cancer res 1970 weinstein 724 8

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1970;30:724-728.Cancer Res

Gerald D. Weinstein and Phillip FrostCell Proliferation in Human Basal Cell Carcinoma

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(CANCER RESEARCH 30, 724 728, M arch 1970]

Cell Proliferation in Hum an Basal Cell Carcinom aGerald W einstein and Phillip FrostD e pa rt m en t o f D e rm a to lo gy U n iv er si ty o f M i am i S ch oo l o f M e di ci ne M i am i F lo ri da 3 31 36

SUMM RY

Inform ation about cell proliferatio n kinetics in humancance rs has been lim ited by the inaccessibility of m ost tumors. B asa l c ell c arc in om as a re v ery c ommon , slowly g rowing, locally invasive tum ors of skin that have been studiedb y a uto ra dio gra ph ic te ch niq ue s u sin g tritia te d th ym id in ein vivo The reproductive cycle of the germinative cellpopulation w as analyzed. The D NA synthesis period w asfound to be 20 hr; the G ; period, 188 hr; the Gperiod,7 hr; the m itotic period, 1.5 hr; and the total germ inativecycle, approxim ately 217 hr. These results indicate thatthere is m ore cell proliferation activity in a basal cell carcinoma than suggested by the clinically apparent slowgrow th of the tumor. Therapeutic aspects of basal cellcarcinom a are discussed relating to chem otherapeuticdrugs.INTRODUCTION

In recent years the kinetics of cell proliferation in norm al and tum or tissues has been activ ely studied for patho-physiological and chem otherapeutic clues. A lm ost allth ese in ve stig atio ns h av e b ee n p erfo rm ed in e xp erim en ta lanim al tumor system s rather than in human tum ors, because of the difficulties in ob taining m ultiple tissue samplings (1). W ith thymidine- H as a DNA marker most ofthe studies in hum ans have provided only a single determ ination of th e labeling index , the p roportion of p rolifer-ative cells synthesizing D NA at 1 tim e (15). An analysiso f o th er c ell c yc le p arame te rs re qu ire s, h ow ev er, m ultip lespecim ens taken at different tim es after exposure to thym idine-1 H in vivo. In hum ans, the few studies of the complete cell cycle have been perform ed on the hem atopoi-etic system , gastrointestina l tract, neoplastic effusions,solid tum ors, and psoria sis (3 , 5, 7, 1 2, 14 , 18 . 20).BCCV are one of the most common cancers known tom an. W hile having a negligible rate of m ortality and m e-ta sta tic a ctiv ity , th ey n on eth ele ss a re p ro gre ss iv ely enla rging, locally invasive tum ors. W hen 1 or m ore cutaneoustum ors are present in an individual patient, m ultiple biopsy sam ples ca n be easily o btained fo r stud ies. T he tech-1This investigation w as supported by N IH Research G rants CA -10292 and AM-11559 and a grant from the John A . H artford F oundatio n. T he fac ilitie s o f the G en eral C linica l R esea rch C en ter su ppo rtedby N IH G rant FR 0261 w ere used.J T h e a bb re via ti on u se d is : BCC, b as al c ell c ar cin om a.R ec ei ve d J un e 1 0, 1 96 9; a cc ep te d Aug us t 1 1. 1 96 9.

niques used in this report hav e perm itted a detailed an alysis of cell proliferation kinetics of B CC in vivo w ith relativ ely sm all am ounts of thymidine- H (19).M TERI LS ND METHODS

