364

THE EARLY DIAGNOSIS OFGENITAL CANCER BY CYTOLOGY

By ERICA WACHTEL, M.D.Institute of Obstetrics and Gynaecology, Hammersmith Hospital

The cytological diagnosis of malignant diseaseis based on the fact that malignant as well asnormal epithelia shed their cellular constituentsand that these exfoliated cells can be recovered formicroscopic examination from body fluids andsecretions. The method was first described byG. N. Papanicolaou in I927 at The Third RaceBetterment Congress, but did not arouse anygeneral interest until 1943, when Papanicolaou andTraut published *their standard textbook onUterine Cancer Diagnosis by Vaginal Smear, afterperfecting their staining technique. Since then,the method has been extended to cancer detectionin other organs, foremost to the diagnosis of lungcarcinoma from the sputum.The diagnosis of uterine malignancies is made

by examination of vaginal and cervical secretionsor by direct scraping of the squamo-columnarjunction with a wooden spatula. The lattermethod was described by Ayre and is known as the' surface biopsy.' It has the obvious advantageof avoiding dilution of the material and providesan excellent means of examining directly any'suspicious-looking ' area of the cervix uteri.On the other hand, this very precision of themethod carries the disadvantage that'it is directedonly against the cervix and does not allow forcollection of material from other sites Qf thegenital tract.

Papanicolaou's original technique of aspiratingmaterial from the posterior fornix collects cellsfrom the entire genital tract and thus enables thecytological detection of unsuspected malignanciesin the cervix as well as in the uterine body andoccasionally also in the ovaries. This method,(though more troublesome for the cytologist owingto the great deal of dilution due to the wide areafrom which material is collected), is more reliablein our opinion for cancer detection in symptomlesspatients, and recommends itself strongly forroutine use. Surface biopsy should be reservedas an additional method of investigation and shouldcertainly be employed in any patient presentingcervical pathology.

Aspiration is carried out with a piece of slightlycurved glass tubing to which a rubber bulb isattached at one end (Fig. i). The free end of thetube is introduced into the posterior fornix whilethe bulb is compressed; when in position, thepressure on the bulb is gradually reduced whilethe tube' is slowly. moved sideways so that thefluid from the entire area is collected. Thisaspiration should be done prior to manual orspeculum examination as contact with lubricantsmust be avoided.The introduction of the glass tube and the

subsequent aspiration are completely painless tothe patient and it is such an easy procedure thatpatients can be taught to take 'their own smears,which is a great help wherever serial smears arerequired. (As in hormonal assessments.)

Surface biopsies necessitate the use of aspeculum. The cervix is exposed and a woodenspatula similar to those used for throat examina-tions, but trimmed into a suitable shape (Fig. 2)is drawn round the squamo-columnar junction,using slight pressure so that the surface epitheliumis scraped off.Whatever method of collection of material is

used, the subsequent procedure is identical.Thin, even smears are made on glass slides whichare immediately, when still wet, immersed in thefixing fluid where they remain for a minimumperiod of 15 minutes but may be left for as long as7-8 days. In suitable portable fixing jars with ascrew-cap top, they can be posted to a cytologicallaboratory. Immediate wet fixation of the slidesis an important detail as air-dried smears do notabsorb the stains. Fixing fluids in use are ether-alcohol, Schaudinn's solution or a mixture of 7parts tertiary 'butyl alcohol and 3 parts absolutealcohol, which is the most stable of the three.The staining of the slides is carried out in the

laboratory and Papanicolaou's original method,with only minor individual variations accordingto the predilection of the cytologist, is still in use.For details of the technique the reader is referredto 'Papanicolaou and Traut's book (I943). The

copyright. on M

ay 4, 2022 by guest. Protected by

http://pmj.bm

j.com/

Postgrad M

ed J: first published as 10.1136/pgmj.30.345.364 on 1 July 1954. D

ownloaded from

July 1954 WACHTEL: The Early Diagnosis of Genital Cancer by Cytology 365

Fill Fijil

FIG. i.-Apparatus for posterior fornix aspiration.FIG. 2.-Wooden spatula for 'sturface biopsy.'

stained and mounted slides are ready for micro-scopy.

