1565

CLINICAL PRACTICE

Prognostic importance of occult axillary lymph nodemicrometastases from breast cancers

INTERNATIONAL (LUDWIG) BREAST CANCER STUDY GROUP

Serial sectioning of ipsilateral axillary lymph nodesjudged to be disease-free after routine histologicalexamination revealed micrometastases in 83 (9%)of 921 breast cancer subjects. These patients had apoorer disease-free (p=0·003) and overall

(p=0·002) survival after 5 years’ median follow-upthan did patients whose nodes remained negativeon serial sectioning. The presence of axillary lymphnode micrometastases correlated with the

presence of vascular invasion and tumour size

(p<0&middot;0001 and p=0&middot;02, respectively). The

presence of occult micrometastases remainedstatistically significant after adjusting for otherprognostic factors. The detection of thesemicrometastases in lymph nodes may identify ahigh risk "node-negative" population and shouldbe considered as part of the routine pathologyexamination.

Introduction

In 1981 the Ludwig Breast Cancer Study Group started aninternational trial (Trial V) in patients 65 years of age or lesswith proven breast cancer with or without ipsilateral axillarylymph node involvement and without detectable distantmetastases. One of the aims was to ascertain whether

chemotherapy given in the immediate postoperative periodinfluenced the outcome not only in node-positive patientsbut also in those considered to be node-negative afterhistological examination and staging by the local

pathologists. 1.2 -All pathological material was reviewed centrally. One

aspect of the central review was to examine serial sections of

ipsilateral axillary nodes that were categorised as negativeafter routine examination locally and centrally. 1275 patientswere classified as node-negative by routine examination,and nodes of 921 of these were serially sectioned.There have been several reports3-8 of a high incidence of

occult nodal disease on re-examination, usually on further

sectioning of the histological material. However, there is nounanimity as to the prognostic significance of such occult,especially micrometastatic, nodal disease.6-12 We describehere our assessment of the prognostic importance oflymph-node micrometastases.

Subjects and methodsAll patients entered into trial V had their breast cancers removed byeither total mastectomy with axillary clearance or modified radicalmastectomy. To be eligible, patients must have had unilateraldisease of clinical stage Tla, T, T 2a’ T2b, or T 3a’ No or Nl, andMo according to the TNM classification.

Patients were randomised to one of three groups, two of whichreceived one cycle of perioperative chemotherapy (intravenouscyclophosphamide, methotrexate, and 5-fluorouracil on days 1 and8) starting within 36 h of mastectomy. Two-thirds of those classedas node-negative by the local pathologist received perioperativechemotherapy. All node-negative patients received no othertreatment. Two-thirds of node-positive patients also received

perioperative chemotherapy, half with no additional therapy(perioperative alone) and half with six additional cycles of

chemotherapy (perioperative plus conventionally-timedchemotherapy). The group of node-positive patients that receivedno perioperative chemotheapy received six cycles of conventionally-timed chemotherapy. The results at 42 months median follow-up ofthe node-negative2 and node-positive1 groups have been reportedelsewhere.The local pathologist was responsible for the histological

preparation of surgical specimens. The tissues were fixed inbuffered formalin, embedded in paraffm, sectioned, and stainedwith haematoxylin and eosin (H&E) as a standard procedure. Acomplete set of stained sections from the primary tumour (twosections were taken at right angles to each other whenever possible),the nipple, the remaining quadrants of the breast, and all lymphnodes identified in the specimen was then sent to the Ludwig BreastCancer Study Group Coordinating Centre in Bem, Switzerland,for review. According to the pathology protocol, all paraffin blocks

’Writing Committee: R. Bettelheim, K. N. Price, R D. Gelber, B. W. Davis,M Castiglione, A. Goldhirsch, A. M. Neville Correspondence to Dr A.Goldhirsch, International Breast Cancer Study Group, Konsumstrasse13, 3007 Bern, Switzerland

1566

TABLE I-NODE STATUS CONVERSION AND EFFECT ON 5-YEAR

DISEASE-FREE SURVIVAL

*1 medullary, 1 papillary.tPentumoral vascular/lymphatic invasion.#NS= >0 05

containing lymph nodes originally found to be free of tumour wereforwarded to Central Pathology Review for further study. When itwas the policy of the clinic to keep all paraffin blocks on file, stainedand/or unstained serial sections of such lymph nodes, cut asdescribed below, were submitted.

