Predictive value of the fraction of cancer cells immunolabeled for proliferating cell nuclear...

$: Predictive value of the fraction of cancer cells immunolabeled for proliferating cell nuclear antigen or Ki67 in biopsies of head and neck carcinomas to identify lymph node metastasis:$
PREDICTIVE VALUE OF THE FRACTION OF CANCERCELLS IMMUNOLABELED FOR PROLIFERATING CELLNUCLEAR ANTIGEN OR KI67 IN BIOPSIES OF HEADAND NECK CARCINOMAS TO IDENTIFY LYMPH NODEMETASTASIS: COMPARISON WITH CLINICAL ANDRADIOLOGIC EXAMINATIONS

Ming Liu, MD,1 Georges Lawson, MD,1 Monique Delos, MD,2 Jacques Jamart, MD,3

Christophe Ide, MD,4 Emmanuel Coche, MD,5 Birgit Weynand, MD,6

Gauthier Desuter, MD,7 Marc Hamoir, MD,7 Marc Remacle, MD, PhD,1

Etienne Marbaix, MD, PhD6

1 Department of ENT and Head and Neck Surgery, University Hospital of Mont-Godinne, Yvoir, B-5530, Belgium2 Department of Pathology, University Hospital of Mont-Godinne, Yvoir, B-5530, Belgium3 Center of Biostatistics and Medical Documentation, University Hospital of Mont-Godinne, Yvoir,B-5530, Belgium4 Department of Radiology, University Hospital of Mont-Godinne, Yvoir, B-5530, Belgium5 Department of Radiology, Saint-Luc University Hospital, Brussels, B-1200, Belgium6 Department of Pathology, Saint-Luc University Hospital, Brussels, B-1200, Belgium. E-mail:[email protected] Department of ENT and Head and Neck Surgery, Saint-Luc University Hospital, Universite Catholique deLouvain, Brussels, B-1200, Belgium

Accepted 9 August 2002Published online 12 December 2002 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/hed.10218

Abstract: Background. Neck metastasis is a major prognos-tic factor of head and neck carcinoma, but its preoperative de-tection is currently unreliable. Molecular markers of the meta-

static potential of a carcinoma would help to avoid unnecessaryneck dissection in patients with nonmetastatic cancer.

Methods. The fractions of cancer cells immunostained forproliferating cell nuclear antigen (PCNA) and Ki67 were deter-mined in 80 preoperative biopsy specimens of head and neckcarcinomas. The value of both indexes to detect metastasis in thesubsequent neck dissection was compared with that of the clini-cal and radiologic examinations.

Results. PCNA and Ki67 indexes correlated with neck me-tastasis. Cutoff points were determined for both indexes to dis-criminate metastatic from nonmetastatic carcinomas. By multi-variate logistic regression, these indexes were significant

Correspondence to: E. Marbaix

Presented in part as an oral presentation at the 5th Research Workshopon the Biology, Prevention, and Treatment of Head and Neck Cancer,McLean, Virginia, August 27–30, 1998.

© 2003 Wiley Periodicals, Inc.

280 PCNA/Ki67 in Metastatic Carcinoma HEAD & NECK April 2003

predictors of metastases, together with clinical T stage and neckpalpation.

Conclusion. The fraction of cancer cells immunolabeled forPCNA or Ki67 in preoperative biopsy specimens is helpful topredict neck metastasis. © 2003 Wiley Periodicals, Inc. HeadNeck 25: 280–288, 2003

Keywords: PCNA; Ki67; carcinoma; metastasis; head and neck

Lymph node metastasis is the most powerfulprognostic factor of patients with laryngeal, pha-ryngeal, or oral carcinoma,1 being associated witha low 5-year survival rate, but its preoperativedetection remains currently difficult. Clinical ex-amination (ie, palpation of neck lymph nodes) isan easy and inexpensive method but is associatedwith high false-negative or false-positive rates.2,3

