Anaplasia in unilateral Wilms' tumor: A report from the National Wilms' Tumor Study Pathology Center

Anaplasia in Unilateral Wilms' Tumor: A Report From the National Wilms' Tumor Study Pathology Center CRAIG W, ZUPPAN, MD, J, BRUCE BECKWITH, ME), AND DENNIS W. LUCKEY,PHD Fifty-eight unilateral anaplastic Wilms' tumors entered on the Third National Wilms' Tumor Study (NWTS-3) were reviewed. The favorable outcome o f stage I anaplastic tumors was confirmed. In stage II-IV tumors, the extent and severity of anaplasia were not o f prognostic importance. However, two histologie features were correlated with extremely poor outcome; anaplasia in extrarenal tumor sites (P = .016) and predominantly blastemal tumor pattern (P -- .020). The importance of these features in predicting relapse and death for children with anaplastic Wilms' tumor was supported by the study of 47 additional anaplastic Wilms' tumor cases from NWTS-2. These results emphasize the importance of examining sites of extrarenal spread in anaplastic Wilms' tumors. Cytologic criteria for anaplasia and clues to its recognition are reviewed, and the potential clinical and biological implications of our findings are discussed. HuM PATHOL

19:1199--1209. 9 1988 by W.B. Sannders Company. A n a l y s i s o f t h e First N a t i o n a l W i l m s ' T u m o r S t u d y ( N W T S - 1 ) i d e n t i f i e d e x t r e m e n u c l e a r pleom o r p h i s m (anaplasia) o f t u m o r cells as the m o s t powe r f u l p r e d i c t o r o f p o o r p r o g n o s i s in Wilms' t u m o r s (Fig 1). 1 T h e overall survival f o r patients with anaplastic W i l m s ' t u m o r in t h a t s t u d y was a p p r o x i m a t e l y 50%, a f i g u r e t h a t has i m p r o v e d o n l y m o d e s t l y with c u r r e n t intensified therapies. Despite specific definitional criteria, t h e r e is a wide r a n g e o f variability in the e x t e n t a n d severity o f anaplasia a m o n g these tum o r s , a n d a diversity o f cell types are affected. T o ascertain whether specific cytohistologic features m i g h t d e l i n e a t e clinically distinct s u b g r o u p s , a n d to d e t e r m i n e w h e t h e r existing cytologic criteria f o r anaplasia s h o u l d be m o d i f i e d , we r e v i e w e d all anaplastic W i l m s ' t u m o r s r e g i s t e r e d in t h e T h i r d N a t i o n a l W i l m s ' T u m o r S t u d y ( N W T S - 3 ) . I n t h a t study, patients with a n a p l a s t i c W i l m s ' t u m o r w e r e t r e a t e d more aggressively than were patients with nonmetastatic W i l m s ' t u m o r lacking anaplasia.

From the Department of Pathology, The Children's Hospital, Denver; the Department of Pathology, Loma Linda (CA) University; the Department of Pathology, University of Colorado School of Medicine, Denver, and the Department of Pediatrics, the Children's Hospital Kempe Research Center, Denver. Accepted for publication February 22, 1988. Supported in part by grant CA 11722 from the United States Public Health Service, Key words: kidney tumor, Wilms' tumor, anaplasia, tumor cytology. Address correspondence and reprint requests to J. Bruce Beckwith, MD, Department of Pathology, The Children's Hospital, 1056 E 19th Ave, Denver, CO 80218. 9 by W.B. Saunders Company. 0046-8177/88/1910-0011 $5.00