All the paraffin blocks containing lymph nodes from the casesreported locally and centrally as node-negative were seriallysectioned at six levels, with six 3 [t sections being cut at each level. Atleast ten sections between levels were cut for regular spacing andthen discarded. The six serial sections from each level weremounted on glass slides; two at each level were stained with H&Eand examined by the Central Review pathologists. The remainingfour sections from each level were filed for future

immunohistological studies. The median number of nodes perpatient examined was 14. The number of node blocks prepared forexamination varied between 1 and 45 per patient; the average was12.

Only the presence of tumour cells within the body of the lymphnode was accepted as a metastasis. Tumour emboli in the

subcapsular sinus or in any endothelial-lined space within the nodewere noted but were not counted as a metastasis. However, it is

(a) Disease-free and (b) overall survival for 921 node-negativebreast cancer patients whose lymph nodes were seriallysectioned by the central review pathologist.

Survival for 83 patients who became node positive (occult micro-metastatic deposits found by the review) compared with that for 838patients who did not convert. Median follow - u for the study cohort is fiveyears.

important to note that tumour cells were found within the substanceof the nodes on further examination in all the cases in which initiallyonly a subcapsular, sinusoidal, or vascular metastasis was noticed.The breast cancers were classified histologically according to

conventional WHO criteria. Tumour size, the presence or absenceof peritumoral vascular invasion, and histological grade13 wererecorded. Carcinomas were classified as non-invasive, intraductwith minimum stromal invasion, limited invasive (50% of thetumour consisting of intraduct carcinoma), invasive ductal, invasivelobular, mixed ductal and lobular, or those with pure specialfeatures such as tubular, mucous, papillary, squamous, and

medullary carcinomas. If another histological type of cancer otherthan lobular carcinoma was present as well as infiltrating ductcarcinomas, the tumour was placed in the invasive ductal category.

Differences in incidence of conversion (of node status fromnegative to positive) according to patient characteristics wereevaluated using X2 tests, and log-rank tests were used for

comparisons of disease-free survival. Estimates of 5-year disease-free survival and their standard errors were calculated by theKaplan-Meier method and Greenwood’s formula, respectively.Cox proportional hazards regression models were used to evaluatethe results, with adjustment for various prognostic factors. Themedian follow-up at the time of these analyses was 5 years. Allp-values were two-sided.

1567

Results

Material needed for central review was available from 1203of 1275 cases classified as lymph node-negative. In 46 casessections already examined by the local pathologist containeda tumour metastasis. Material from 921 of the remaining1157 cases was serially sectioned; in the others serial sectionswere not cut because the lymph node material was notforwarded to the Central Review laboratory (219 patients) orwas misplaced in the Cental office (17). The 5-yeardisease-free survival for the 921 cases serially sectioned wasthe same as that for the 354 cases not assessed (72%).

Serial sectioning revealed occult micrometastatic depositsin 83 (9%) of 921 patients (table I). Conversion wascommoner among patients with larger tumours (p = 0 02),those with peritumoral vascular invasion (p < 0-0001), andthose less than 50 years old (p < 0-0001). Ductal and lobularcarcinomas were equally associated with the presence ofoccult nodal micrometastases.

There was a definite outcome disadvantage for those whoconverted to the node-positive classification in 5-yeardisease-free survival (58% [SE 6] vs 74% [2], p = 0-003, fig[a] and table l) and in overall survival (79% [5] vs 88% [1],p =0002, fig [b]). The difference was most obvious amongpostmenopausal patients and those with invasive ductalcarcinomas. The difference was also noted between the twotreatment groups, especially among patients givenperioperative chemotherapy.

Adjustment in Cox proportional hazards regressionmodels for patient age, menopausal status, oestrogen

TABLE II-PERCENTAGE (SE) PATIENTS WITH FIVE-YEARDISEASE-FREE SURVIVAL BY TREATMENT AND NUMBER OF

POSITIVE NODES FOUND ON SERIAL SECTIONING COMPAREDWITH PATIENTS CLASSIFIED AS NODE POSITIVE BY LOCAL

PATHOLOGIST

Numbers of patients given in square brackets.

TABLE III-REPORTED FREQUENCY OF OCCULT AXILLARY NODE

MICROMETASTASES

*All these cases were infiltrating lobular carcinomas.tDetected by immunohistochemical methods and not serial sectioning.

receptor status, tumour size, vascular invasion,histopathological grade, and adjuvant treatment showed aprognostically significant correlation of nodalmicrometastatic involvement with treatment (p = 0 02) andhistopathological grade (p = 0 05), and especially with thepresence of vascular invasion (p = 0 00002) and tumour size(p = 0-002).

