Prognostic value of histopathology in advanced neuroblastoma: A report from the Childrens Cancer Study Group

Prognostic Value of Histopathology in Advanced Neuroblastoma: A Report From The Childrens Cancer Study Group JANE CHATrEN,MD, HIROYUKISHIMADA,MD, HARLANDN. SATHER,PHD, KWAN Y. WONG, MD, STUARTE. SIEGEL MD, AND G. DENMAN HAMMOND, MD We report the histopathologic findings in 420 patients with stage III and IV neuroblastoma enrolled in Childrens Cancer Study Group trials conducted from 1980 to 1983. A prospective study of individual cytohistologic features showed that outcome was related in a statistically significant manner to mitotic rate, multinuclearity, foam cells, ganglion cells, necrosis, and calcification, but only the latter was consistent for both stages. A similar test of four selected published classifications indicated the greatest prognostic value for the system developed by Shimada et al to distinguish favorable from unfavorable tumors. I This classification proved significant in both stages and on examination of both primary and metastatic sites. Concordance in histologic assignment of prognosis by two observers was 83%. We conclude that the Shimada classification is valid and reproducible, and that it may be useful in planning therapy in advanced neuroblastoma. Selected cytohistologic parameters and the other classifications were less strongly predictive of outcome, but are worthy of continued study. HUM PATHOL 19:1187--1198. 9 1988 by W.B. Saunders Company.

I n the course o f designing prospective t h e r a p e u tic trials o f c h i l d h o o d solid t u m o r s , the C h i l d r e n s C a n c e r Study G r o u p (CCSG) has included centralized p a t h o l o g y review f o r the p u r p o s e o f quality control. M a n y h a v e v o l u n t e e r e d as reviewers o f these trials, anticipating that the c o n v e r g e n c e o f histopathologic materials f r o m a large, consistently treated g r o u p o f patients with a single t u m o r type would inspire useful pathologic classification or insight into the natural history o f these diseases. T h i s r e p o r t describes the review e x p e r i e n c e with two r e c e n t n e u r o b l a s t o m a studies, testing a catalog o f cytohistologic f e a t u r e s a n d f o u r p u b l i s h e d classifications; Stout, 2 Beckwith a n d Martin, ~ Makinen, 4 a n d S h i m a d a et al.~ It confirms the value o f the S h i m a d a classification a n d ex-

MATERIALSAND METHODS

Patients Patients aged 0 to 18 years with neuroblastoma were entered in a series of CCSG studies from January 1980 to November 1983. Studies involved in this report are 373P and 373 for newly diagnosed stage III (88 evaluable) and 371P and 371 for newly diagnosed stage IV (332 evaluable) neuroblastoma patients. 5 These studies were designed to test the efficacy of multi-agent chemotherapy programs, to identify prognostic variables at diagnosis, and to evaluate the effect of radiation and chemotherapy on resectability. In general, survival did not differ significantly among the various treatment regimens, nor did it differ from previous trials by the CCSG and others. 6 The overall estimated survival rates of the patients in these studies were 60% (2-year) and 55% (3-year) for stage I I I disease, and 26% (2-year) and 18% (3-year) for stage IV disease. Prognostic variables other than histology have been identified in these studies (age, stage, serum neuron specific enolase, serum ferritin, N-myc oncogene copy number) and some of these have been reported elsewhere. 7-9

Specimens

From the Department of Pathology, Childrens Hospital of Philadelphia; the CCSG Pathology Center, Childrens Hospital of Columbus; the University of Southern California School of Medicine, Los Angeles; the Division of Hematology-Oncology, Childrens Hospital Medical Center, Cincinnati; and the Division of Hematology-Oncology, Childrens Hospital of Los Angeles. Accepted for publication February 22, 1988. Supported by grants from the Division of Cancer Treatment, National Cancer Institute, National Institutes of Health, Department of Health and Human Services. Key words: neuroblastoma and ganglioneuroblastoma, classification of mitotic rate in calcification. Address correspondence and reprint requests to the Childrens Cancer Study Group, 199 N Lake Ave, Third Floor, Pasadena, CA 91101. 9 1988 by W.B. Saunders Company.