Before discussing the criteria of malignant cellsit is necessary to say a few words about the normalcells present in all smears and td describe certainabnormalities other than malignant features. It isessential to become thoroughly familiar with thenormal vaginal cytology and its variations ininflammatorv conditions before proceeding to thedetection of malignant cells.The vast majority of cells found in normal

vaginal smears come from the squamous epith-elium. Scrapings from the cervix also containnormally a large number of columnar cells fromthe endocervix. Endocervical cells in aspirationsmears are unusual and generally indicate cervicalpathology such as cervicitis, cervical polypi orerosion. Endometrial cells in posterior fornixaspirations are seen physiologically during orshortly after the menstrual period, but arepathological at any other phase of the cycle,indicating an abnormal endometrium.

Squamous cells occur in 3 varieties accordingto the layer from which they have exfoliated.A healthy vaginal epithelium in a young womanshows a superficial, an intermediate and a basallayer. Normally in young women only cells fromthe 2 upper layers are recovered in the aspiration.In cases of severe infection, however, where thesurface layer has become eroded by the inflam-matory process, basal cells from the lowest layerwill also be seen and their percentage indicates

the severity of the inflammatory destruction.Conversely, thinning of the epithelium with lossof surface layers, occurs with failing ovarianfunction and can be recognized by the presence ofbasal cells in the smear in the absence of infection.

Superficial squames are the largest cells foundin vaginal o'r cervical smears. They are poly-hedral cells with a clear cytoplasm staining blue-green or red with the acidophilic or basophilicstains of the triple dye. Some authors regard thestaining reaction as a function of oestrogenactivity, describing acidophilic cells as oestro-genized, but we consider the staining reactionto be dependant on the pH of the vagina and makeno attempt to correlate it to the oestrogen level.The nuclei of these superficial (and intermediate)squames are, however, of primary importance inhormone estimations. They have either a vesi-cular nucleus which is small and, under normalconditions, does not exceed I/50 of the cellvolume, or a punctate, pyknotic nucleus. Webelieve that nuclear pyknosis is an oestrogen effectand work out the percentage of pyknotic nuclei(Cornification Index= CI) to establish the oestrogenlevel. The CI in a given patient is not a stablefigure but varies with the stage of the cyclereaching a peak value at ovulation.' Cornificationindices after the menopause are normally extremelylow.

Progesterone effects on these cells are alsorecognizable, i.e., the cells which int the proli-ferative and ovulatory phase appear clear cut withsmooth, sharply defined edges, cluster togetherand show edge curling (envelope effect) undetprogesterone influence.

Intermediate squames are smaller and rounderthan superficial cells and have' a somewhat largernucleus. They are specifically increased inpregnancy when they may be boatshaped (navi-cular cells of pregnancy).

Basal cells are the smallest cells of the squamousepithelium. They are round or oval, have alarge round nucleus which does not exceednormally j of the cell volume, and usually stainblue-green. They very often show degenerativechanges, i.e., vacuolization of the cytoplasm,perinuclear halo formation and karrhyorrhexis..Since they indicate failing ovarian function, theyare prevalent in postmenopausal women. Theirpercentage in any given smear, provided there isno infection present, gives a fair measure of theregressive changes occurring in the patient (Fig. 3).The columnar epithelium seen in smears differs

very strikingly in size from the squamous cells.Endocervical cells are pear-shaped cells, about 3times as big as a lymphocyte with a rounded lowerpole and a flattened upper end bearing cilia. Thenucleus stains dark, fills about j of the cell and

copyright. on M

ay 4, 2022 by guest. Protected by

http://pmj.bm

j.com/

Postgrad M

ed J: first published as 10.1136/pgmj.30.345.364 on 1 July 1954. D

ownloaded from

366 POSTGRADUATE MEDICAL JOURNAL July I954

lies eccentrically in the lower pole. The cytoplasmis pale, clear and stains blue-green. These cellshave an unfortunate habit of rapid disintegrationso that often only clusters of dark staining nucleiwith little or no cytoplasm are found. Thesecould be misinterpreted as malignant nuclei ifone were not familiar with their appearance,especially since they often show inflammatorychanges. Characteristic for their innocent natureis the smooth nuclear outline, the uniformity ofnuclear size and shape, and the even or finelygranular nuclear structure (Fig. 4).