Cell p ro li fe ra ti on k in et ic s a s s tudi ed by autor ad iogr aph icanalysis, w ith thym idine- H as a DNA label, has beenre viewe d in se ve ra l p ub lic atio ns (1 ,9 ). A da pta tio n o f th esem ethods to study cutaneous diseases has been describedin previous reports by us and is briefly review ed here (18,20).The 9 patients studied to determ ine the duration ofD NA synthesis had m ultiple nodular and/or superficialB CC that perm itted an average of 10 biopsy specim ens tobe obtained from each patient. Additional single specimens were taken from other patients to determ ine 1-hrlabeling indices. A ll patients w ere over 40 years old w ithclinically diag nosed and histolo gically p roven BCC s. Inthe first group of patients m ultiple sites of tum or w ere injected superficially with 0.1 m l (5 /iCi) thym idine- H(specific activity, 1.9 C i/mmole , S chw arz B ioR esea rch,Inc., Orangeburg, N . Y .) and the sites were carefullymarked. At serial intervals between 1 and 33 hr, 4-mmpunch biopsies w ere obtained, fixed in B ouin s solution,a nd p re pa re d h is to lo gic ally fo r a uto ra dio gra ph y. In je ctio nschedules were arranged to perm it all biopsies to betaken between 9 a.m . and 9 p.m . The slides were dippedin Kodak NTB-3 emulsion and exposed for 1 to 4 weeks.B lac k silve r grains from tritium emissions o ver nuclei indicate that those cells were synthesizing DNA (S phase)during the pulse labeling period of the thymidine-3Hinjection.F or a s ho rt in te rv al fo llowin g in je ctio n, o nly u nla be le dmitoses are seen within the tumor (Fig. 1). W ithin a fewhours, labeled m itoses appear, representing the m ovement of cells labeled previously in the S phase as theym igrate through the Gperiod into the m itotic period.F rom sequential specim ens the ratio of la beled m itoses tototal m itoses is determ ined and plotted in a classical labeled mitoses versus time after injection curve. The Sphase is m easured as the interval (in hr) betw een the 37points on the ascending and descending limbs of the labeled m itoses curve. The use of 37 intercepts w as suggested as a theoretical analysis by Quastler (10) whileother w orkers m ore c ommonly use 50 intercepts for them easurem ent of the S and G 2 p eriods. In the several studies we have perform ed on hum an cutaneous tissues, the

7 CANCER RESEARCH VOL. 30

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Cell P r olife ra tio n in BCC

Fig . 1 . a, B CC 1 hr after the intrale sio nal injection of thy midine -'H . Labe le d c ell s are s catte re d thro ug ho ut the tumor, b. hi ghe r magnification of similar specimen showing unlabeled mitoses.labe le d m ito sis c urv e ne ve r re ache s 100% pre sumably fo rtec hnical reaso ns as discussed below. The use o f the 3 7%intercepts therefo re has appeared to be a more reasonable level at w hich to determine the S periods for comparativ e purpo ses in human cutaneo us tis sues. The tim efrom the thymidine-'H injectio n until the appearance o fa significant number of labeled mitoses (defined as the37% point on the ascending limb) is the mean G> period.The g erm inativ e c ell c yc le duration (from mito sis to m itos is ) i s calculated as fo llows:

(A )

100-o80~>_Q

Dt0coO^

40oS-20:

/-x/T/I-M\: r / Nj^i0136 9 12 15 18 21 247Time In H ours Afte r Inje ctionfTritidted ThymidmeM0J\1^30 33

Ns = no. of cells in S pha se7, = duration of S phaseNgc = tota l no. of germina tive cellsTgc = dura tion of entir e germina tive cell cycleN,/Ngc = la beling index ( )

Chart 1 . Labe led m ito ses curv e to de te rmine the duratio ns of theDNA synthes is and G2 premi to ti c pe ri ods. The curv e i s drawn throughpoints ( ),each of which represents the mean of the percentage labeledm ito se s n patie nts . An av erag e o f 1 14 m ito se s was c ounte d at e ac h i nte rv al f or e ac h pati ent. V erti cal l ine s, 8 0% conf ide nc e l im its o f e ac hmean. O O. corrected data considering the thickness of sectionsas described in tex t.T, is obta ined fr om the la beled mitoses cur ve (C ha rt 1)whic h represents the cumulativ e data from 9 patients. Aunifo rm distributio n o f labeled cells in tumo r areas was aprerequi si te f or de te rmining the ratios quanti tative ly , l abeled cells to total cells and labeled mitoses to total mitoses. In some specimens all tumor areas did not showlabe le d c ells w ith unifo rm dis tribution. This has be en assumed to be a te chnical arti fac t f rom inadequate di stribu

tion of the labeled compound at the time of injection orthe bio psy being o btained slig htly o ff center from the inje ction s ite . An alte rnate e xplanation is that the re are is olate d are as o f tumor whic h c ontai n no ac tiv e c ell pro life ration, but this is doubtf ul s inc e m ito tic f igure s are pre se ntin all areas. Since the data reported here are taken fromare as s ele cte d fo r unifo rm labe ling the re sults may represent the max imum DNA-synthe si zing ac tivi ty w ithin the setumors , rather than the average activi ty .The number of germinative cells (N gc) i n the B CC w asobtained by counting the individual viable cells in enlarged photomicrographs o f sel ec ted we ll -l abe led i slandsof tumor. In each of these areas, the number of labeledcells (yvs) w as counted and related to the N gCto obtainthe labeling index (N,/Ngc). The labeling index wasmeasured in specimens taken from 18 patients 30 to 60m in afte r thymidine -'H inje ction (Table 1 ).