0046-8177/88/1910-001055.00/0

tends its applicability to metastatic sites. It also indicates s o m e cytohistologic f e a t u r e s that either corres p o n d to a n d s u p p o r t that classification o r indicate o t h e r directions for study.

Requirements in each study included submission of a slide or slides considered representative by the contributing pathologist to a reviewer (J.C.) through the CCSG Pathology Center. Histologic materials for stage III patients were obtained before chemotherapy or radiotherapy. For stage IV patients, positive bone marrow aspirates or biopsies coupled with elevated urinary catecholamine levels were considered adequate for enrollment in the therapeutic studies, but not for histologic analyses. The first available nonmarrow samples in some stage IV cases were therefore obtained at variable times after initiation of treatment, whether from primary or metastatic sites. Histologic sections were retained in the repository unless their return was requested by the contributing pathologist at entry.

Histologic Review

Cytohistologic catalog study. A catalog of cytohistologic observations was constructed and applied by observer no. 1 (/.C.) to 61 stage III and 46 stage IV cases as they were entered on study. Items in the catalog described components of nuclear and cytoplasmic maturation, 4 mitotic ac-

1187

HUMAN PATHOLOGY S'[ROMA-R ICH

STROt'~-POOR

DISTRIBUTION OF Ii~"IATURE CELLS

i

Volume 19, No, 10 (October 1988)

AGE UNFAVORABLE

8~s,

ISOLATED

CLUMPED

,l tivity (above or below 1/high power field [hpf]), the presence of foam cells, pigmentation, multinuclear tumor cells, anaplasia, TM hemorrhage, necrosis, inflammation or calcification, and the architectural features perceived in the distribution of the cells in neuropil and stroma. Frequency of each item and its estimated prognostic effect on survival were analyzed. T h e architectural features proved too idiosyncratic for such analysis and are not reported. Classifications in the literature. Three published classifications were applied to each of the cataloged cases on enrollment. Stout's classification distinguishes between diffuse and composite tumors among ganglioneuroblastomas. 2 Beckwith and Martin establish grades 1 to 4 based on the degree o f histologic maturation? Makinen considers any degree of maturation evidence of favorable prognosis. 4 The survival rates of the subsets of these classifications and their prognostic importance were examined. The Shimada classification was published in 1984 and added to the study retrospectively. The sizes of the patient groups differ because this review soon supplanted the catalog study, and some cases differ because of availability of slides. Because of our positive results, and because our recent review experience indicates several areas of confusion, this classification is described and discussed in detail, diagrammed in Fig 1, and summarized in Table 1. According to this system, tumors are first divided into stroma-rich and stroma-poor categories. Extensive growth of Schwannian spindle cell stroma, imparting a neurofibromatous pattern, is the distinguishing feature of stroma-rich tumors, generally called ganglioneuroblastomas. This Schwannian stroma should not be confused with the usually scanty fibrous con-

TABLE 1. Shimada Classification System Favorable Histology Stroma-rich Stroma-poor Age < 18 m Age 18-60 m

Age > 5 y

Unfavorable Histology

Well-Differentiated Intermixed

Nodular

MKI < 200/5,000 MKI < 100/5,000 and differentiating None

MKI > 200/5,000 MKI > 100/5,000 or undifferentiated All

Abbreviation: MKI, mitotic-karyorrhectic index. 1188

18-60Mos, FIGURE 1.