Endometrical cells are the smallest epithelialconstituents found in smears. They are notbigger than one and a half to twice the size of alymphocyte, which they resemble to a certainextent. They are round cells with a relativelylarge nucleus filling i to j of the cell volume and aclear cytoplasm, rather more dense than that ofendocervical celfs, which usually stains blue-green.They possess the same tendency to rapid disin-tegration as endocervical cells and appear alsovery frequently as clusters of nuclei only. Again,this may present diagnostic difficulties.When looking at a series of smears through the

microscope one finds two groups of films: the" clean " and the " dirty " patterns. The formerare composed of normal epithelial cells only ormay show Doederlein bacilli or a few leucocytesas well. They are characterized by an essentiallyuniform pattern throughout the entire film, canbe screened quickly and -present no difficulties indiagnosis. They rarely, if ever, contain malignantcells.The second group, " the dirty film," is composed

of clusters of cells and there is an excess ofleucocytes and bacteria and often also of red bloodcorpuscles. There is no uniformity of appearancein any two microscopic fields and screening thesefilms is a long and wearisome task. Such patternsmay indicate a great variety of conditions. Theyare physiological during menstruation, or patho-logical in inflammatory or malignant disease.

Inflammatory nuclear changes present anextremely complex appearance including varia-tions in size, shape, staining properties, hyper-chromasia and granular structure. Howeverabnormal the nuclei may appear the differentiatingcharacteristics from malignancy are:

i. Although the nuclei may be enlarged, theyare smaller than J of the cell 'Volume.

2. Their granules are fine and even and donot show variations inside the same nucleus.Malignant films are almost always " dirty"

films. They show clumping. of the " normal"epithelium, a great many abnormal cells withinflammatory nuclear changes exfoliated fromsites adjacent to the heoplastic area, malignant

cells from the tumour, leucocytes and bacteria inexcess and usually, though not necessarily so,many red blood corpuscles. The cornificationindex of a " malignant smear" is usually high,which is especially striking in cancer patients wellpast the menopause.The number of malignant cells found in a

smear depends on the exfoliative properties of thetumour and the degree of dilution occurring. Asa rule, the younger the carcinoma, the morereadily does it exfoliate and the easier the recoveryof these cells in the smear. Well establishedcarcinomata of long standing often fail to shedrecognizable cancer cells into the vaginal secretions,probably because these tumours have ulceratedsurfaces with superimposed infection. Howeverthese cancers are clinically obvioms and cytologicaldiagnosis would, in any case, be of academicinterest only. It is far more important from apractical point of view, that the early neoplasmusually exfoliates readily and that its cells can bediscovered by routine screening at a time beforeclinical signs and symptoms are apparent.

Cancer cells, irrespective of their origin, areidentified by their nuclei. The pathognomonicfeatures of malignancy are the coarse granularstructure of the nucleus and the variation in, sizeand shape of the individual granules inside thesame nucleus. Other features suggestive, but notdiagnostic, of malignancy are nuclear enlargement,irregularity of nuclear outline and hyperchromasia.

Well differentiated malignant cells resemblethe epithelium of their origin and thus indicate,not only the presence of malignant disease but alsothe source and type of the tumour. No difficultyarises in differentiating cytologically betweensquamous- and columnar-celled cancers, if thenew growth is composed of at least moderatelywell differentiated carcinoma cells. Very im-mature and anaplastic tumours may, however,present great diagnostic problems as to the tissuefrom which they are derived, although the recogni-tion of their malignant character is easy. Similardifficulties are encountered in histological sectionsof such cases.

Differentiated squamous cancer cells occur in3 varieties according to the epithelial layer fromwhich they originate:

i. Malignant tadpole cells (corresponding tosuperficial squames). Fig. 5.They are the largest malignant cells found in

smears. Their appearance resembles a tadpole.Note the- large nucleus filling more than iof thecell, its granular structure and the variation insize and shape of the individual granules.

2. Malignant fibre cells, derived from theintermediate layer, are thin fibre like cells with

copyright. on M

ay 4, 2022 by guest. Protected by

http://pmj.bm

j.com/

Postgrad M

ed J: first published as 10.1136/pgmj.30.345.364 on 1 July 1954. D

ownloaded from

)'uly I954 WACHTEL: The Early Diagnosis of Genital Cancer by Cytology 367

.. .. ..