Many malignant nodes have been found to have adiameter less than 1 cm,4 and even during theoperation, surgeons may be led astray by a reac-tive hyperplastic node whereas other less inflam-matory neighboring nodes are, in fact, sites of me-tastases.5

Imaging investigations such as CT, MRI, orultrasound-guided fine-needle aspiration cytologyhave been reported to be superior to physical ex-amination in the detection of lymph node metas-tasis of head and neck cancer.6–8 However, themajor limitations of these techniques rely on thediagnostic criteria themselves, which are basedon size and evidence of necrosis. Neither border-line-sized nodes without necrosis nor extracapsu-lar spread can be reliably differentiated from re-active or normal nodes.9,10 Moreover, a centralarea of low attenuation surrounded by an irregu-lar wall is suggestive of necrosis in a metastaticlymph node but can also occur in an abscess or acyst.11

A better way to predict lymph node metastasishas thus long been awaited, and recent interesthas begun focusing on molecular factors with pre-dictive potential with a view to categorizing tu-mors more effectively and making treatmentschemes more accurate.

Cell proliferation has been investigated for itsrelationship with tumor invasion and metastasis.The proliferating cell nuclear antigen (PCNA), aDNA polymerase–associated protein needed forDNA replication and repair,12 and Ki67, a largemolecular weight protein, which is only expressedduring the G1 to M phases of the cell cycle,13 werefound to be potential prognostic biomarkers.14

The expressions of PCNA and Ki67 were signifi-

cantly increased both in less-differentiated tu-mors and in those with lymph node metastasescompared with those without metastases. In ad-dition, high PCNA immunolabeling was corre-lated with shorter patient survival.15–20

These studies indicated that PCNA and Ki67may be independent prognostic factors in headand neck squamous cell carcinoma, and their im-munolabeling could be useful in identifying pa-tients with clinically negative lymph nodes whoare at considerable risk of occult metastases andmay benefit from elective neck dissection. How-ever, an association between PCNA immunolabel-ing and nodal metastasis was not found in all re-ports,21 and the efficacy of PCNA and Ki67immunolabeling on preoperative biopsy speci-mens has not yet been investigated.

The objectives of this study were thus first, toinvestigate the value of these biomarkers in pre-operative biopsy specimens as predictors of lymphnode metastases from squamous cell carcinomasof the larynx, pharynx, and mouth, and second, tocompare the results with those of preoperativeclinical and imaging assessments.

MATERIALS AND METHODS

Patients. A cohort of 489 patients with preopera-tive biopsies of squamous cell carcinomas of thelarynx, pharynx, or mouth was prospectively re-cruited in the files of the Pathology Departmentsof the University Hospital of Mont-Godinne (154cases from February 1993–June 1999) and ofSaint-Luc University Hospital (335 cases fromMay 1986–September 1998). The patients whowere not operated, whose operative specimen wasnot available (324 cases), or who were operatedbut without a lymph node dissection (64 cases)were excluded, as well as those whose clinical andradiologic files were not available (21 cases).There thus remained 41 patients from Mont-Godinne University Hospital and 39 patientsfrom Saint-Luc University Hospital.

The patients characteristics are summarizedin Table 1. The mean age of the patients was 58years (range, 38–81 years). They had either car-cinoma of the larynx (n � 70: 4 subglottic, 22glottic, 44 supraglottic), of the pharynx (n � 6: 2hypopharyngeal, 4 oropharyngeal), or of the baseof the tongue (n � 4) and were operated by eithertotal (n � 35) or partial (n � 41) laryngectomy, orby pelviglossectomy (n � 4), with unilateral (n �11) or bilateral (n � 69) functional or radical neckdissection. None of them had previous oncologic

PCNA/Ki67 in Metastatic Carcinoma HEAD & NECK April 2003 281

treatment such as chemotherapy and/or radio-therapy. All patients were examined clinically be-fore surgery by a head and neck surgeon andevaluated with imaging techniques that were in-terpreted by radiologists.