Diagram of Shimada classification. Endpoints of favorable histology classes are underlined, Endpoints of unfavorable histology classes are in shaded box,

nective tissue stroma or with large mats of neuropil sometimes seen in stroma-poor tumors (Fig 2). A m o n g the stroma-rich tumors are two patterns of prognostic importance. The favorable pattern is diffuse, wherein immature cells are uniformly distributed, either in an isolated fashion ( " w e l l - d i f f e r e n t i a t e d " ) o r in m i c r o s c o p i c c l u s t e r s ("intermixed") t h r o u g h o u t the tumor. T h e unfavorable stroma-rich pattern is "nodular," wherein one macroscopic encapsulated focus (or very few foci) o f much less mature and stroma-poor tumor is seen in a stroma-rich Schwannian background. Stroma-rich tumors tend to occur in older patients, but age does not modify their prognostic classes. The stroma-poor tumors lack significant amounts of Schwannian stroma by routine light microscopy. They are grouped as favorable or unfavorable on the basis o f three variables: neuroblast differentiation, mitosis-karyorrhexis index (MKI), and age at diagnosis, as shown in Fig 1. Age is thus integrated into the classification of stroma-poor tumors rather than treated as an independent variable. Stated another way, there is a different prognostic classification for each of three age groups with stroma-poor tumors. The degree of differentiation is determined by the extent to which the neuroblasts have developed nuclear and cytoplasmic features of ganglion cells regardless of their elaboration of processes in the form of acellular "neuropil." T h e tumors are considered "differentiating" if at least 5% of the cells have a vesicular nucleus and a distinct rim o f cytoplasm (as in the Beckwith and Martin classification3). Thus, the "differentiating stroma-poor neuroblastoma" is much less m a t u r e t h a n the " w e l l - d i f f e r e n t i a t e d s t r o m a - r i c h ganglioneuroblastoma." The other histologic observation required to subclassify the stroma-poor tumors is the MKI. This index is the sum o f cells with mitotic figures plus those with malformed, lobulated, or minutely pyknotic nuclei plus those with overt individual cell necrosis per 5,000 cells examined in viable areas (Fig 3). Thus, karyorrhexis is used as a summary term for a variety of nuclear abnormalities indicating borderline viability or disturbed mitotic activity which may be difficult to distinguish histologically. MKI is defined as "high" if it exceeds 100/5,000 in patients over 18 months old or exceeds 200/5,000 in younger patients. To summarize, the Shimada classification recognizes f a v o r a b l e histology c o n s i s t i n g o f s t r o m a - r i c h , welldifferentiated and intermixed tumors, and the favorable stroma-poor subsets, and unfavorable histology consisting

NEUROBLASTOrV~ HISTOLOGYAND PROGNOSIS [Chatten et al]

FIGURE 2. Stroma [A] is composed of spindle cells in a deeply eosinophilic, wavy fibrous ma#ix. Neuropil [B] is acellular, pale-staining, and finely fibrillar or granular. [Hematoxylin-eosin stain', magnification xl00.] of stroma-rich n o d u l a r and the unfavorable stroma-poor subsets (Table 1). T h e original r e p o r t o f this classification 1 was based on histologic material from p r i m a r y lesions before chemotherapy and/or radiation therapy. In this study, however, we reviewed all cases in the repository without r e g a r d to site or t r e a t m e n t status at the time of sampling. Observer no. 1 (J.C.) classified 60 stage I I I tumors (all untreated; 56 primaries a n d four lymph node metastases) and 132 stage IV tumors (65 u n t r e a t e d primaries, 29 untreated metastatic sites, 31 treated p r i m a r y sites, and seven treated metastatic lymph nodes). T h e u n t r e a t e d metastatic lesions were 26 l y m p h nodes, two overt bone lesions, and one liver lesion with open biopsy. W h e n concurrent p r i m a r y and metastatic sites were available, classification by observer no. 1 was based on the p r i m a r y lesion only. T h e results were tabulated a n d the survival experience o f the two prognosis groups (favorable a n d unfavorable histology) were analyzed. Reproducibility of the Shimada classification. Since observer no. 2 (H.Sh.) had m a d e all the histologic observations in the original report,~ a n d observer no. 1 m a d e all those in the present study, it was possible to test observer variation. In the final phase, therefore, observer no. 2 i n d e p e n d e n t l y classified 128 o f the stage IV cases which had been classified by observer no. 1 and r e t u r n e d to the Columbus repository. T h e concordance between the two observers was analyzed.