MATERIALS AND METHODS O u r initial study population consisted of those patients entered on NWTS-3 f r o m its inception in May 1979, through May 1984, allowing for at least a 2-year follow-up. O f 1,700 entries in the randomized and followed categories during this period, 75 (4.5%) were diagnosed as having anaplastic Wilms' tumor, of which 64 were unilateral. O u r study was limited to unilateral tumors because bilateral (stage V) cases were not treated uniformly. At least two slides o f tumor in the NWTS Pathology Center were required for inclusion of a case in the present study. All slides were reviewed by two pathologists (C.W.Z. and J.B.B.), who were unaware of outcome. T h e presence of anaplasia was verified, and 13 additional clinical and pathologic variables were analyzed. Clinical information was obtained from summaries provided by the NWTS Data and Statistical Center and from institutional pathology reports. T h e definition of anaplasia presently used by the NWTS was the basis for inclusion of cases in this study (Table 1). After all criteria were met for a given case, the presence of only one was required when evaluating the distribution and extent of anaplastic change. All quantitative studies were conducted using a Zeiss standard 14 binocular microscope with 10• field eyepieces. T h e extent o f anaplasia was expressed in three ways: the number and proportion of tumor slides (those with at least 0.25 cm 2 o f t u m o r ) c o n t a i n i n g anaplastic ceils; whether more or fewer than 10% of high power fields showed anaplasia (the present criterion for distinguishing focal from diffuse anaplasia); and the n u m b e r of scanning (3.2 x objective) fields required to encompass all foci o f anaplasia. Severity of anaplasia was coded as mild in borderline cases that barely met the definitional criteria, typical in the more usual cases, and extreme when dense fields of profound cytologic atypia were present. Sites of anaplasia were coded as near the margin if within one scanning field of the t u m o r margin; extrarenal if within tumor infiltrating the renal sinus,* in metastatic tumor, or in tumor extensions beyond the renal capsule (including tumor thrombus in the renal vein or inferior vena cava); and intravascular if within intrarenal blood vessels. T u m o r pattern was recorded as blastemal, epithelial, or stromal when one of these elements comprised 65% or more of viable tumor sectioned. A mixed pattern was assigned when no single component predominated to that *For staging purposes, our current practice is to accept minor degrees of sinus invasion as still intrarenal (stage I). However, for evaluating sites of anaplasia in this study, we considered the renal sinus to be outside the kidney in view of its rich vascularity and minimal barrier to tumor spread.

1199

HUMAN PATHOLOGY

Volume '19,No. 10 [October 1988]

FIGURE I. Striking anaplasia is exhibited by this tumor, with an abnormal mitotic figure and clustered hyperchromatic, greatly enlarged nuclei. [Hematoxyiin-eosin stain; magnification x350.]

level. Likewise, the predominant cell type affected by anaplasia was recorded when 65% or more of the anaplastic cells were confined to the blastemal, epithelial, or stromal cell types, regardless of overall tumor pattern. Other histopathologic variables examined included the maximum density of anaplastic cells in a low power (10 x objective) field, the background nuclear size in regions of anaplasia (estimated by comparing with the diameter of adjacent RBCs), the maximum mitotic rate adjacent to anaplastic foci (averaging at least 3 high power [40 x objective] fields), and the ratio of multipolar mitotic figures to enlarged hyperchromatic nuclei. T u m o r stage was determined from histologic sections, in conjunction with institutional pathology reports, using NWTS-3 critera. 2 We devised a subjective grading system to quantitate the background nuclear enlargement, cytologic atypia, and histologic disarray manifested by anaplastic Wilms' tumor, which we will refer to as "unrest." In this scheme, grade 1 unrest reflects minimal disorder with nuclear diameters approximating those of RBC profiles (Fig 2), while grade 3 connotes striking background atypia and disarray just short of anaplasia (Fig 3). Grade 2 is intermediate. Following analysis of data from NWTS-3, we studied selected histologic features of anaplastic Wilms' tumors entered on the Second National Wilms' T u m o r Study (NWTS-2) in an attempt to validate or refute our preliminary findings. NWTS-2 differed from NWTS-3 in that most patients with anaplastic tumors were treated the same as patients with non-anaplastic tumors of comparable stage. 3 Of the 58 anaplastic Wilms' tumors entered on NWTS-2, 11 were excluded for reasons similar to those in Table 2. The remaining cases were staged according to NWTS-3 criteria, yielding 12 stage I tumors and 35 stage II-IV tumors. Follow-up of at least 1 year was available for

all relapse-free survivors. The results presented below refer to the NWTS-3 cases, unless specified otherwise. The Wilcoxon rank sum test was used to obtain twosided P values for all continuous variables. Categorical data were subjected to Chi square analysis. Multivariate analysis was performed using logistic regression techniques. 4

RESULTS Fifty-eight cases with follow-up i n f o r m a t i o n were c o n f i r m e d as h a v i n g unilateral anaplastic Wilms' tumors. E x c l u d e d cases are s u m m a r i z e d in T a b l e 2. As h a d b e e n previously n o t e d in N W T S - 2 , 5 patients with stage I t u m o r s did not s h a r e the p o o r o u t c o m e o f those with h i g h e r stage anaplastic t u m o r s . Only o n e o f 17 stage I patients in N W T S - 3 e x p e r i e n c e d relapse. T h e analyses to follow are t h e r e f o r e limited to the r e m a i n i n g stage I I - I V t u m o r s , unless specified otherwise. Follow-up r a n g e d f r o m 2 to 75 m o n t h s , with a m e a n o f 23 m o n t h s . All b u t t h r e e r e l a p s e - f r e e survivors h a d m o r e t h a n 2 years o f d o c u m e n t e d follow-up. T h e t h r e e exceptions w e r e alive a n d well at 6.5, 8.5, a n d 20.5 m o n t h s f r o m diagnosis. M e a n time to relapse was 5.5 m o n t h s , a n d all relapses o c c u r r e d within 12 m o n t h s o f diagnosis. T h e two survivors followed for less t h a n 12 m o n t h s w e r e e x c l u d e d f r o m final analyses relating to survival, e x c e p t in Fig 4, since K a p l a n - M e i e r logistics p e r m i t inclusion o f cases with short follow-up. Clinical a n d Histological Features