In the analysis of cell proliferation in the B CC, it hasbeen assumed that the viable cells of the tumor are a homogene ous pro life rating c ell population in a s te ady s tatew ith unifo rm phases in the cell c ycle. Ev idence fo r theseas sumptions and poss ibl e variations are di scus sed be low .Since the histo logical sections w ere cut 6 hick (human skin c anno t re liably be cut much thinne r) and tritium72 5



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G er ald D . We instein a nd P hillip F r ostTable lLa b el in g in de x i n BCC

Pat i ent ABCDEKGH1J KL MNOPQRAve r ageS. D. / Ve r m na t i v ec e l l s 1 8 6 9 1 9 3 0 1 2 1 7 1 4 9 5 1 8 3 4 3 3 9 0 1 3 65 1 2 2 1 1 3 5 9 5 4 1 9 6 7 0 4 1 9 0 2 7 4 9 1 5 1 3 23 6 5 1 6 8 5 1 8 4 5 1 0 9 0 N / N l a b e l i n gn d e x ) 5 . 0 5 . 1 6 . 9 13 . 2 7 . 2 6 . 9 12 . 8 9 . 6 8 . 6 8 . 7 11 . 7 5 . 6 11 . 0 10 . 3 5 . 9 8 . 2 16 . 8 11 . 5 9 . 2 3

Labe li ng i nde x c orre cte d f or thi ckne ss o f s ec tio ns by f ac to r o f 1 .4(see text) .' Superficial multicentric BCC's.emissions have a range of only 1.5 to 2.0 , a correctionfactor of 1.4 is used to compensate for visible nuclei co ntaining tritium that are to o far below the pho to graphicemulsio n for their emissions to produce grains (6). Thesame fac to r presumably co ntributes to the failure o f thelabe le d m ito se s c urv e to re ach 100%. Bec aus e s ome m itoses containing thymidine-' H are more than 2 belo wthe emulsion they are spurious ly re corded as unlabe ledmito ses. A c orrected curv e is also presented in Chart 1 . If50% intercepts are used for analysis on the correctedcurve, the S and G2 period values remain the same.The duration of mitosis (Tm) in B CC is obtained by relating the number of mitoses to the number of labeledcells in 1-hr specimens. Tm c an then be calculated by thefollowing equation:

TV .T, NmTm (B )Nm = No. of mitosesTm = dura tion of mitotic per iodR SU TS

The duration of the D NA synthesis period (7S) in B CCw as obtained from a curve of the mean percentage of labeled mitoses at each hour tested in 9 patients. A n averag e o f 1 14 mito ses w ere co unted on each bio psy specimenat each time interval. The S phase, measured by the distance betw een 3 7% po ints o n the labeled m ito ses curv e, is20 hr. A similar value is found on the corrected curvewhen using the 5 0% po ints, as is o ften use d in animal studies w here thinner sections can be obtained. The median

G2 period obtained from the curve is 7 hr. The labelinginde x de te rm ined from tumors i n 18patie nts is 9 .2 3 .2%co rrected (Table 1 ). When the B CC's are separated intosol itary nodular and superf ic ial , mul ti centri c type s, the irlabeling indices are 8 .4 2.9% and 1 0.6 3.7% respe ctive ly . The s li ghtly higher labe ling index in the superf ic ialB CC may be accounted for by a greater ease in penetration of thy midine-'H throug hout the smaller islands ofthes e tumo rs o r may reflect a small difference in the pro -life rativ e ac tiv ity o f the se 2 type s o f BCC .The duration o f the germinative cell cycle in the B CCcalculated from Equation A is 217 hr. Multiple microscopic fields of labeled tumors were examined in 5 pati ents to obtain Nm and TVSTable 2 ). The mito ti c durationis calculated to be 1.5 hr. The d period is determinedfrom the following equation:

Tgl = Tgc - (T, Tm) (C)The Gpe riod is approximate ly 188 hr fo r the av erag eB CC cell. The entire cell cy cle is summarized in Chart 2 .

DISCUSSIONThis report provides, to our know ledge, the most detai le d i nfo rmation available re garding the kine tic s o f c ell

Table 2Ra ti o o f m it os es to DNA- syn th es izi ng c el ls i n BCCPatientRPKSJAverage

S.D.N

mitoticcells2423152114N. labeledcells3073412372241777.6%

1.4N~/N.