Preoperative biopsy specimens were removedendoscopically and fixed for variable times (4–24h) in formalin followed by 1 h in Bouin’s liquid forthe biopsies performed in Mont-Godinne Univer-sity Hospital, in formalin or in Bouin’s liquid forthe biopsies performed in Saint-Luc UniversityHospital, and embedded in paraffin. The differen-tiation of the carcinomas and the presence oflymph node metastasis were assessed by patho-logic examination of the surgical specimens. ThepTNM stage was determined according to theWHO classification system.22

Clinical and Imaging Examinations. Any palpablelymph node in the neck was clinically consideredmetastatic.

The imaging techniques used in the study in-cluded 69 spiral CTs, 3 MRIs, and 8 ultrasonog-raphies. Spiral CTs were performed mainly on To-moscan LX (Philips Medical Systems, Eindhoven,The Netherlands) and Somaton S+ (Siemens

Medical System, Erlangen, Germany) after intra-venous injection of contrast medium. MRI wasperformed on a MRI 1.5-T scanner (Gyroscan NT,Philips Medical Systems) with a cervical coil.Noncontrast axial T1- and T2-weighted and axialT1-weighted images with gadolinium enhance-ment were recorded. Ultrasonographic examina-tion was mainly performed by a 128 XP ultra-sound machine (Acuson, Mountain View, CA)using a 7-MHz flat transducer.

The criteria for identifying lymph nodes asmetastatic by the imaging techniques were: (1)nodal width larger than 10 mm; (2) multiplicity ofthe nodes (>10); or (3) evidence of necrosis.

The clinical and imaging results were ana-lyzed retrospectively in the patients files.

Immunohistochemical Staining. Immunolabelingwas performed on the preoperative biopsy speci-mens using the antibodies described in Table 2.

In brief, 4 �m-thick histologic sections weredeparaffinized, hydrated, and washed with TRIS-buffered saline (pH 7.2). Microwave pretreatmentin citrate buffer (pH 5.7) was performed twice at650 W for 5 min, and endogenous peroxidase ac-tivity was blocked by incubation of the slides inwater with 0.3% H2O2 for 30 min. After the sec-tions were treated with normal goat serum (1/10)for another 30 min to reduce nonspecific binding,they were incubated overnight at 4°C with theprimary antibody (Table 2). Biotinylated anti-mouse IgG (Dako, 1/500) was applied as a second-ary antibody for 30 min, followed by peroxidase-conjugated streptavidin (Dako, 1/500) for 30 min.Immunolabeling was visualized with diamino-benzidine tetrachloride (Dako, 0.5 mg/ml). Con-sistent negative controls were obtained by omis-sion of the primary antibodies or their replace-ment by irrelevant antibodies of the same isotype(Table 2).

Table 1. Patient characteristics.

Patient characteristics Number

Age (yr)<60/60–69/�70 40/29/11

GenderMale/female 73/7

Tumor differentiationWell/moderate/poor 36/27/17

Tumor stageClinical: cT1/cT2/cT3/cT4 11/36/29/4Pathologic: pT1/pT2/pT3/pT4 11/31/23/15

Lymph node metastasisAbsent (pN0)/present (pN+) 51/29

Table 2. Antibodies used.

Antibody Company Clone Ig-classDilution

(concentration)Stainingpattern

PCNA *DAKO A/S PC10 IgG2a 1:400 (1.5 µg/mL) NuclearKi67 *DAKO A/S MIB-1 IgG1 1:100 (1.5 µg/mL) NuclearNegative controls†ER ‡Novocastra CC4-5 IgG1 1:40 (unknown) Negative§NC *DAKO A/S DAK-GO5 IgG2a 1:40 (2.5 µg/mL) Negative

*Glasstrup, Denmark†Estrogen receptor.‡Newcastle-upon-Tyne, UK.§Negative control (anti-aspergillus niger glucose oxydase).


Evaluation of Immunohistochemical Stainings andStatistical Analyses. Distinct brown staining ofthe nuclei was considered as positive immunola-beling for PCNA and Ki67.