Statistical M e t h o d s Life table analyses were used to examine outcome in the various subgroups. T h e Kaplan-Meier estimate 1~ was used in these calculations. C o m p a r i s o n of outcome between subgroups was done with the log r a n k statistic for life table data. 12a3 In some comparisons in which m o r e than two groups were c o m p a r e d and a specific o r d e r i n g o f the subgroups with respect to outcome was hypothesized, the log rank test for linear t r e n d was used. ~3,~4 Stratified log rank tests x3,~4 were used to test for overall effect o f a candidate factor after adjustment within the stage subgroups. This a p p r o a c h was also used to adjust for the effect o f age in the Shimada classification system. All the life table analyses used survival time as the disease e n d p o i n t o f interest. T h e use of other endpoints such as progression-free survival in advanced stage neuroblastoma typically give very similar results for comparison o f subgroups, so only the survival data are p r o v i d e d in this report. T h e m e d i a n follow-up periods for the surviving patients are 43 months for stage I I I and 48 months for stage IV. Although there were differences in t r e a t m e n t received within stage I I I and stage IV subgroups, survival outcome within stage is very similar for each treatment. Thus, all treatments within stage have been combined for r e p o r t i n g purposes. Life table analyses were p e r f o r m e d for the 228 en-

1189

HUMAN PATHOLOGY

Volume 19, No. 10 [October 1988]

FIGURE 3. This photomicrograph compares siToma-poor tumors. [A] Undifferentiated with a low MKI. [B] Differentiating with a high MKI. [Hematoxylin-eosin stain; magnification x200.]

rolled patients who did not have histologic material analyzed in this study. The outcome for each stage was very similar to those with pathologic review. This suggests that there was no selection bias related to outcome involving patients available for this report.

(P = .03) for multinuclearity. All five patients with foam cells in their tumors died after relatively short follow-up (1, 7, 16, 16, and 17 months), giving a significant result for this factor when both stages are combined, but the small number of patients involved make this result more tenuous than the previous

RESULTS OF HISTOLOGIC REVIEW

ones.

Three factors gave significant results in stage III disease, but showed no prognostic effect in stage IV patients. Those factors were mitoses, necrosis, and ganglion cells. Presence of the first two factors imparted a poor prognosis in stage III patients, while the third suggested a good prognosis.

Cytohistologic Catalog Study Results of the application of selected catalog items in 107 cases are enumerated in Table 2. Although various items showed statistically significant effects on survival, the effects were not always consistent in both stages III and IV. The factor showing the greatest and most consistent effect for both stages was calcification (stage III, P = .004 and stage IV, P = .04; Fig 4). Presence of calcification was associated with a favorable prognosis and the combined stage adjusted significance of this factor showed a 2.76 time s greater mortality rate in those patients without calcification (P = .0005). Presence of multinuclearity suggested some benefit for survival in both stages, but this was at only a borderline significant level in stage III (P = .09) and a nonsignificant level in stage IV (P = .20). However, combining both groups in a stagestratified analysis did give an overall significant result

Classifications in the Literature The results of applying the three older published classifications to these cases showed some interesting results (Table 3). Although Stout's diffuse v composite subdivisions 2 proved of no prognostic significance, both Beckwith and Martin's grading ~ and Makinen's 4 differentiation classification seemed to distinguish prognostic subgroups significantly in stage III (P = .0002 and .02, respectively). T h e Makinen system failed to show much prognostic effect for stage IV disease (P = . 16), while the Beckwith and Martin system did (P = .05) (Fig 5). Parenthetically, it should be

1190

NEUROBLASTOM/kHISTOLOGYAND PROGNOSIS[Chatten et all

TABLE 2. Resultsof Catalog Study Category Nuclei Differentiation None Some Mature Variable Mitoses over 1/hpf Anaplasia present Multinuclearity present Cytoplasm Quantity None Tags Circumferential Variable Foam ceils Pigmented cells Ganglion cells present Necrosis present Calcification present

Stage I I I Frequency*

25% 42% 8% 26% 61% 11% 44% 26% 49% 13 % 12% 3% 0% 25% 45% 30%

Stage IV Frequency

( N = 61)