(X 100%)7.86.76

zo

88 2 8 2 5 2 7H ours fro m e nd of previou s m ito sisChart 2 . A sche matic repres entatio n o f the ge rminativ e cell cy cleand component parts in the av erag e BCC cel l.

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Cell P r olife ra tio n in BCCproliferation of a human tumor. B CC's are very slow lygrowing , local ly invas ive tumors that rare ly metas tas iz e.Multiple tumors of the nodular or superficial types frequently o ccur, demons trating the multic entric o rig ins o fthis type o f cancer. They appear mo st frequently on theface and upper trunk w ith some relatio n to sun-ex po sedareas. BCC's also appear in the basal cell nevus syndrome, as a delayed response to arsenic intake, and inxeroderma pigmentosum, a disease in w hich an abnormality o f the DNA repair mechanism o ccurs (4 ).The utilizatio n o f small amounts o f iso to pe (5 iCi)o rintrale sional (i.d.) inje ctions has be en a us eful to ol fo r theinv estig atio n o f cutaneo us disease s (1 8). A comparablestudy o f cutaneo us tumo rs w ith thymidine-'H administered i.v . w ould require 200 times more isotope than required in the pres ent study (5 ). Thymidine-'H is co nsidered to be a physiological marker o f D NA sy nthesis andw e have not found evidence that the local injection technique inte rfe re s w ith normal pro li fe rativ e ac ti vity o f thecell po pulatio ns being studied. The similarity o f labele dm ito se s c urv es in 9 patie nts w ith BCC and the reproduc i-bility of curves in specimens from psoriatic patients (inw hich there is a much shorter S period) supports the validity o f the experimental technique (18). To obtain appro ximately 1 0 bio psie s fo r the S phase determinatio n ineach patient, several different tumors w ere usually required. A ll tumors in a subject w ere assumed to be similar with respect to cel l proli feration kinetics .In a s tudy in which 2 0 mCi thymidine-'H were injec tedi .v . into 2 pati ents w ith BCC, thereby e liminating poss ibl eeffects of trauma from the intralesional injection, an Sperio d duratio n o f 1 9 hr was fo und, co nfirm ing the present results.The analy sis o f tumor grow th mus t take into c ons ide ration the fo llowing fac to rs : (a) the durati on o f the c ell c yc leand its component parts in the pro liferativ e cell po pulation, (b) the proportion of proli ferating and nonprol iferat-ing cells (grow th fraction) w ithin the tumor, and (c) therate of cell death (13). In determination of the cell cycletime, the duratio n of the S period and the labeling indexare the primary facto rs ex amine d. While the labeling index is a function of the size of the proliferativ e cell pooland is, therefore, subject to less than perfect measurement, the S phase is more easily and reliably obtained.The duratio n o f the S phase in B CC is sig nificantly lo ng erthan in normal epidermis (about 1 6 hr, unpublished observations ) and psoriati c epidermis ( 8.5 hr) , ( 18 ). In o therhuman cancers, S values have been reported as 17 to 60hr f or ne oplas tic e ffus ions and 20 hr fo r le ukemic c ell populations (3, 12). The few other values available for Sphas e in normal human tis sue s are 13 to F4 hr fo r e ry thro -cyte and granulocyte precursors and 10 to 15 hr fo r gastrointestinal tract epithelium (7, 14). The data for theBCC provide furthe r e videnc e that in c ertain human cancers S phase may be longer than that of the tissue fromwhich the canc er o rig inates. This is co ntrary to o bserv ations in some animal tumor systems in w hich all parts ofthe cell cy cle are sho rte ned in the neo plastic cell po pulation c ompared to its no rmal c ounte rpart (2 , 1 1).