The percentage of PCNA- or KI67-immuno-labeled cancer cells was determined by pointcounting, using a 42-point Weibel grid overlyingthe microscopic field at 40× objective magnifica-tion on a Leitz microscope. At least 420 cells(mean ± SD � 503.3 ± 61.1, n � 160) werecounted in each immunohistochemical section.

To verify the reproducibility of the analysis, asecond counting was performed on 22 of thosesame slides, either labeled for PCNA or Ki67, af-ter a 2-month interval by the same examiner. Theabsolute difference between the two countingswas 5.1% ± 2.8% (mean ± SD, n � 22) and theSpearman rank correlation coefficient betweenthe two measurements was 0.97.

A second immunolabeling for both PCNA orKi67 was performed on 20 randomly selected bi-opsy specimens from Mont-Godinne after an8-month interval using the same technique. Im-munolabeled cancer cells were counted by thesame examiner. The absolute difference betweenthe two immunolabelings was 12.4% ± 12.8%(mean ± SD, n � 40), and the Spearman rankcorrelation coefficient between the two measure-ments was 0.82.

Numerical variables were expressed as medi-ans with 25th and 75th percentiles and rang, andcompared between groups by the Wilcoxon ranksum test. Association between categorical param-eters and correlation between numerical or ordi-nal parameters were assessed by the chi-squaretest and the Spearman rank correlation coeffi-cient. Nonparametric receiver operating charac-teristic (ROC) curves23 were constructed using

SPSS statistical software (SPSS Inc., Chicago,IL), and areas under the curves were expressedwith their standard errors. A multivariate logisticregression was performed with the Wald test toselect the best predictors of lymph node metasta-sis. All statistical tests were two-tailed.

RESULTS

Accuracy of Clinical and Imaging Examina-tions. Preoperatively, 22 patients had been con-sidered to have lymph node metastases by neckpalpation (Table 3). However, only 15 of these 22patients actually had metastases, whereas the 7remaining patients showed no metastasis in theneck dissection surgical specimens. Pathologic ex-amination, in fact, revealed that 29 of the 80 pa-tients had neck metastases, although 14 of themhad initially been judged negative by clinical ex-amination. Thus, the false-negative rate of clini-cal examination caused by occult metastasis was48%.

Twenty-seven patients were staged as havingneck metastases by imaging techniques, but inonly 13 of them were the metastases confirmed bypathologic examination. Furthermore, neck me-tastases were not identified in the other 16 meta-static patients, and the false-negative rate causedby occult metastases was thus 55%.

Carcinomas of the pharynx and of the tonguewere more frequently associated with lymph nodemetastases (67% and 75%, respectively) than car-cinomas of the larynx (31%). Among laryngealcancers, 39% of the supraglottic, 18% of the glot-tic, and 25% of the subglottic carcinomas weremetastatic. Thus, carcinomas of the pharynx or ofthe mouth were found to be more likely to metas-tasize than laryngeal carcinomas (p � .018),

Table 3. Comparison of clinical examination, imaging findings and immunohistochemical indexes in the prediction of metastasis.

Metastasis predictions (+/−)

Sensitivity(%)

Specificity(%)

Accuracy*(%)

Positive predictivevalue† (%)

Negative predictivevalue‡ (%)

pN+(n = 29)

pN0(n = 51)

Palpation (22/58) 15/14 7/44 52 86 74 68 76Imaging (27/53) 13/16 14/37 45 73 63 48 70Clinical T-stage: III or IV

vs I or II (33/47) 16/13 17/34 55 67 63 48 72Tumor location: pharynx or

mouth vs larynx (10/70) 7/22 3/48 24 94 69 70 69PCNA (31/49) 24/5 7/44 83 86 85 77 90Ki67 (35/45) 22/7 13/38 76 75 75 63 84

*Overall probability of a correct decision.†Probability that a positive prediction is correct.‡Probability that a negative prediction is correct.


whereas there was no significant association be-tween metastases and the primary site of laryn-geal carcinomas.

