NEW PERSPECTIVES IN CANCER CYTOLOGY

1)r. CLAUDIO BAKIGOZZI" and nr. (;I~JSE:PPE DELL.hPIANE.t

M OST of the neoplasins offer such histo- logical fe'itures as gencially to permit a clear- cut diagnosi5, the difficulty of discriminating between hyperplastic and neoplastic growth being limited to iaie special cases.

l ' he histological peculiarities ol cancerous growth are the icsult of new capacities, my$- teriousl) acquired by the cells, to rcpi otluce theniselves and to fall into necrosis at an abnormal rate. I t can be said that the neo- plastic tissue acquires an exalted degree ol lability.

It is clear, therefoie, that since the causes of cancel are still unknown, a new light could be thrown on the problem by a better knowl- edge of cellular reproduction and of those phenomena that lead to cell necrosis.

This aim has been pursued by a great num- ber of investigations, which have never suc- ceeded in showing what change is occurring in the cell when cancel begins.

Although niariy workers have laid emphasis on metabolic changes, it tannot be said that we habe a clear pictuie ol the physiology of the malignant tell. Eken more obvxre is the microscopic pattern of the cancer cell, the cliai actelistic feature of which escaped the most carckul examinations imtil a lew years ago. It seeins piobable that the malignant cell docs not differ lrom the noi n i ~ l one as a whole, arid the old cytologists were not able to find definite criteria capable of providing a cyto- logical diagnosis. We may quote only the old observations b y Pianese, who pointed out the increase of tlie nucleolar mass, and the abnor- malities in the number of the chromosomes observed by niany authors. T h e former obser- bations on the nucleolus have bcen confiimed by more recent investigations, while the chro- mosome number seems to be absolutely noi ma1 in inany canccr mitoses, thus appearing as an at least inconstant charactei for cancer.

An inteicsting new step has been made by Papanicolaou's school, which showed many distinct chai actcrs of the cell that, although not always present, ncvcrthcless can provide

* Professor of Genetics, University of hfilan, Italy. -I- Professor of Obstetrics and Gynecology, UniLersity

Received for publication, June 13, 1950. of Turin, Italy.

some information on cancer. Among these, ernpliasis has been laid on "anisocytosis," or difterenccs in cell pattein and size that are not present in normal tissue. Big nucleoli and abnorrnal milotic figures could also be found by Papanitolaou, together with high stain- ability. 'Therefore, actoi ding to this method, the cytological diagnosis ol cancer is reached through the hndings of abnormal cell shapes and abnormal associations of cell gioups, in niatcrials obtained from body fluids as, for exainplc, those of the vagina, of the bronchus, etc.

Starting froin quite different points of view, far 11 om those of classical histology, Caspersson and Santcssoii succeeded in finding a new method to differentiate malignant from nor- mal cells. Using monochromatic ultraviolet light, they were able to show that in the malig- nant tunlor inass two different kinds of nuclei t an be found (the proliferating and the pre- nccrotic ones, labeled as A and R; see Figs. 1, 2). The former t)pe appears iich in nucleic acids, which seem to be rnucli less in the second, 01 B type, in which the iemnants of nucleic at ids aie confined to the big nucleolus, only scanty traces being present in the reniain- ing p i t s ol the cell. Both types ale connected by a continuous chain of intermediate stages. In suniniaiy, the most outstanding fact pointed ou t by Caspersson and Santesson is the recogni- tion of a structural heterogenicity in the tu- mor, A and B cells being variously intermixed. T h e same results have been reached by Koller, who was also able to find a difference between proliferating and preiiecrotic (or inatui e) cells, using an easy technique, i.e., the smearing of biopsy material after treatment with carmine or Schiff-Feulgen reagent.

AlLTHOD

I t may be 01 some value to outline briefly tlie technique generally used. The cytoplasm is dissol\ed b y treating with acetic acid arid carmine, while the nucleai inatel ial remains intact and takes up the dyc. The pecu- liar action on the cytoplasm is pi-o\ed by the toniparison of nuclei after treatment with acetic acid with nuclei in the living state

FIG. 1. Type-.\ (proliferating) nuclei troni uterine cancer. (Acetic carmine.)

Fit,. 2. I ) p - B (nonproliferating or mature) nuclei from uterine cancer. (.icetic carmine.)

(plant and aniiiial cells); the clironiatin struc- fixed lor twenty-four hours in acetic-alcohol lures appear pyactically unchanged. 7‘his (two-thirds 95 per cent alcohol and one-third niethod includes tlie so-called “squasli” prepa- glacial acetic acid). T h e material can be pre- rations of the material thus fixed a i d stainctl. served for sc\,eral days in this fixative. This consists ol‘ isolating and flattciiing the nuclei, by pressing the cove1 slip with the needle. T h e dissolved cytoplasm is removed and the naked and flattened nuclei show clearly tlie intcrnal s t r i i~ t~ i re . A necessary re- quirement is the softness of the material, which iiiiist be cut into sniall picc& not exceeding a few cubic millinietcrs. Before handling for staining and squashing, tlie material can be stored for long periods of time (one to two months) in two pai-ts of 70 or 95 per cent alcoliol to one part of acetic acid. Not basically different is tlie Feulgen reaction, which can be applied to squashed and unstained cells (in this case acetic fixative only is used) after rc- inoval of tlie coverslip. The best results are obtained by using slides covered by a thin film of egg a1l)iumin clricd alter smearing and coverslips smeared with animal fat. The chro- matin structures in this nianiicr adhere to the albumin and the coverslip can be detached easily.