TABLE 1. Definition of Anaplasia in Wilms' Tumors

Markedly enlarged and usually multipolar mitotic figures. Threefold nuclear enlargement compared with adjacent nuclei of the same cell type. Hyperchromasia of the enlarged nuclei. (All 3 must be present.)

Patient age r a n g e d f r o m 11 to 138 m o n t h s , with a m e a n o f 62 m o n t h s . T w o thirds o f the anaplastic t u m o r s o c c u r r e d in females. M e a n t u m o r d i a m e t e r was 11.6 cm. T u m o r stage was almost equally divided b e t w e e n II, I I I , a n d IV. Most t u m o r s w e r e o f m i x e d histologic p a t t e r n

1200

ANAPLASTICWILMS' TUMOR [Zuppan et al}

FIGURE 2. Grade 1 unrest characterized by minimal cellular unrest and nuclear size approximating that of RBC profiles. [Hematoxylin-eosin stain; magnification x 350.]

(66%), followed by predominantly blastemal (22%), epithelial (10%), and stromal (2.5%) patterns. Grade 2 or 3 unrest was identified in 93% of cases. The blastemal component was affected by anaplasia in 83% of tumors, stromal elements in 78%, and epithelial elements in 54%. Stromat anaplasia was often the most visually arresting, since nuclei of stromal cells are usually widely separated and thus more conspicuous. The extent of anaplasia was extremely variable (Table 3). Cells exhibiting anaplasia were sometimes clustered, facilitating their recognition, but were more often widely scattered. Several differences emerged when the anaplastic tumors were contrasted with non-anaplastic Wilms' tumors of comparable stage (Table 4). The mean age

at diagnosis was higher (62 v 47 months), as observed in an earlier study. 6 Only one (2%) of the anaplastic tumors was diagnosed before age 2 years, compared with 17% of the non-anaplastic tumors. Also of note was the increased frequency of anaplasia in stage IV tumors (Table 5). Outcome

FIGURE 3. Lossof background orderliness and marked nuclear variabilily and enlargement just short of anaplasia characterize grade 3 unrest. [Hematoxylin-eosin stain; magnification x350,]

1201

Sixteeen of 41 stage II-IV patients with adequate follow-up information (44%) were alive without known tumor recurrence. O f the 17 patients who relapsed (including the one stage I case), 15 died. The two survivors were alive at two days and 27 months following relapse, respectively. Nine additional

HUMAN PATHOLOGY TABLE 2,

Volume 19, No. 10 [October 1988]

Study Cases a n d Exclusions

I00

64 original study cases - - 3 anaplasia not confirmed on available sections - - 1 not Wilms' tumor (neuroblastoma) - - 1 bilateral tumor - - 1 no follow-up available 58 final study group* Outcome of excluded cases: T h r e e alive, two dead, one unknown.

90 80 m 70 7.

deaths resulted from persistent tumor with failure to achieve complete remission. No deaths occurred in the absence of tumor progression. These figures reflect the dismal salvage rate for relapsed anaplastic Wilms' tumor and validate the use of relapse as an outcome endpoint. Relapses most frequently involved the lung (ten of 17 cases), followed by the abdomen and tumor bed (eight of 17) and the liver (two of 17). In 22 of 41 patients (54%), metastases to lung or lymph nodes were present at diagnosis. Thirteen of those (59%) died of tumor. Table 6 lists the histologic and clinical features lacking prognostic significance. O f particular interest was the lack of correlation between extent of anaplasia and outcome using the three quantitative schemes. Neither the severity of atypia nor the level of background unrest provided additional prognostic information beyond that conveyed by the presence of anaplasia. In agreement with previous analysis of NWTS2 data, 7 age and tumor size were not significant. Cases treated with regimen J did better than those treated with regimen DD-RT, although this difference did not reach statistical significance. The possibly increased effectiveness of regimen J is presently under investigation by the NWTS Committee. (Reginaen J was the addition of cyclophosphamide to the TABLE 3. Proportion of Tumor Slides Showing at Least One Marker of Anaplasia [NWTS-3, Stage I-IV] No. of Cases (%)

76%-100% 51%-75% 26%-50% 1%-25%*

24 6 19 9

(41) (10) (33) (16)

* Anaplasia limited to one of 21 and three of 36 tumor slides in two cases.