As expected, large tumors were also morelikely to be metastatic than small ones. Indeed,the pathologic stage and the preoperative clinicalstaging of the tumor were both correlated withthe presence of lymph node metastases (p � .001and p � .029, respectively). The best prediction ofthe presence of metastasis according to the clini-cal stage was actually obtained when discriminat-ing the cT1 or cT2 from the cT3 or cT4 tumors(Table 3).

Immunolabeling of PCNA and Ki67. PCNA andKi67 were immunolabeled within the nuclei of theproliferating normal epithelial cells, exclusivelylocalized in the basal and suprabasal layers of thenoncancerous squamous epithelium surroundingthe tumor. Such immunolabeling of both proteinsin the normal epithelium was used as a positiveinternal control, indicating that the biopsy wastechnically suitable for the study.

In cancer cells, the nuclear immunostaining ofPCNA and of Ki67 (Figure 1) was heterogeneouswithin each tumor and varied widely between tu-mors. Thus, to evaluate a possible relationshipbetween these immunolabelings and the presenceof metastases, the fraction of cancer cells immu-nolabeled for each protein was determined bypoint counting.

When comparing primary tumors associatedwith lymph node metastases with those withoutlymph node metastasis (Figure 2), highly signifi-cant differences were found in the percentages ofcancer cells immunolabeled for PCNA or Ki67,with a higher index of positive cells in the pN+group than in the pN0 group (p < .001 for bothPCNA and Ki67). The fraction of PCNA-immunolabeled cancer cells correlated with thatof Ki67-immunolabeled cancer cells (r � .51, n �80, p < .001).

The fractions of PCNA- or Ki67-immunola-beled cancer cells were also inversely correlatedwith the degree of differentiation (r � .34 forPCNA and r � .25 for Ki67, p � 0.002 and p �0.028, respectively), but no correlation was foundwith the pathologic stage of the tumor. As statedpreviously, the stage of the tumor was higher incase of metastasis (p � .001 for pT and p � .029for cT), whereas the histologic differentiation ofthe tumor was not correlated with metastasis.

Sensitivity and Specificity of PCNA or Ki67 in Predict-ing Lymph Node Metastasis. To be of clinical use,cutoff points needed to be established for the frac-tion of cancer cells immunostained for PCNA orKi67 to distinguish the metastatic from the non-metastatic tumors. To define the optimal cutoffpoints, ROC curves23 were constructed by deter-mining the sensitivities and specificities obtainedfor each fraction of PCNA- or Ki67-immuno-labeled cancer cells (Figure 3).

Areas under the curves (± standard errors)were 0.829 ± 0.051 and 0.781 ± 0.059 for PCNAand Ki67, respectively. The percentages of immu-nolabeled cancer cells that gave the maximalvalue when adding sensitivity and specificitywere 36.6% for PCNA and 10.7% for Ki67. Usingthese cutoff points, 83% of the tumors could becorrectly predicted as being associated withlymph node metastasis by PCNA and 76% byKi67 (sensitivity), whereas 86% of the tumorscould be correctly predicted as being nonmeta-static by PCNA and 75% by Ki67 (specificity)(Table 3).

FIGURE 1. Immunolabeling of nuclei of cancer cells for PCNA(A) and for Ki67 (B) in squamous cell carcinoma of the larynx.Bar = 50 µm.


Multivariate Statistical Analysis. In practice, it islikely that multiple parameters will be taken intoaccount to predict the occurrence of lymph nodemetastasis. A multivariate logistic regression wasthus performed to identify the relevant param-eters for predicting metastasis in this study. Thefractions of PCNA- and of Ki67-immunolabeledcancer cells (p � .005 and p � .035, respectively),the clinical stage of the tumor (p � .040) and neckpalpation (p � .004) were the only significant pa-rameters, whereas the site of the primary tumor,the histologic differentiation of the tumor, and theimaging techniques could not significantly predictthe presence of metastases. Lymph node metas-tases were found in only 1 of the 21 patients show-ing all four significant parameters negative (5%)and in only 2 of the other 21 patients showingthree parameters negative (10%). Two significantparameters were positive in 20 patients, and 9 ofthem were initially seen with metastasis (45%).In contrast, three or four significant parameterswere positive in 14 and 4 patients, respectively,and 13 and all 4 of these patients showed lymphnode metastases (93% and 100%, respectively).