T h e steps of the method used by 11s are as iollows:

P’ixation. Very sinall fragments of tissue, or material aspirated from body cavities, are

Stairiiiig. Very sinall fragments of tissue, of the dimension ol a fraction of millimeter, or siiiall aiiioiints or the sediment in case of exudates or secretions, are placed in a watch glass, or directly on an albuniinated slide, and covered with acetic carmine (acetic orcein can also be used). Alter at least one-half hour, the preparation on the albuniinated slide is covered with a coverslip smeared with animal fat. With dissecting needles, pressure is then applied on the coverslip with gentle tapping movements over the pieces of tissue or aspir- ated material. After blotting the slide, it can be studied immediately under oil immersion or a high dry objective.

The slide is placed o\wnight in a jar with 95 per cent alcohol; then remove tlie coverslip with a razor blade, tlchydi-ate for one minute in absolute alcohol, and clear and irioiint in balsam.

‘Ihis technique is by no means original. It was first devised by Belling as early as 1921, and since that time it has become a routine method in every karyological investigation. Tlic details may be found in Darlington and Lc Cour.

Prepamlio?i of Pci.rna?zent Slide.

1561 C A N C I ~ R J a n u a ~ y 1951

RESULTS

rather than of microchemical significance and must therefore be considered very useful for practical purposes.

Our own investigation moved in the same

plement to the study of the nuclei in the rest- ing stage. Here, again, Koller has studied the

The arc Of ]nost important features, which Cusmano was able to nlixed these, a series of irregularities can be observed, as nondisjunction between chro- matids a t anaphase, anaphase bridges, frag.

tant Cusmano was to the de- poles, and multipolar spindles. Sometimes the scribed by Koller. Cusmano's results agree chromosomes arc overcontracted and sliow a completely, and the following characteristics llaploid nunll)cr. Generally, however, the can be assumed as specific for cancer cells. T h e nlitoscs can be considered as too variable to proliferating elements (type-A cells) arc pro- offer a safe vided with nuclei rich in chromocenters, or Barigozzi and Casabona, and Casabona irregular masses of chromatin; the non- on a series of comparative invest+-

ency to necrosis, are practically devoid of Grattarola transplantable adenocar- chromocenters, and the chromatin threads are visualized with difficulty, while the nucleolus or the liucleoli appear very large and of irregu- nuclear level does not show any dif- lar shape. It is then clear that, even in the absence of any characteristic feature in the

the recognition of a peculiar structure in the cancer cell. It must be noted that these obser- vations by Cusmano have been made only on cancers o f the uterus, and also that the previ- OLIS findings of Kollcr did not include all types of neoplasms. Should it be confirnicd that the special features just mentioned are common to all types of cancer, new viewpoints on the nature of nialigriaiit tumors might appear.

Special attention was paid by one of us (Dcllepiane) to the problem oE differentiating between malignant tumoi s and hyperplasia of' the endometrium. A sharp difierence was found, because in most instances no similari- ties exist between malignant and hyperplastic cells. On the other hand, no differences can be detected between normal and hyperplastic epithelial nuclei.

The investigation is carried out in the fol- lowing ways. T h e slide is carefully examined under ii high-power objective with green light. The investigator must be familiar with the appearance of normal nuclei so that he can make a comparison between normal and patli- ological findings. The structure of normal nuclei, for epithelial cells at least, seems to be very homogeneous. The presence of one or two (seldom three or four) chromatic masses is assumed as a good index of normality. In the proliferating A nuclei of malignant cells, there is generally a very high and 'lot I'recisely determinable number of these masses.

The mitoses were also analyzed, as a corn-

M~~~ mitoses are

path, and the first approach Illadc by Our mentations, irregular lnigrations toward the

for diagnosis.

proliferating (type-B have ' tend- tions on difierent neoplasms of the rat, and

cinonlas of rat and rI1ousc. it call be at fcrerlcc in different animal

Having thus recognized

their findings, that the cytological pattern

existence of a

worth while to use these

cyto~lasnl? the first step has been made toward specific microscopic structure in the malignant- nucleus, it

FIG. 3. Type-K nurlei of different sizes from the vagirial fluid in a case of uterine cancer. (Acetic carmine.)