TABLE 4.

i

60

.i

[

i

i

Risk Factors Absent

g~ 5o

* Two of these patients who were disease-free with follow-up of less than 1 year are excluded from final analyses relating to survival.

Percent of Slides

I-L]

>

?_ g_ 3o 20

iI

i

Risk Factors Present lo t

, , , , 12

, , 24

, , 36

, 48

; , 6

; 7

Mean age at diagnosis Female patients Mean tumor size Metastases at diagnosis~" Stage IV tumors Survival

62 mo 66% 1 1 . 6 cm 52% 31% 41%

8

Months

FIGURE 4. Kaplan-Meier plot of relapse-free survival, Patients whose tumors manifest either a blastemal predominant pattern or anaplasia in extrarenal tumor extensions [lower line] did much worse than those whose tumors lacked both of these features [upper line]. Note the early occurrence of relapses in both groups, all within 12 months of diagnosis. [Tick marks indicate patients alive at last follow-up.] three chemotherapeutic agents used in regimen DDRT [actinomycin D, vincristine, and daunomycin].) Univariate analysis suggested that three histologic markers were of adverse prognostic significance (Table 7). Anaplasia in extrarenal tumor sites, either direct extensions or metastases, was associated with significantly worse outcome. Extrarenal anaplasia was encountered in three of 17 specimens with sections of nodal metastases, and in 11 of 32 cases with sections from non-metastatic extrarenal extensions (renal sinus, three; extracapsular, six; renal vein/IVC, three). One patient had anaplasia in both metastatic and local extrarenal sites. The specific extrarenal tumor site affected was not important (P = .677). This result was confirmed by review of cases from NWTS-2 (Table 8). T u m o r pattern was also significant. Patients with predominantly blastemal tumors had a lower relapsefree survival rate than those with other patterns (11% v 50%). This result was also confirmed in the NWTS2 series (Table 8). The predominant cell type expressing anaplasia, independent of overall tumor pattern, was of borderline significance, as shown in Table 7. Patients with anaplasia predominantly involving blastemal cells

Comparison of Stage II-iV Wilms' Tumors* Anaplastic

,

Non-Anaplastic 47 mo 55% 12.3 cm 39% 19% 91%

TABLE 5. Stage

Frequency of Anaplasia by Stage* No. of T u m o r s Showing Anaplasia (%)

I II III IV

* NWTS-3, first 1,700 entries. t Includes lymphogenous and hematogenous metastases.

* NWTS-3, first 1,700 entries.

1202

17/497 15/285 13/317 13/173

(3.4) (5.3) (4.1) (7.5)

TABLE 6.

Features Lacking Prognostic Significance in Stage II-IVAnaplastic Wilms' Tumor [NWTS-3] Relapsed/Dead

Relapse-Free Survivors (%)

20 3

13 (39) 3 (50)

5 2-14

4

Extent of anaplasia Percent of high power fields involved <10% />10% No. of slides involved Median Range Proportion of slides involved Median Range No. of scanning fields involved Median Range Severity of anaplasia Mild Typical Extreme Background nuclear diameter <1 RBC 1-1.5 RBCs 1.5-2 RBCs >2 RBCs Background cytologic unrest Maximal grade present

P Value .627 .291

1- 17

.104 .73 .27-1

.45 .05-1 .278

14 3~0

10 3-102 .772

1 19 3

1 (50) 14 (42) 1 (25)

1 13 7 2

0 10 4 2

.805 (0) (44) (36) (50) .208

1

2 3 Grade of diffuse unrest

0

2 (100)

16 7

9 (36) 5 (42)

9

9 (5O)

10 4

5 (33) 2 (33)

3

2 (40)

5 3

1 (17) 1 (25)

.573

1

2 3 Grade of unrest in metastases

.682

1

2 3 Maximum density of anaplastic cells (per 10x field)

.321

1

- -

- -

2-5 6-10

11 7

9 (45) 2 (22)

11-20

2

4 (67)

3

1 (25)

0

1 (100)

>20 Ratio of abnormal mitoses to hypertrophic nuclei <.1

.776

91-.3

.3-.7 .7-.9 >.9 T u m o r in intrarenal vessels Anaplasia present Anaplasia absent Anaplasia near tumor margin Present Absent Mitotic rate (per high power field)

2

1 (33)

12 7 2

7 (37) 5 (42) 2 (50)

0 13

1 (100) 6 (32)

.162 .312 19 4

11 (37) 5 (56)

1

4 (80)

10 12

7 (41) 5 (29)

10.5 7-20

12 6-18

56 11-138

65 24-100

6 9 8

7 (54) 4 (31) 5 (38)

17 4

8 (32) 7 (64)

.130

0-1

2-5 >5 T u m o r size (cm) Median Range Patient age (too) Median Range Stage II III IV Therapeutic regimen* DD-RT b jc

.329 .392 .476

.076

Abbreviations: DD-RT, vincristine, actinomycin D, Adriamycin, and radiotherapy; J, DD-RT plus cyclophosphamide. * One patient receiving regimen DD without radiotherapy, one receiving regimen K, and one in whom therapy was unknown are excluded from this table.