DISCUSSION

Despite intensive investigation, detection, andstaging of lymph nodes, metastasis from carci-

noma of the larynx, pharynx, or mouth continuesto present a major challenge to the head and necksurgeon. Clinical palpation and imaging tech-niques are currently not sufficient to reveal neckmetastasis preoperatively. The reported false-negative rate in finding neck metastasis by clini-cal palpation is 8% to 80% according to the subsiteof the tumor,24–28 and intraoperative staging doesnot significantly improve the false-negativerate.5,29 The value of CT, MRI, ultrasonography,or radionuclide scanning in assessing the pres-ence of lymph node metastasis remains contradic-tory.9,24,30

In this series of 80 head and neck carcinomas,the sensitivity of clinical and imaging assess-ments was about 50% only. Such lack of sensitiv-ity is obviously due to the occurrence of micro-scopic metastases that cannot be disclosed bythese examinations. Lymph nodes as small as 5mm in diameter can nowadays be detected withspiral CTs and MRI, increasing the sensitivity ofthe method as high as 98% in one study.31 How-ever, the specificity drops as low as 13% whensuch small lymph nodes are considered meta-static.

Although imaging techniques are needed toappreciate the extension and the staging of theprimary tumor accurately, it seems unwise to relyexclusively on clinical or imaging findings in se-lecting patients for prophylactic neck treatmentbecause of the high rate of occult metastases andresulting false negatives with the currently usedcriteria. More sensitive methods to detect meta-static disease are clearly required, and new his-topathologic analyses have been investigatedwith that purpose.32 Identification of tumor bio-markers of lymph node metastasis from head andneck carcinomas will challenge conventional ex-amination and be extremely helpful to better cat-egorize tumors.

The cell kinetics of many human tumors havea possible prognostic significance on the meta-static process, and the immunolabeling of PCNAand of Ki67 in the primary tumor has been re-lated to the presence of nodal metastasis.15 How-ever, the value of such markers in predicting theoccurrence of metastasis has not yet been inves-tigated in preoperative biopsy specimens.

In this study, we observed that a high index ofPCNA- or Ki67-immunolabeled cancer cells inpreoperative biopsy specimens was significantlycorrelated with lymph node metastasis. Both in-dexes were correlated with one another. However,the percentage of immunolabeled cancer cells for

FIGURE 2. Whiskers-boxes presentation of the percentages ofPCNA- and of Ki67-immunolabeled cancer cells in biopsy speci-mens of patients with ( ) or without (�)lymph node metastasis.Medians are represented by the horizontal lines inside the boxeswhich correspond to the 25th–75th percentiles. Extremes are atthe end of the whiskers. Significant differences were found be-tween � and for PCNA (p < .001) and Ki67 (p < .001).


Ki67 was consistently lower than that for PCNA.This could be due to a difference in antibody af-finity, to the expression of PCNA in nonprolifer-ating cells and therefore not expressing Ki67, orto a combination of both factors.12,13 Indeed,PCNA is necessary for DNA repair with excisionand replacement of the abnormal nucleotides andis thus expressed in nonproliferating cells repair-ing DNA alterations. Aggressive, poorly differen-tiated tumors that are prone to metastasize arerich in DNA alterations and could thus expressPCNA in many cells that are not engaged in thecell cycle and therefore not expressing Ki67. Inour series, the percentage of PCNA- or Ki67-immunolabeled cancer cells was inversely corre-lated with the degree of differentiation, in agree-ment with previous studies.15–20

According to the ROC curves, cutoff pointswere determined for PCNA and Ki67 fractions(37% and 11%, respectively), showing high sensi-tivity (83% and 76%) and specificity (86% and75%) for each protein in discriminating patientswith or without neck metastasis. The sensitivityof both analyses was much higher than for theclinical and radiologic examinations (52% and45%, respectively), whereas their specificity wasroughly equivalent.