NEW PERSPECTIVES I N CANCER CYI OI.OGY . Bar-igozzi G. Dellefiiaiie [157

obsei vdtioris for diagnostic ~ x n p o ~ e s . I t m a y be noted that this new approach appeals i o be coniplcrnentary to, and not in conflict with, Papanicolaou's views. \Vliile Papanicoldou does not use special techniques for the stud) 01 tlie chromatin stinctnres, our method, on the othei hand, deals with the iecognition of what anisocytosis, or better, what anisohai yo- 4 s is.

FIG. 4 . c arrnine.)

No1 ma1 nucleus from vaginal fluid. (.Iretic

One of us (Dellepiaiie) collcctcd nearly four hundred cases of women affected b y different gynecological conditions. In each patient, the general clinical symptoms as well as tlie his- tological structure of the uterine inucosa and the cytology of the vaginal fluid were analyzed. The results obtained by the study of the vaginal fluid give an error not exceeding 7 per cent. 'This error includes both false-positive arid false-negative cases, with a slight tendency for false positives to prevail; this deserves par- ticular consideration. In some women, in the absence of any synlptoin of neoplasm, only a small minority of A and B nuclei were found aniong thousands of normal elements. Such cases may suggest the possibility of a very early stage of cancer that can vanish later. Research along this line is progressing. Among the false negatives, sonic cases were seen in which the solid structure of the tumor or its localintion in the upper part oi the uterus prevented the vaginal fluid receiving any malignant cells.

I f karyological analysis is performed on biopsy material, the error falls to nearly 1 per cent: this is the true error of the specificity of the nuclear structure, and it seeins so low that the use of this method can be considered a good lest for the detection of cancer.

This study has now been extended to two further fields: the effect of roentgen rays and other ionizing radiations and nitrogen mustard and stilbenic compounds on chro- matin. Cusmano iouiid that, after irradiation with roentgen rays or treatment with radium, a decrease occurs in the number of A cells,

follo~vetl b y an increase ol B cells, which later disappear. I t seeins, therefore, that the most striking effect of ioniring particles on inalig- nant cells is to kill the proliferating element, which dies while it is passing through the U stage.

T h e effect of nitrogen mustard (Barigozzi and Casabona)' seem \'cry active on the mitotic chroniosomes, which become sticky and de- spiralized. Stilbenic coinpounds determine a high degree of stickiness that manifests itself at anaphase as false bridges. All this proves that a response to chemical agents can be detected in tlie chrornatin and is not limited to dis- tinct points, but extends to the entire corn- plex.

The analysis of all these points offers new ideal paths to future investigations. T h e first concerns the possibility of extending the obser- vations collected to all malignant tumors. For instance, we cannot yet state with certainty that what has been shown to be true for the uterine niucosa is valid also for the bladder, lungs, or other organs. Investigations are in progress. Meanwhile, it can now be said that our observations on mamniary carcinoma, rectal carcinoma, and on neoplasms of the bladder arid larynx support the view oE the existence of a typical pattern in the different organs.

The possibility of improving the cytological diagnosis is strictly correlated with the speci- ficity of the nuclear pattern, and both re- searches must progress conteniporaneously.

Yet another field is offered to this method and, more precisely, that of the differentiation between benign and malignant tumors. In

FIG. 5. Mitotic figure in anaphase showing stick- ing chrommomes. From a case of sarcoma. (Acetic carmine.)

Frc;. G. hlult ipolar spindle ol a liyperploitl pro- liferating cell in a case of transplantable sarcoma o f the r a t (Gnlliern sarcoma). (4cetic carmine.)

fact, there are many neoplasms in which his- tological criteria alone are not sufficient for a definite classification as to their expected clinical behavior. To give some examples, we can mention the frequent difficulty encoun- tered in the classification of adenoina versus aclenoma malignum, fibroma versus fibrosar- coma, leioniyoina versus leiomyosarcoma, et c. If our method were to be successful in this respect, it would certainly represent a step forward in the microscopic evaluation of tumors.

Another vei y intcrcsting point that should be inkestigatcd is that of the so-called “precan- cerous stagc” ant1 ol eat ly di‘ignosis.

\Vhat happens at the very bcginning of can- ceriLation is unknown, since the uwal methods nidke recognition of a turnor possible only when i t has already grown to a certain s i x T h e detection of a single malignant cell makes i t possible to recogni7e the neopladc devia- tion in a small number of elcrncnts, which are parts 01 a cell population that is still pre- dominantly normal. If this point ot view is accepted, one could assume that the precan- cerous stage corresponds to the picsence of caricerized cells at a very low frequency. T h e beginning of true neoplasia (eai ly diagnosi?) could be ieprescnted by a somewhat higher rate of cancerizcd cells. Finally, a predomi- nance of A and B cells in a sample should mean that a fully developed malignant neo- plasm is present.

It is our hope that furthcr study, while lead- ing to a clearer undcrstanding of the nuclear stiucture in benign and malignant cells, will also supply the diagnostic laboratories with new tools for the early recognition of canccr and for a mole accurate differentiation of benign from malignant tumors.

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