Unlike no rmal e pidermis, pso riatic epidermis, o r ich-thy otic epidermis , o r in squamous cell carcinoma, thereis li ttle e videnc e his to lo gic ally o f c ell diffe re ntiati on andmaturation wi thin the BCC and, there fore , the v iable c el lshav e usually been considered to be a pure population ofg erm inativ e c ells . Furthe rmore , thymidine -l abe le d c ellsand m ito tic figure s (1 6) are found dis tribute d in a randommanne r throughout the tumor mas s rathe r than c ompartmentalized in any reg io n o f the tumo r such as the periphery , where palisading cells are seen. We hav e, therefo re ,cons idered the BCC a re lative ly homogeneous populationo f pro li fe rative c el ls f or the purpose o f de te rmining i ts c el lproliferation kinetics.The results of this study show that the average cell ofthe BCC reproduces every 9 days. If, however, a BCCwere not a homogeneous population of cells but contained 2 or more c ell populations , i.e ., pro life rativ e c ells ,di ff erentiated cel ls , and dead cel ls , the true labe ling indexwould be g reater and the Gpo rtio n o f the g erminativ ecell cy cle w ould be pro po rtio nately sho rter, because o f adecrease in the size o f the g erm inativ e cell po pulatio n. A tpresent, it is no t y et po ssible to ascertain how homogeneous thi s c el l population i s, but further s tudies to de te rminea g rowth fractio n, as described by Mendelso hn (8 ), are inprogre ss . Obv ious de ad pykno tic c ells on his to lo gic al e xamination w ere not included in the labeling indices andthe que stionable pre se nc e o f diffe re ntiate d c ells c anno tbe eliminated w ith certainty. The data obtained are believed to be a reasonable estimate o f the cell cycle in theBCC with avai lable techniques.Clinical experience has show n that the B CC is an extremely slowly g rowing tumo r which may take months o reven years to double in size. This does not confo rm w iththe experimentally determined cell doubling time of 9days. The concept of cell loss must then be consideredas a significant factor in the net grow th of this tumor.Three hypotheses concerning cell loss should be con-

: , S 3 V - * -* >-v ?{;--; Fi g. 2 . H is to lo gi cal appe aranc e o f a BCC showing py kno ti c nuc le isuggestive of cell death.

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G er ald D . Weinstein a nd P hillip F r ostsiderea to account for the differences betw een the do ubling times of individual cells and the tumor itself (1 3).(a) The B CC is a superficial tumor and a sig nificant number o f c ells co uld be lo st by desquamatio n at the surface.In actuality, very little desquamation is seen and manytumors are c omple te ly surrounded by c onne cti ve tis sue ,thereby preventing cell loss at the skin surface, (b) Ametastasizing tumor w ould experience cell loss by thedissemination of cells in the body through the vascularand lymphatic channels, but in the BCC such an occurrence is rare, (c) W ithin a tumo r there is cell death whichmust occur at a rate almost equal to that of cell division,permitting o nly a v ery slig ht increase in cell numbe r andtumor size. Cell death is difficult to pro ve; in mo st histol gica samples there are some cells that appear to besmall in size w ith staining characteristics of py knoticnuclei o f dy ing cells (Fig . 2 ). This o bserv atio n canno t beproven or measured w ith reliability at this time, but itremains the most likely explanation to account for thedifference between the clinical doubling time of theB CC and the doubling tim e o f the indiv idual cells.The treatment o f B CC's has bee n restricted almo st entirely to the modalities of surgery and X-ray. In mostinstances, these forms of therapy provide excellent results. How ever, in the few situations of deep extensivetumor invas ion o r numerous supe rfic ial multic entric tumors systemic or topical chemotherapy would be adv antag eo us. One published repo rt rev eals the failure o flarge systemic doses of methotrexate given at w eeklyinte rvals to produc e a s atis fac to ry c linic al re spons e (17).B ased on the germinative cell cycle data in the presentreport, only 9% of cells in the tumor population in Sphase would be affected by the DNA poison, methotrexate, during the several hours of a significant bloodlevel attained from each w eekly injection. While themechanism by whic h metho tre xate achie ve s a bio lo gic alresult (the death o r decreas e in the number o f pro lifera-tive cells in a tumor) is not fully understood, the abovedrug schedule w ould not be expected to be effective because such a small percentage of the cell population isattacked by methotrexate. Even if the drug was completely effective in producing a lethal effect in this 9%group of cells, they w ould still be replaced several-foldw ithin 1 w eek. Mo re frequent exposure to chemother-apeutic agents that block D NA synthesis like methotrexate and 5-fluorouracil, on a daily basis by topical orsystemic administration, w ould theoretically be moreefficacious.REFERENCES

1 . B ase rg a, R. The Relationship o f the Ce ll Cy cle to Tum or Grow thand Contro l o f C el l D iv is io n: A Re view. Canc er R es ., 2 5: 5 81 -5 95 ,1965.