1203

HUMANPATHOLOGY Volume19, No. 10 [October 1988] TABLE7. Histologic Features of Prognostic Significance [MWTS-3] Relapsed/Dead

Relapse-Free Survivors (%)

11 10

2 (15) 13 (60)

8 0 1 14

1 (11) 4 (100) 0 (0) 11 (44)

8 3 5 7

2 (20) 5 (63) 8 (62) 1 (12)

Extrarenal tumor sites* Anaplasia present Anaplasia absent Tumor pattern Blastemal Epithelial Stromal Mixed Predominant cell type expressing anaplasia Blastemal Epithelial Stromal No predominance

P Value .016 .020

.040

* In three patients, extrarenal tumor sites were not histologically evaluable.

fared less well than those in whom anaplasia predominantly affected stromal or epithelial cells. Tumors in which anaplasia was prominent in more than one cell type also behaved aggressively. However, four of the eight cases with unfavorable outcomes in this latter group had other unfavorable features (anaplasia in an extrarenal tumor site or a predominantly blastemal tumor pattern). Analysis of cases from NWTS-2 confirmed the poor outcome associated with predominantly blastemal anaplasia, but failed to support an adverse prognostic significance for anaplasia affecting multiple cell types (Table 8). Multiple logistic regression analysis showed extrarenal anaplasia to be the most significant independent predictor of relapse. Blastemal tumor pattern provided additional prognostic information at a borderline level of statistical significance (P = .06). Blastemal predominance of anaplasia was not a significant independent predictor of relapse (P = .39). Therefore, extrarenal anaplasia and blastemal predominant tumor pattern would appear to be the principal histologic factors predictive of relapse in patients with anaplastic Wilms' tumor. Patients manifesting either or both of these features experienced a relapse-free survival rate of 17%, compared with 61% -if both were absent (P = .004; Fig 4). (The slightly higher survival rate when these features are considered together [17%] rather than separately [11% and

15%, respectively] reflects overlap of these categories among relapsed patients but not among survivors.) Data from NWTS-2 followed the trends seen in NWTS-3, and are shown in Tables 8 and 9. Eleven of 12 NWTS-2 patients with stage I tumors were relapse-free 2.4 to 9.6 years from diagnosis. Analysis of stage II-IV tumors showed the same three factors to be associated with relapse and death in this group as in the NWTS-3 cases (Table 8), although the number of survivors was too small to establish independent statistical significance, with the exception of extrarenal anaplasia (P = .017). The modestly better outcome for children with anaplastic tumors in NWTS-3 was largely due to increased survival rates for patients with stage IV disease (Table 9). DISCUSSION Cytologic Criteria for Anaplasia

The arbitrary definition of anaplasia used in the histologic analysis of cases from NWTS-1 was established before analyzing the results from that study, and was selected because it was consistently reproducible by of one of us (J.B.B.). Difficulties sometimes occur in the application of this definition; the following paragraphs are intended to help clarify these criteria for the pathologist in practice.

TABLE 8. Relapse Risk Factors~Comparison of NWTS-2 and NWTS-3 Relapse-Free Survival

Extrarenal anaplasia Present Absent Tumor pattern Blastemal Epithelial Stromal Mixed Predominant cell type expressing anaplasia Blastemal Epithelial Stromal No predominance

NWTS-2(%)

NWTS-3(%)

Combined(%)

0/11 (0) 6/15 (40)

2/13 (15) 13/23 (60)

2/24 (8) 19/38(50)

0/4 (0) 0/1 (0) 3/3 (100) 7/27 (26)

1/9 (11) 4/4 (100) 0/1 (0) 11/25 (44)

1/13 (8) 4/5 (80) 3/4 (75) 18/52(35)

1/9 (11) 0/2 (0) 5/11 (45) 4/13 (31)

1204

2/10 5/8 8/13 1/8

(20) (62) (62) (12)

3/19 5/10 13/24 5/21

(16) (50) (54) (24)

ANAPLASTICWILMS' TUMOR [Zuppan et al] TABLE 9.