Since the trial had a cohort of node-positive patientstreated with the same perioperative regimen,l a comparisonwas made with that group in table 11. Most (70/83) of thepatients with conversion of node status had only one positivenode, whereas most (279/381) of those recognised to be nodepositive initially by the local pathologists had more than oneinvolved node. Patients with one affected node had similar

prognosis whether the metastasis was found by serial

sectioning or by routine examination. The beneficialinfluence of perioperative therapy was also evident. Too fewpatients had more than one node involved for inferences tobe drawn about effect of multiple node involvement.

Discussion

The proportion of patients in this study with occultlymph-node micrometastases (9%) is lower than that inmost other reports (table ill), perhaps because fewersections were examined than in the other studies. We

expected occult disease to be of only microscopicproportions but, as in another report,9 this was not alwaysthe case. We found more than one affected node in 13 of 83

cases, and in 12 a considerable part of the node was replacedby carcinomatous deposits.Not unexpectedly large tumour size and peritumoral

vascular invasion were more likely than other factors to beassociated with conversion. Only vascular invasion was ofrelevance in another study,5 in which tumour size, locationin the breast, and grade showed no correlation. No specifichistological type of tumour was more likely than others to beassociated with occult disease in our study, althoughpapillary and infiltrating lobular carcinomas have beenreported to be frequent associations.9,1oThe serial sectioning of multiple axillary lymph nodes is a

time-consuming practice and should be confirmed to be ofprognostic value and therapeutic importance before beingrecommended for routine use. Our finding that nodalmicrometastases confer a disease-free and overall survival

disadvantage accords with that reported by Friedman et al,8but is at variance with that reached by others .4,6, ’7 Our studydiffered from the others in that it was prospectively plannedand included a large number of subjects who were recruitedto a single clinical trial in a short time (4 years).

Wilkinson et al9 noted that nodal metastases overlooked atthe initial examination but detected without further node

sectioning at a later review carried a poorer survival

prognosis than did disease detected after serial sectioning. Inour study micrometastases were detected simply by re-examination of the initial material for 46 patients; theirprognosis (61% [8] disease-free survival) was lessfavourable than that for node-negative patients but wassimilar to that for patients with metastases detectedon serial sectioning. Among those who usedimmunohistochemical methods to detect occult nodalmetastases without recourse to serial sectioning, Trojani etallO,l1 reported that nodal micrometastases were associatedwith higher recurrence and poorer survival rates.Thus, from the most recent and present data, detection of

occult metastatic disease in the axillary nodes is important

1568

since it carries a poorer prognosis. Perioperativechemotherapy for this group of patients gave an addeddisease-free survival advantage, especially when only onenode was involved. The beneficial effect of this form of

therapy reported for 42 months’ follow-up for node-

negative disease is shown in this study to be maintained at 5years.The detection rate of occult nodal micrometastases in this

series (9%) is well short of the total percentage of

node-negative subjects who will relapse within 5 years ofsurgical removal of the tumour. Accordingly, it may beadvisable to divide each axillary node into 2 mm slices, assuggested by Wilkinson et al,9 and to stain the histologicalsections by immunohistochemical methods. 14,15 Such anapproach, together with a search for marrow

micrometastasesl6-18 and measurement of functional

primary tumour properties (eg, growth factors, proto-oncogenes, ploidy), may serve to identify those poor riskpatients in the "presumed node-negative" group. Thepathological examination of a single H&E stained nodesection is probably no longer clinically tenable.

We thank the patients, the doctors, especially the pathologists, the nurses, thedata managers who made this research possible. We also acknowledge theLudwig Institute for Cancer Research who initiated the project and the Swissand the Ticino Cancer Leagues, the Swiss Group for Clinical and

Epidemiological Cancer Research (SAKK), and Frontier Science &

Technology Research Foundation for generous fmancial support to enable itscompletion.