The most widely adopted therapeutic manage-ment of head and neck cancer nowadays is to per-form a neck dissection, because it is the onlyavailable method to accurately assess the meta-

static or nonmetastatic status of neck lymphnodes in patients with head and neck carcinoma.However, this practice faces the objection that upto 70% of cases are operated wrongly or uselessly(64% in this series), and only 30% of the patientsbenefit.33

A more accurate prevision of occult metastaseshas long been anticipated. The immunohisto-chemical staining of PCNA and Ki67 on biopsyspecimens of head and neck cancers is an easytechnique that can routinely be performed inmost current pathologic laboratories. Assessingthe fraction of immunolabeled cancer cells foreach protein surely requires tedious work for thepathologist (taking about 1 hour for both immu-nostainings in our hands), but this analysis mayprovide a rather good prognostic indication for therisk of occult metastasis. Of course, the cutoffpoints reported in this study rely on the immuno-histochemical and on the fixation procedures usedfor the biopsy specimens, and it is likely that thecutoff points should be adapted according to thetechnical procedures used within each laboratory.

In our series, about 75% to 80% of patientswith an index of PCNA or Ki67 higher than thecutoff point need neck dissection, and 75% to 85%of patients with an index lower than the cutoffpoint need a wait-and-see attitude. Actually,PCNA and Ki67 indexes together with clinical Tstage and neck palpation are the significant pre-dictors of neck metastasis, whereas the radiologic

FIGURE 3. Receiver operating characteristic (ROC) curves showing sensitivity and specificity of thefractions of cancer cells immunolabeled for PCNA and Ki67. The fractions for which the sum ofsensitivity and specificity was maximal are represented by a black dot for PCNA and a white dot forKi67, and correspond to 36.6% and 10.7%, respectively. Sensitivity (Se) and specificity (Sp) corre-sponding to these fractions are indicated.


examinations, the primary site of the tumor, andthe histologic differentiation are of no value ac-cording to a multivariate statistical analysis. Inpractice, the immunohistochemical investigationswill not replace but will rather be complementaryto the clinical and radiologic examinations.

Indeed, when a lymph node is clinically or ra-diologically suspicious of being metastatic whileboth fractions of PCNA- and Ki67-immunolabeledcancer cells in the tumor biopsy are below thecutoff points, the risk of true metastasis is ratherlow. Fine-needle aspiration cytology of the suspi-cious lymph node would be a useful adjunct insuch a situation to help identify the metastaticlymph node that would not have been suspectedby the immunohistochemical indexes.

When no lymph node is palpated in the neck,the finding of fractions of cancer cells immunola-beled for PCNA and Ki67 in the biopsy specimenshigher than their cutoff points increases the like-lihood of occult metastases in cT3 and cT4 tumorsand should encourage the surgeon to perform aprophylactic lymph node dissection. In contrast,when these indexes are below their cutoff pointsand when there is no clinical nor radiologic sus-picion of metastasis, a wait-and-see attitude couldbe proposed.

In conclusion, our study has shown that thefractions of cancer cells immunolabeled for PCNAand Ki67 were significantly correlated with neckmetastasis from head and neck carcinomas andinversely with the degree of cancer differentia-tion. The use of defined cutoff points on specifi-cally stained preoperative biopsy specimens mayhelp toward a more accurate identification of pa-tients with lymph node metastasis. To our knowl-edge, this is the first time that a pathologicmethod with cutoff points is proposed with theaim to predict the occurrence of lymph node me-tastasis from head and neck carcinomas thanks tothe analysis of preoperative biopsy specimens.Further study is needed to confirm the validity ofthis procedure.

Acknowledgments. The authors are grateful toP. Thurion, H. Merckx, and Ph. Camby for theirtechnical assistance, to S. Lagasse for his photo-graphic work, and to Dr. C. Craddock-de Burburefor revising the manuscript.

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Predictive value of the fraction of cancer cells immunolabeled for proliferating cell nuclear...

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