Comparison of Relapse-Free Survival by Stage* Anaplastic Wilms' Tumor

Stage

I II III IV Totals II-IV I-IV

Favorable Histology Wilms' Tumor?

NWTS-2 (%)

NWTS-3 (%)

NWTS-3

]]/]2 5/8 4/14 1/13

(92)

16/17 (94)

(63) (29) (8)

7/13 (54) 4/13 (31) 5/13 (38)

93% 89% 80% 74%

10/35 (29) 21/47 (45)

16/39 (41) 32/56 (57)

83% 87%

* For uniformity, NWTS-3 staging criteria were used for all cases.

t Favorable histology Wilms' tumor as of July 1986.

To meet our requirement for nuclear enlargement, there must be at least a threefold enlargement of the nucleus in two perpendicular axes. This will avoid overinterpretation of simple nuclear elongation in one axis. When there is doubt as to the baseline from which the degree of nuclear enlargement is estimated, nearby erythrocytes may be used to approximate the size of a typical diploid Wilms' tumor nucleus. Care must be taken not to confuse multinucleation with macronucleation (Fig 5). This can be difficult when nuclei are overlapping. Poor fixation or staining, manipulation artifact, and overly thick sections greatly compound the problem. Neoplastic skeletal muscle frequently has multiple nuclei and may also express considerable nuclear atypia. These changes may be distinguished from true anaplasia by the lack of accompanying multipolar mitotic figures and the limitation of the atypia to mature or nearly mature skeletal muscle. Other artifacts that may mimic anaplastic nuclei include smeared masses of DNA (Fig 6), calcification,

stain precipitate, and basophilic extracellular mucin imitating enlarged nuclei. The enlarged nuclei of anaplasia are usually apparent u n d e r low magnification, but confirmation with higher power objectives is important in view of the many imitators of anaplasia. The criterion of nuclear hyperchromasia should be considered an expression of total nuclear chromatin content. Though most of the enlarged nuclei do show increased density of staining, increased chromatin content is also implied when a markedly increased nuclear volume is associated with normal density, and such nuclei may be accepted as meeting our criteria for anaplasia. On the other hand, enlarged nuclei with decreased chromatin density do not qualify as anaplastic. Multipolar mitotic figures (Fig 7) are the most fundamental and least ambiguous feature of anaplasia, and must be identified before this term can be applied with confidence. This finding implies either polyploidy or extreme hyperdiploidy, presumably near or above the triploid range. Large prophase c h r o m o s o m e spreads, resulting f r o m grossly increased chromosome number, are therefore equivalent to multipolar mitotic figures. Mitotic aberrations that do not denote a marked increase in chromosome content should be ignored. Problems may arise when evaluating tripolar or Y-shaped mitotic figures, as in Fig 8. This configuration can be produced by a diploid anaphase in which one edge of the metaphase plate separates before the rest. Similarly, adherence of chromosomes near the center while the edges are separating can produce an X-shaped mitotic configuration that might be mistaken for a tetrapolar mitotic figure. This error will not occur if one requires that each segment of the Y or X contains at least as much chromatin material as do adjacent, presumably diploid metaphase plates. For purposes of this study, we required that each

FIGURE 5. Multinucleation, as evidenced here in rhabdomyoblasts, must be distinguished from the real nuclear enlargement of anaplasfic tumors by confirmation under high dry magnification. [Hematoxylin-eosin stain; magnification x 350.]

t205

HUMAN PATHOLOGY

Volume 19, No. 10 [October 1988]

FIGURE 6. Smeared masses of DNA may mimic the enlarged nuclei of anaplasia, but can be distinguished by their lack of nuclear detail and lack of associated cytoplasm under higher power examination. [Hematoxylin-eosin stain; magnification x 350.]

case meet all criteria of the definition in Table 1. However, circumstances may dictate some liberalization of these criteria in practice. For example, when limited biopsy material is available, it seems reasonable that an unequivocal multipolar mitotic figure alone should be sufficient grounds to justify the diag.nosis of anaplasia, even when enlarged hyperchromlc nuclei cannot be found. This approach is supported by the relatively unambiguous character of multipolar mitotic figures and their nearly universal accompaniment by hypertrophic nuclei in adequately sampled material. Since nearly half of the tumors in

this study had evidence of anaplasia in at least 75% of tumor sections, it is reasonable to expect that anaplasia will often be detected in a limited biopsy. On rare occasions, we have accepted as anaplastic a tumor with multiple unequivocal muhipolar mitotic figures in which hyperchromic nuclei with threefold enlargement could not be found. Except in limited samples, a minimum of two multipolar mitotic figures should be required in the absence of obvious nuclear enlargement and hyperchromasia, since even in nonneoplastic cells a rare mitosis may be multipolar. Obtaining additional sections, or deeper sectioning of

FIGURE 7. Multipolar mitotic figures, the most fundamental feature of anaplasia, can exhibit varying forms, all of which are characterized by multiple metaphase axes. [Hematoxylin-eosin stain; magnification x800.]