International Breast Cancer Study Group: Participants and Authors

(Pathologists in bold type):

A. Goldhirsch, M. Castiglione (Study Coordinators), R. Bettelheim,A. M. Neville, B. Davis, W. H. Hartmann (Study Pathologists), D. Zava,S. Misteli: Operation Center, Bem, Switzerland.R. Gelber (Study Statistician), K. Price, M. Zelen: Harvard School of

Public Health & Dana-Farber Cancer Institute, Boston, MA, USA.M. Isley, M. Parsons, L. Szymoniak: Frontier Science & Technology

Research Foundation, Amherst NY, USA.R. G. Kay, J. Probert, B. Mason, H. Wood, E. G. Gifford, J. F. Carter,

J. C. Gillman, J. Anderson, L. Yess, 1. M. Holdaway, G. C. Hitchcock, M.Jagusch. Auckland Breast Cancer Study Group, Auckland, New Zealand.

G. Marini, E. Simoncini, P. Marpicati, U. Sartori, A. Bami, L. Morassi,P. Grigolato, D. DiLorenzo, A. Albertini, G. Marinone, M. Zorzi. SpedaliCivili & Fondazione Beretta, Brescia, Italy.

A. Hacking, D. M. Dent, J. Terblanche, A. Tiltman, A. Gudgeon, E.Dowdle, P. Palmer. Groote Schuur Hospital, Cape Town, Rep of SouthAfrica.

C. G. Schmidt, K. H&ouml;ftken, L. D. Leder, R. Callies, A. E. Schindler,University of Essen, West German Tumor Center, Essen, Germany.

P. Faber, H. G. Schniirch, H. Bender, H. Bojar, University ofDiisseldorf, Dusseldorf, Germany.C.-M. Rudenstam, J. Save-Soderbergh’, E. Cahlin, S. Nilsson, J.,

Fomander, H. Salander, Ch. Johnsen, 0. Ruusvik, G. Ostberg, L. Mattsson,C. G. Backstrom, S. Bergegardh, G. Ekelund, Y. Hessman, 0. Nelzen, S.Dahlin, G. Wallin, L. Ivarsson, 0. Thoren, L. Lundell, U. Ljungqvist. WestSwedish Breast Cancer Study Group, Goteborg, Sweden.

J. Lindtner, J. Novak, D. Erzen, M. Sencar, J. Cervek, 0. Cerar, B.Stabuc, R. Golouh, J. Lamovec, J. Jancar, S. Sebek. The Institute ofOncology, Ljublijana, Yugoslavia.H. Cortes-Funes, F. Martinez-Tello, C. Mendiola, F. Cruz-Caro, M. L.

Larodera, F. Calero, A Suarez, F. Pastrana, S. Cruchaga, B. Rodriguez.Madrid Breast Cancer Group, Madrid, Spain

J. Collins, R. Snyder, R. Bennett, W. 1. Burns, J. Forbes, J. Funder, T.Gale, L. Harrison, S. Hart, V. Humenuik, P. Jeal, P. Kitchen, R. Lovell, R.McLennan, R. Reed, I. Russell, M. Schwarz, L. Sisely, P. Williams, H.Ritchie. Anti-Cancer Council of Victoria, Melbourne, Australia.M. Byrne, P. M. Reynolds, H. J. Sheiner, S. Levitt, D. Kermode, K. B.

Shilkin, R. Hahnel, G. van Hazel. Sir Charles Gairdner Hospital, Nedlands,Western Australia.SAKK (Swiss Group for Clinical & Epidemiological Cancer Research),

Switzerland:

K. Brunner, G. Locher, E. Dreher, K. Buser, K. Biirki, M. Walther,R. Joss, H. Burgi, M. Spreng, U. Hermann: Inselspital, Bem.H. J. Senn, W. F. Jungi, W. W. Rittmann, M. Stanisic, Th.

Hardmeier, K. Luscher, G. Delmore, U. M. Luthotf, U. Haller, 0.Schildknecht: Kantonsspital, St. Gallen.

F. Cavalli, H. Neuenschwander, W. Muller, C. Sessa, P. Luscieti, E.Passega, P. Rey, S. Martinoli, E. Pedrinis, M. Varini, G. Losa, M.Ginier. Ospedale San Giovanni, Bellinzona.

J. P. Obrecht, F. Harder, H. Stamm, U. Laffer, A. C. Almendral, U.Eppenberger, J. Torhorst. Kantonsspital, Basel.

P. Siegenthaler, V. Barrelet, R. P. Baumann. Hopital des Cadolles,NeuchateL H. J. Schmid. Kantonsspital, Luzern.

P. Alberto, P. Schafer, F. Krauer, M. Aapro, R. Egeli, R. Megevand,E. Jacot-des-Combes, A. Schindler, F. Misset. Hopital Cantonal,Geneva.