FIG. 3.-Normal vaginal epithelium of a menopausalwoman. (a) Superficial squame. (b) Intermediatesquame. (c) Basal cell. x 400.

a large elongated nucleus filling at leastI

of thecell volume.

3. Malignant basal cells (Fig. 6) derivedfrom the deepest epithelial layer, usuallyexfoliate in clusters. These are the mostcommonly found squamous malignant cells insmears.Malignant endocervical and endometrial cells

closely resemble their benign cells of origin buthave " malignant " nuclei.

Fig. 7. Malignant endocervical cells.Fig. 8. Malignant endometrial cells.It is frequently stated in the literature that

it is much more difficult to recognize malignantcolumnar cells than squamous cancer cells. Thisis a fallacy. Apart from the difference in cell-and consequently also in nuclear size, the criteriaof malignancy are identical. In addition, endo-metrial as well as endocervical carcinomata tendto exfoliate in clusters and notwithstanding theirrapid cytoplasmic degeneration the accumulationof malignant nuclei in heaps renders thediagnosis easy. It is, of course, of paramountimportance to bear in mind the size-relationshipand to screen the smaller cellular elements of thefilm with great care.

....................

....

FIG. 4.-Normal perfectly preserved endocervicalcells. (Note cilia at upper pole). X I,000.

The role of vaginal smears in cancer diagnosisis still much debated. We hold that cytologyplays a definite part in gynaecology when used byspecialists with a critical mind, well aware of itslimitations. It is the most valuable means wehave at the present moment of recognizingmalignant disease in its initial stages. It is notcustomary to submit the patient who seeksgynaecological advice for symptoms which do notsuggest malignant disease of the genital tract andin whom no cliniical signs are as yet apparent tocurettage or biopsy. Cytological investigationscan, however, easily be included into the routinetests and if cancer cells are present in the smear,this finding justifies further histological examina-tion and enables the gynaecologist to providetreatment at a very early stage of the diseiase.

Cytological cancer diagnosis is by no means aninfallible procedure. Mistakes do occur owing tofailure of exfoliation or to error of judgement, butdespite these shortcomings the sum total of correctdiagnoses does not compare badly with the resultsof other laboratory methods. It must be borne inmind that biopsy is also not an infallible diagnostictest, since in the patient without clinical evidenceof malignant disease, the removed tissue may have

copyright. on M

ay 4, 2022 by guest. Protected by

http://pmj.bm

j.com/

Postgrad M

ed J: first published as 10.1136/pgmj.30.345.364 on 1 July 1954. D

ownloaded from

368 POSTGRADUATE MEDICAL JOURNAL Yuly 1954

N ~N~

,HM:xs:.

FM~~

:LsiOM.

FIG. 5.-Tadpole cell. (Note enlarged hyperchromaticnucleus with coarse irregular granulation). x ,ooo.

;.......:.......a

FIG. 6.-Malignant basal cells. (Showing enlarged,hyperchromatic, coarsely granular nuclei withirregular outlines. X I,000.

s.s}>.gi:...........-T

:t.::}:~~~~.:}o.'o.....

i.:}'............. !eS,..........:tS::.S.:.:

=<>i:i:y8:-°' E ....:.s..:. :...:........... ..........:.. .. ...}..Y:.....

<8 8;~~~~~~~~. .-::.::.:.... .... .... .... :.. .... ...

:::3.::... :}::}i':.:.::.:!.i::.::.

vs::sE w iiWW-n'~~~~~~~~~~~..U...... .. ........e.

... .:rl:..-le: :} z :: e.. 3.. :....>.:..:..: ....:.:i .::i::}:!::}:!::::.... :..:. ..::.: :: >; 6:: :: ::::: :u:. .:. : ot.t...:.. ... ..! .i:.:. .....!. ..:

... ... ... .... ... ...ew ~~~~~~~.. d.. .i,...'-.:.. ...}:.}:~~~.. ............ .. .. ... '.: :'i:....... :i..i:'..}:i::: .....:: ...:: - -: : ::.::;::::::.:::::::::::: :::::: --::::::- :::::::|w != ijEXii|j "..j'..2.j....... t0

ibi-E 1 E i w S N i.. i;

FIG. 7.-Malignant endocervical cells. X I,OOO.