1206

ANAPLASTICWILMS' TUMOR [-Zuppan et al)

FIGURE 8, Pseudomultipolar mitotic figures arising from incomplete metaphase "unzipping" without obvious increase in ploidy, Such mitotic figures should not be considered truly multipolar unless each arm contains at least as much chromatin material as adjacent normal-appearing metaphase plates. [Hematoxylin-eosin stain; magnification x800.]

available tissue blocks, will usually obviate the need to depart from traditional diagnostic criteria. C l u e s t o t h e R e c o g n i t i o n of A n a p l a s i a

Several features may facilitate the recognition of tumors that are likely or unlikely to harbor anaplasia. This change is extremely u n c o m m o n in tumors removed from patients less than 2 years of age. Conversely, the incidence of anaplasia is increased in Wilms' t u m o r in older children, women, non-white patients, and those with high-stage tumors. 6

T h e presence of moderate cytologic atypia (unrest) identifies tumors deserving careful study for anaplasia, although the difficulty of consistently recognizing this feature limits its usefulness. T h e large blastemal cell type depicted in Fig 9 is one pattern of unrest seen frequently in anaplastic Wilms' tumors. Extremely high mitotic rates, smudging of stromal nuclei, and a peculiar degenerative p h e n o m e n o n resulting in cytoplasmic hyaline eosinophilic globules resembling Russell bodies (Fig 10) are additional nonspecific features c o m m o n l y n o t e d in anaplastic Wilms' tumor. Anaplasia is often most obvious in stromal nuclei, perhaps because the paler background

FIGURE 9. [A] Large blastemal cells are frequently associated with anaplasia, [B) Diffuse small cell blastema. Anaplasia is seldom seen in tumors composed exclusively of the latter pattern. [Magnification x350.]

1207

HUMAN PATHOLOGY

Volume 19, No. 10 [October 1988]

FIGURE 10. Smearing of overlapping stromal cell nuclei [arrows] and eosinophilic cytoplasmic hyaline globules [arrowheads] are non-specific but seen with increased frequency in anaplastic Wilms' tumor.(Hematoxylin-eosin stain', magnification x 350.]

provides increased contrast, enhancing recognition of nuclear abnormalities. In contrast to the results from NWTS-1, x there was no strong predilection of anaplastic cells for metastatic tumor sites in this study, although their presence in these sites was prognostically important. One of the goals of this study was to establish the number of sections of Wilms' tumor that should be taken in order to detect or exclude the presence of anaplasia. With meticulous examination, nearly half the NWTS-3 cases exhibited one or more markers of anaplasia on at least 75% of tumor slides, and 84% of cases had anaplasia on more than 25% of tumor slides (Table 3). However, in nearly 20% of the cases, anaplasia was limited to three or fewer scanning fields. Our results suggest that the current NWTS recommendation (a minimum of one tumor section per centimeter of maximum tumor diameter) remains a reasonable guide for initial sampling, since this strategy would have detected anaplasia in 55 of 58 cases in this series. With multicentric tumors, each individual lesion should be sampled using the same guideline. Biologic I m p l i c a t i o n s

This study provides some insight into the biology of anaplastic Wilms' tumors. Anaplasia in stage I Wilms' tumor does not have particularly ominous connotations, in contrast to stage I rhabdoid tumor of kidney and clear cell sarcoma of kidney. While most stage I anaplastic tumors in our NWTS-3 population were treated with aggressive therapy, more recent stage I entries on NWTS-3 and all stage I tumors on NWTS-2 were treated with standard therapy for favorable histology Wilms' tumor, without apparent increase in relapse rates. It is presumed that stage I anaplastic Wilms' tumor does not share the propen-