2 . B resc iani, F.: A Co mpariso n of the Ce ll Generativ e Cy cle in N orm al, Hy pe rplastic and N eo plastic M amm ary G land o f the C3 HMous e. In: C el lul ar Radi ati on B io lo gy . Univ ers ity o f Te xas , M . D .Ande rs on Hos pi tal and Tumor Ins ti tute at Hous to n, pp. 5 47 -5 57 .Bal timore : The Wil li ams & Wilkins Company, 1 964.3. Clarkson. B .. Ota. K.. Ohkita, T., and O'Conner A . Kinetics ofProliferation of Cancer Cells in N eoplastic Effusions in M an.Cance r, 1 8: 1 189-1213 . 1 965.4 . Cl eav er, J. E. D ef ec tiv e Re pai r R epl ic ati on o f DNA in Xero de rmaP igmento sum. Nature , 2 18 : 6 52 656 , 1 968.

5 . Frinde l. E., M alaise , E.. and Tubiana, M . Cell Pro liferatio n Kineti cs in F iv e Human So lid Tumors . Cance r, 2 2: 6 11 -6 20 . 1 968.6 . Iv ers en. O . H ., and Eve ns en, A . Expe rim ental Skin Carc ino ge ne -s is i n M ic e. A cta Patho l. M ic ro bio l. S cand., 1 56 (Suppl .): 9 7, 1 96 2.7 . L ipkin. M. Cel l Pro li fe rati on in the Gas tro in te sti nal Trac t o f Man .Fede rati on Pro c. , 2 4: 1 0 15. 1 965.8 . Mendelsohn. M. L. Autoradiographic Analys is o f Cel l Pro li ferationin S pontaneous B reast Cancer of C3H M ouse. III. The Grow thFrac ti on . J. Nati . Cance r Inst. , 2 8: 1 015 1029, 1 962.9. M ende ls ohn. M . L. The Kinetic s o f Tum or Cell Prolife ratio n. In:Ce ll ular Radi atio n B io lo gy . Uni ve rs ity o f Te xas , M . D . Ande rs onHos pital and Tumor Ins titute at Hous to n, pp. 4 98 5 13 . B al tim ore :The Wil li ams & Wilkins Company, 1 964.1 0. Quastler, H. The A naly sis o f Cell Po pulatio n Kinetic s. In: L. F.Lamerto n and R . J. M . Fry ( eds .) , C el l Pro li fe rati on, p. 1 8. Phi lade lphia: F. A . Dav is Company , 1 96 3.11. Reiskin, A . B ., and M endelsohn, M . L. A Comparison of the CellCyc le in Induce d Carc ino mas and Their N orm al Co unte rpart.C anc er R es .. 2 4: 1 13 1 1 13 6, 1 96 4.12. S aunders, E. F., Lampkin. B . C., and M auer, A . M . V ariation ofPro l iferative Activi ty in Leukemic Ce ll Populations o f Patients wi thA cute L eukemia. J. C li n. Inv es t., 4 6: 1 35 6- 13 63 , 1 96 7.13. Steel, G. G. Cell Loss as a Factor in the Grow th Rate of HumanTumors . European J. Cance r, 3 : 3 81 -3 87 , 1 967.14 . S try ckm ans. P., Cro nkite. E. P., Fache , J., Fliedne r, T. M ., andRamos, J. D eoxyribonucleic A cid S ynthesis Time of Erythro-po ie tic and Granul opo ie tic C ell s in Human Being s. N ature , 2 11 :

7 17 7 20 , 1 96 6.15. Titus, J. L., and S horter, R. G. Labeling of Human Tumors w ithTri ti ated Thymidine . Arch. Pathol . 7 9: 3 24 -3 28 , 1 965.1 6. V an Sco tt, E . J. D ef ini ti on o f Epide rmal Canc er. In: W . Monto ng aand W . C. Lo bitz (eds.). The Epide rmis, pp. 5 73 -5 86. N ew Y ork:Academic Press , 1964 .17. V an Scott, E. J., S haw , R. K., Crounse, R. G., and Condii, P. T.E ffe cts o f Metho tre xate o n Bas al Ce ll Carc inomas . A rc h. D erma-to l., 8 2: 7 62 -7 68 , 1 960.1 8. Weins te in, G ., and Fro st, P . Abnormal . Cel l Pro li fe rati on in Pso ri as is . J. Inve st. De rmato l., 5 0: 2 54 -2 59 , 1 968.1 9. W einstein, G., and Fro st, P. Cell Pro life ratio n Kine tics in B asalCel l Carc inoma (Abstract) . Clin. Res. , 6 :260, 1968 .2 0. Weins te in, G ., and Fro st, P. C ell Pro li fe rati on K ine tic s i n B eni gnand Mal ig nant Ski n D is eas es i n Humans . N ati. C anc er Ins t. Monog raph, 3 0: 2 25 -2 46 , 1 969.

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