'.1*

4 4.

.

...........44.

.4.7

*..4'.v ..

...k,....

4. -.

-4

. 4. ..

FIG. 8.-Malignant endometrial cells. X I,OOO.

copyright. on M

ay 4, 2022 by guest. Protected by

http://pmj.bm

j.com/

Postgrad M

ed J: first published as 10.1136/pgmj.30.345.364 on 1 July 1954. D

ownloaded from

July I954 WACHTEL: The Early Diagnosis of Genital Cancer by Cytology 369

been taken from an area free of new growth. Ifboth methods are employed the overall cancerdetection will be increased. Errors in judgmentare mainly responsible for' false positive ' reports,and it is, therefore, imperative to confirm ' posi-tive smears ' by histological methods -before anytherapeutical measures are taken. ' Positivesmears ' which are unsupported by histologyshould be repeated; if follow up smears continueto show cells believed to be malignant, moreextensive repeat biopsies must be carried out, buton no account should a patient be submitted tomajor surgery or radiotherapy on the strengthof one positive smear report.A survey of the 3 years' ending December

31, 1952, routine cytological screening carried outat the Hammersmith Hospital gave the followingresults: The total number of patients sufferingfrom malignant disease of the uterus seen inthe Gynaecological Department was 64; 30 ofthese were squamous and 34 columnar-celledmalignancies.Of the 30 squamous cancers 26 were diagnosed

correctly by routine smear procedure, 3 or ioper cent., were missed and for one an unsuitablesmear had been submitted. This compares wellwith American figures of 'false negatives.'Of the 34 adenocarcinomata 31 were diagnosed

correctly, two were missed and for one anunsuitable film had been submitted. This givesan incidence of error of only 5.8 per cent., which ismuch lower than the corresponding Americanfigures.More important than these absolute figures is

the fact that out of these 64 carcinomata 8 wereentirely unsuspected clinically and would not havebeen diagnosed and treated at this early stage hadcytological investigation not been carried out.The incidence of false positive reports has been

regrettably high, but as the only criterion ofcomparison is the histological report, which isregarded as the ultima ratio although it is sometimesbased on a single biopsy only, it is possible thatcases now listed as cytological errors, may, in thecourse of time, prove to have been correctlydiagnosed cytologically. One case included inthe 64 carcinomata had been listed as a ' falsepositive' for i8 months until the patient wasre-admitted with secondary vault metastases.(Martin and Kenny I950 and Martin I953).

In the light of our present knowledge, however,and accepting the histological report as the finaljudgement, we have had 9 ' false positive' reports,all. of which concerned erroneous diagnoses ofcolumnar-celled malignancies; most of these camefrom patients with ulcerating polypi, one from apatient with a sloughing endometrium and onefrom a woman diagnosed as an atypical glandularhyperplasia.To sum up: of our 62 carcinomata investigated

cytologically, omitting the two cases with unsuit-able smears, 57 or 9I.9 per cent. were diagnosedcorrectly, 5 or 8.7 per cent. were ' false negatives,'and 8 were detected by the smear. Cytology isa valuable aid to cancer diagnosis and an easilyapplicable method of cancer detection in theunsuspected case.

BIBLIOGRAPHYAYRE, J. ERNEST, (i9si), Cancer Cytology of the Uterus, New

York, Grune and Stratton.MARTIN, R. T. and KENNY, M. (i95o), 'Changes in the Cervix

Uteri simulating Carcinoma in Two Successive Pregnancies,'J. Obstet and Gynaec, Brit. Emp., 7, 6o8.

MARTIN, R. T., Y. Obstet. and Gynaec., Brit. Emp., (in press).PAPANICOLAOU, G. N. and TRAUT, H. F. (r943), Diagnosis

of Uterine Cancer by the Vaginal Smear, New York, TheCommonwealth Fund.

VINCENT MEMORIAL HOSPITAL (I95o), The CytologcalDiagnosis of Cancer, Philadelphia, W. B. Saunders Co.

WACHTEL, E. and PLESTER, J. A. (1952), 'The Vaginal Smearas an Aidto Diagnosis of GenitalTract Malignancies in Women.'J. Obstet. and Gynaec., Brit. Emp., 3, 323.