sity for early micrometastases manifested by rhabdoid tumor of kidney and clear cell sarcoma of kidney. Are the monstrous cells that identify anaplastic Wilms' tumor responsible for the clinical aggressiveness, or might they be merely effete by-products of altered cellular biology? The bizarre appearance of anaplastic nuclei and the frequency with which degenerating multipolar mitoses are seen leads us to doubt that these monstrous cells are often capable of effective cell replication. We propose that the bizarre cells are markers of increased genetic mutability and multiple mitotic errors. I f these cells are merely markers rather than primary determinants of altered biology, their numbers might be of limited significance. This could account for the fact that none of the quantitative variables of this study were correlated with outcome. An increase in genetic lability co~nld facilitate the emergence of drug-resistant tumor clones in treated tumors. Support for the concept of genomic lability comes from a study by Douglass et al, s in which multiple complex chromosomal rearrangements, rather than hyperdiploidy, correlated most closely with treatment failure in children with anaplastic Wilms' tumor. If increased mutability is the basis for the resistance of anaplastic Wilms' tumor to therapy, it would seem reasonable to design new therapeutic approaches attempting maximum initial tumor kill before the emergence of resistance. Physical agents, such as ionizing radiation, and combinations of many agents with differing modes of action delivered over a short time period would seem to be reasonable approaches in attempting to improve survival rates of these patients. Also of possible relevance is recent work regarding the role of multiple drug resistance genes in h u m a n tumors, 9 since this mechanism involves the agents commonly used in the treatment of

1208

ANAPLASTICWILM~ 11JMOR [Zuppan et al]

Wilms' tumor. Therapeutic strategies directed against the resistance mechanism of multiple drug resistance genes might prove effective if these genes are overexpressed in anaplastic Wilms' tumors. The markers of extremely adverse outcome that we have identified in this study might be helpful in the preliminary testing of new approaches. Our discovery that blastemal predominant anaplastic tumors are associated with adverse outcome is of interest. Blastemal predominant Wilms' tumors of "favorable histology" tend to present as advanced stage lesions. This reflects the ability of blastemal ceils to infiltrate aggressively and metastasize early, perhaps because they are less cohesive than the more mature epithelial and stromal elements of Wilms' tumor. This inherent aggressiveness on the part of untreated blastemal cells, coupled with factors that enhance resistance to therapy, may well account for our findings.

Summary and Conclusions Our results reemphasize the importance of careful sampling and study of Wilms' tumors, since small foci of anaplasia appear to have the same significance as florid diffuse anaplastic change. Existing NWTS criteria for sampling (at least one tumor section per centimeter of maximum tumor diameter) would have detected anaplasia in 55 of 58 study cases. Our results also emphasize the importance of careful sampling of sites of tumor extension beyond the kidney, including the renal sinus. This study confirms the view that stage I anaplastic Wilms' tumors do not share the unfavorable outcome of higher stage lesions, and can be safely treated with favorable histology regimens. Therefore, it is critical to verify the staging of anaplastic Wilms' tumors that seem to be confined to the kidney. Finally,

we have identified histologic features that characterize a subset of anaplastic Wilms' tumors associated with extremely low likelihood of survival, defining an appropriate population on which to test new therapeutic approaches.

Acknowledgment. We are deeply indebted to the innumerable clinicians and pathologists whose collaboration through the National Wilms' Tumor Study has advanced our knowledge of Wilms' tumor to its present state and also provided the large groups of patients treated in relatively uniform fashion, allowing us to conduct this study. We also gratefully acknowledge the assistance of Barbara Nesmith from the NWTS Data and Statistical Center, who provided most of the statistics regarding patients with favorable histology Wilms' tumors during the period of this study. REFERENCES 1. BeckwithJB, Palmer NF: Histopathology and prognosis of Wilms' tumor. Results from the First National Wilms' T u m o r Study. Cancer 41:1937-1948, 1978 2. D'Arlgio GJ, Evans AE, Breslow NE, et al: Biology and management of Wilms' tumor, In Levine AS (ed): Cancer in the Young. New York, Masson, 1982, pp 633-662

3. D'Angio GJ, Evans A, Breslow N, et al: The treatment of Wilms' tumor: Results of the Second National Wilms' T u m o r Study. Cancer 47:2302-2311, 1981 4. Lachenbruch P: Discriminant Analysis. New York, Hafner, 1975, pp 27-29 5. Beckwith JB: Wilms' tumor and other renal tumors of childhood: An update. J Urol 136:320-324, 1986 6. Bonadio JF, Storer B, Norkool P, et al: Anaplastic Wilms' tumor: Clinical and pathologic studies. J Clin Oncol 3:513-520, 1985 7. Breslow N, Churchill G, Beckwith JB, et al: Prognosis for Wilms' tumor patients with nonmetastatic diseases at diagnosis-Results of the Second National Wilms' T u m o r Study. J Clin Oncol 3:521-531, 1985 8. Douglass EC, Look AT, Webber B, et al: Hyperdiploidy and chromosomal rearrangements define the anaplastic variant of

Wilms' tumor. J Clin Oncol 4:975-981, 1986 9. Pastan IP, Gottesman M: Multiple-drug resistance in human cancer. N Engl J Med 316:1388-1393, 1987

1209