced disease-free ~ u r v i v a l ~ ~ ' ~nda short overall ~urvival.'~urthermore, recent publicationsshowed that a positive BMM status in follow -up aspirations,as well as the expression of the urokinase plasminogenactivator receptor on the disseminated tumor cells, weresignificantly correlated with a shorter d is e a s e -f ~ e urvivalin g astrointestinal ~ a n c e r ? ~ - ~ ~Comparison of the selected studies regarding the prognos-tic relevance of the presence of epithelial cells in bonemarrow can only be performed with reservation because ofthe varying size of the patient cohorts, their heterogeneity inthe extent of tum or disease (TNM c lassification) and tumorresection (R classification), and the differences in methodol-ogy. The sample size of the studies ranged from 24 to 727patients, and only seven studies included more than 100patients, although according to the recom mendations of theinternational cancer comm ittees, evaluation of a new prog-nostic fac tor requires sufficient numbers of patients to avoidpremature judgments derived from statistical e r ~ o r s . ~o

overcome this problem, some investigators increased thenumber of cancer p a t i e n t ~ 7 . 1 1 ~ ~ ~ ~ ~ ~ ~ ~ 8 . 2 6 - 3 ~ ~ ~ 8nd control pa-t i e n t ~ , ~ ~ . ~ ~ . ' ~espectively, including patient series alreadyanalyzed for BMM status without reflecting the fact ofmultiple testing by using appropriate statistical approachesaimed to adjust the P value. Nevertheless, some discrepan-cies between individual studies remain unsolved, which bestcan be exemplified in non-small-cell lung cancer, in which a21.9% detection rate of epithelial cells in bone m arrow wasreported in 1993, and in the same, but enlarged patientcoho rt, an increased rate of 59.7% was published in 1996.'8.26

Furthermore , differences in m ethodology m ake it difficultto compare the selected studies. A large spectrum ofantibodies directed against different epithelial antigens ofthe cytoskeleton and/or located on the cell surface was usedin various staining procedures, often w ithout testing specific-ity and sensitivity. All antibodies are directed againstepithelial-associated antigens, but the expression pattern ofnone of the recognized antigens is exclusively restricted toepithelial cells. As there is yet no detection tool with a 100%specificity for epithelial tumor cells available, more represen-tative and independently recruited series of control patientshave to be investigated to come to a reliable estimation offalse-positive results. In addition, use of a panel of antibod-ies as a c o ~ k t a i l , ~ . ~ . l ~ J ~s compared with a single antibodyfor testing, creates another variable that may influence theresults. Furthermore, only speculations are possible on therate of false-negative patients. Possible reasons are thelimitation of the sample size (Table 5). the loss of positivecells during procession, or the failure of the applied antibodyto recognize carcinoma cells with aberrant antigen expression.

Table 5. StatisticalModelb Dehminehe Numbwof Bone Manow-DeriuwlCdlrThatMustk ExaminednAchiew aDefinedSensitivity Level

Sensitivily/ h i n e d b n a Marrow Cab x lo6106Cellr 0.1 0.5 0.8 1 3 5 10

NOTE.Theprobability (%)of detectinga minimum of 1 positive bone marrowcell, eg, a positive siutus, depends on the size of the sample (examinedbone morrow cells)and the concentrotion of positive cells in the bone marrow. Ifa positive status shouldbe b a d n o cancentration of 2 1 positive cells in 106bone marrowderived cdlr (sensitiviw level), it needsa sample size of 3 x 106cells todetectat least I p6sitive cdl with 95%probability.

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4 FUNKEAND SCHRAUMost conspicuous, the number of, mononuclear cells

X lo 5 and 5.0 X 106ever, there is no proof for the choice of the given

statistical model was established onhighly sensitive antibody

cells in a pure bone marrow sam pleX lo6 bone marrow-

f 95% (Table 5). This model corresponds wellcalcula tions perform ed by Osborne et al.993100Never-

determined which level of sensitiv-In fact, in practice, bone marrowdiluted with variable proportions of peripheral

these dilutions, although suggestions for calculating theerythrocyte concentration in bone marrow and peripherablood have been made.33

In conclusion, the presence of epithelial cells in bonemarrow has to be validated as an independent factor for pooprognosis in cancer patients by further studies with standardized procedures before its official acceptance in the TN Mclassification.

ACKNOWLEDGMENTWe are indebted to Dr B. Mayer for critical and inspir ing discus sion

of the manuscript, and to Professor Dr F.W. Schildberg for con tinuouand generous support of our work. We dedicate this manuscript to ouscientific teacher Professo r Dr G. Riethmiiller, who always urges us tobe critical.

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