Continuation of Bibliography-H. P. Ferreira, M.D., from page 359.BELLERBY, C. W. (X934a), Nature, 133, 494.BHADURI, J. L. (i05I), Proc. 38th Ind. Sci. Congress, Pt. II, 171.BINET, L., VERNE, J., and LUXEMBOURG, F., C.R. Soc.

Biol., Paris, xI6, 1241.BISHOP P. M. F. (1934), Brit. med. J., i, I 86.BROUHA, L., and HINGLAIS, H. (I93I), Gynec. et Obstet., a4, 42.BRUHL, R., and REIKOFF, W. (1935), Z. Geburtsh. Gynak.,

112, I.BUNDE, C. A. (x947), Amer. J. Obstet. Gynec., 53, 317.COWIE, A. T. (1948), 'Pregnancy Diagnosis Tests: A Review.'ELEK, S. D. (19s3), Personal Communication.FARRIS, E. J. (i944), Amer. Y. Obstet. Gynec., 48, 200.FRAZER, J. F. O., and WOHL7ZOGEN, F. X. (I950), Brit. med. 7.,

ily 330'.FRIEMJ4AN, M. H. (I929a), Proc. Soc. exp. Biol., N.Y., 26, 720.GALLI MAININI, C. (/947a), Sem. med., 54 (I), 337.GALLI MAININI, C. (1948), j. Amer. med. Ass., 138, 121.GEOGHEGAN, F., and McGRATH, J. (I944), Irish J. med. Sci.,

6'm Ser., No. 225, 509.HAINES, M. (I948) Lancet, ii, 255, 923.HAINES, M., and F1ERREIRA, H. P. (1954), in the Press.HOBSON, B. M. (19S2), Y. Obstet. and Gyn. Brit. Emp., 59, 3, 352.HOGBEN L. (1930), Proc. Roy. Soc. Afr., S, I9.HOUSSA+, B. A., and LASCANO GONZALEZ, J. M. (1929),

Rev. Soc. argpnt. Biol., 5, 77.HUMMEL, K. P. (I942), Endocrinology, 30, 74.

KONSULOFF, S. (I934), Klin. Wschr., I3, 776.KRAUS, E. J. (I932), Munch. med. Wschr., 79, 214.KUPPERMAN, H. S., and GREENBLATT, R. B. (I946), Sth.

med. 7. (B'gham, Ala.), 39, I8.KUPPERMAN, H. S., GRENBLATT, R. B., and NOBACK,

C. R. (I943), 7. clin. Endocr., 3, 548.LANCET (1948), ii, 977.LANDGREBE, F. W. (I939), . exper. Biol., I6, 89.LANDGREBE, F. W. (1948), Proc. Roy. Soc., Edinburgh, 63, 213.LAW,J.W.((I99), Bull. Inst. Med. Lab. Tech., 14, II, I75.OWEN, S. E. (I936), Endocrinology, 20, 214.POU DE SANTIAGO, A. (1947b), Arch. Moguayos Med. Arng.

Espec., 30, 457.RAZA, S. H., and SPURRELL, W. R. (I937), Y. PhYsiol., 90, 429.REIPRICH, W. (I933), Klin. Wschr., 12, 1441.REIPRICH, W. (it34), Z. Geburtsh. Gynak., I09, 285.SALMON, N. J., WEST, S. H., SALMON, A. A., and FRANK,

I. L. (1942), J. cin. Endocr., 2, I67.SCHWABACHER (i9Si), Med. Press, 226, 82.SCOTT, L. D. (1940), Brit. 3. exp. Path., 21, 320.SHAPIRO, H. A., and ZWARENSTEIN, H. (0934a), Nature,

133, 339-SIMOLA, P. E., and RIVAS, L. (1936), Suom Kemist., 9, B, 24.WALKER, T. F., and WALKER, D. V. H. (1938), 7. Amer. med.

Ass., II, 1460.ZONDEK, B. (193oe), Klin. Wschr., 9, 964.

copyright. on M

ay 4, 2022 by guest. Protected by

http://pmj.bm

j.com/

Postgrad M

ed J: first published as 10.1136/pgmj.30.345.364 on 1 July 1954. D

ownloaded from