S. Leyvraz, P. Anani, F. Gomez, D. Wellmann, G. Chapuis, P. DeGrandi, P. Raymond. Centre Hopitalier Universitaire, Lausanne.W. Weber, G. Noseda. Swiss Cancer League, Bern.

M. N. H. Tattersall, A. Coates, D. Hedley, D. Raghavan, F. Niesche, R.West, S. Renwick, J. Donovan, P. Duval, R. J. Simes, A. Ng, T. Foo, D.Glenn, R. A. North, J. Beith, R. G. O’Connor, M. Rice, J. Grygiel, J. Stewart,R. Sillar. Urnversity of Sydney and Royal Prince Alfred Hospital, Sydney,Australia.

REFERENCES

1. Ludwig Breast Cancer Study Group. Combination adjuvantchemotherapy for node-positive breast cancer. Inadequacy of a singleperioperative cycle. N Engl J Med 1988; 319: 677-83.

2. Ludwig Breast Cancer Study Group. Prolonged disease-free survivalafter one course of perioperative adjuvant chemotherapy for node-negative breast cancer. N Engl J Med 1989; 320: 491-96.

3. Saphir O, Amromin GD. Obscure axillary lymph-node metastasis incarcinoma of the breast. Cancer 1948; 1: 238-41.

4. Pickren JW. Significance of occult metastases. A study of breast cancer.Cancer 1961; 14: 1266-71.

5. Huvos AG, Hutter RVP, Berg JW. Significance of axillarymacrometastases and micrometastases in mammary cancer. Ann Surg1971; 173: 44-46.

6. Fisher ER, Swamidoss S, Lee CH. Rockette H, Redmond C, Fisher B.Detection and significance of occult axillary node metastases in patientswith invasive breast cancer. Cancer 1978; 45; 2025-31.

7. Apostolikas N, Petraki C, Agnantis NJ. The reliability of histologicallynegative axillary lymph nodes in breast cancer. Path Res Pract 1989;184: 35-38.

8. Friedman S, Bertin H. Mouriesse H, et al. Importance of tumor cells inaxillary node sinus margins ("clandestine" metastases) discovered byserial sectioning in operable breast carcinoma. Acta Oncol 1988; 27:483-87.

9. Wilkinson EJ, Hause LL, Hoffman RG, et al. Occult axillary lymph nodemetastases in invasive breast carcinoma: characteristics of the primarytumor and significance of the metastases. Pathol Ann 1982; 17: 67-91.

10. Trojani M, de Mascarel I, Bonichon F, Coindre JM, Delsol G.Micrometastases to axillary lymph nodes from carcinoma of breast:Detection by immunohistochemistry and prognostic significance. Br JCancer 1987; 55: 303-06.

11. Trojani M, de Mascarel I, Coindre JM, Bonichon F. Micrometastases toaxillary lymph nodes from invasive lobular carcinoma of breast:Detection by immunohistochemistry and prognostic significance. Br JCancer 1987; 565: 838-39.

12. Rosen PP, Saigo PE, Braun DW, Weathers E, Fracchia AA, Kinne DW.Axillary micro- and macrometastases in breast cancer. Ann Surg 1981;194: 585-91.

13. Bloom HJG, Richardson WW. Histological grading and prognosis inbreast cancer. Br J Cancer 1957; 2: 359-74.

14. Wells CA, Heryet A, Brochier J, Gatter KC, Mason DY. Theimmunocyotchemical detection of axillary micrometastases in breastcancer. Br J Cancer 1984; 50: 193-97.

15. Bussolati G, Gugliotta P, Morra I, Pietribiasi F, Berardengo E. Theimmunohistochemical detection of lymph node metastases frominfiltrating lobular carcinoma of the breast. Br J Cancer 1986; 54: 631.

16. Redding WH, Monaghan P, Imrie SF, et al. Detection ofmicrometastases in patients with primary breast cancer. Lancet 1983; ii:1271-73.

17. Cote RJ, Rosen PP, Hakes TB, et al. Monoclonal antibodies detect occultbreast carcinoma metastases in the bone marrow of patients with earlystage disease. Am J Surg Path 1988; 12: 333-40.

18. Berger U, Bettelheim R, Mansi JL, Easton D, Coombes RC, Neville AM.The relationship between micrometastases in the bone marrow,histopathologic features of the primary tumor in breast cancer andprognosis. Am J Clin Pathol 1988; 90: 1-6.