P Valuet

( N = 46) P Valuer

30% 48% 15% 7% 72% 26% 43%

.75 .0011 .6-F .092 --

Combined Comparisons

.34

.58

.68 .76 .20

.041 .5---8 .032 -

24% 50% 11% 15% 7% 2% 39% 48% 37%

.19 .26 -.0092 .0--~.0-042

-

.84

,59

.081 .9---5 .65 ,69 .042

.04 a .95 .15 ,30 .00052

* Proportion of each category found in the reviewed cases. t P value examining survival outcome for prognostic significance (underlined when statistically significant or marginally significant). aPresence of the item tends to unfavorable outcome. 2presence of the item tends to favorable outcome, $ Combined stage III and IV survival outcome using stratified analysis. P value represents the "independent" prognostic effect of the factor after adjusting for any prognostic effects due to stage of disease.

noted that markedly undifferentiated tumors or "small round cell tumors," corresponding to the authors' interpretation of Beckwith and Martin grade IV, occurred in only 13 of the 107 tumors graded.

1.00

3.75

Shimada Classification

Prognostic value. Figure 6 displays the Shimada classification and outcome of each patient by age categories, excluding 11 surviving patients with followup less than 3 years. Both stages and all sites are included. The better survival of younger patients and of favorable histologic types is evident and further analyzed below. The samples from previously treated patients (indicated by triangles) show a higher proportion of stroma-rich tumors (50% v 26%) and a higher proportion of deaths in favorable histologic categories (65% v 38%) than the untreated tumors. Statistical analysis of the data is presented in Table 4, and actuarial survival data is presented in Fig 7. Sixty stage III tumors demonstrated a highly significant difference in survival based on histologic class (P = .0001). The estimated 3-year survival rates for favorable and unfavorable histology patients are 85% and 33%, respectively (Fig 7A). Included in this group are four stage III cases classified on the basis of metastatic lymph nodes, of which the patient with favorable histology is alive at 35 months, while the three patients classified as having unfavorable histology died 6, 12, and 13 months after diagnosis. The analysis for stage IV cases also showed a significant difference between the two prognosis groups (P = .0001; Fig 7B). Three-year survival rates are estimated to be 46% for favorable histology and 13 % for unfavorable histology. This prognostic effect was stronger for the 29 patients with tumor samples 1191

~ "

Survival

-

Stage IiI

L~Calcification Present (N=I8)

-(4) Calcification Absent

~

9.50 l

(6)

0.25 0.00 f~

1.00

gO Sw.,,~u=Z

4~ -

sO , r ~ e .

S~g# IV

o. 75 Icification Present (N=IO) 9.50

(4) Calcification Absent (N=35)

0.25

(3)

0.00

FIGURE 4. Survival from diagnosis by presence or absence of calcification. [Parentheses at end of life table curves indicate the number of patients surviving more than 60 months.] [A] Stage III patients. [B] Stage IV patients.

HUMAN PATHOLOGY TABLE 3.

Volume 19, No. 10 [October 1988]

Comparison of Classifications in the Literature

Stage IlI

Stout Diffuse Composite Unclassified Beckwith and Martin Grade I

No. of Patients

Survival* (%)

33 5 23

56 t 80 )

4

100 "l

II III IV Ungraded Makinen Differentiating Undifferentiating Not evaluated

8 30 10 9

62 I 48 0

31 28 2

65 I 32

Total

61

Stage IV P Valuer

.27

No. of Patients

Survival* (%)

34 8 4

27 t 25 )

5oI

4

.0002 (trend)

.02 --

9 19 3 11

56 16 0

28 16 2

30 t 19 )

Combined Comparison;

P Valuet

.73

.37

.05 (trend)

<,0001 (trend)

.16

.009 1

46

* Life table estimated proportion of patients surviving 3 years from diagnosis. t P value for life table comparison (log rank test) of survival outcome across the entire time period of patient follow-up. A test for ordered trend was used for the Beckwith and Martin classification. :~Combined stage III and IV survival outcome using stratified analysis. P value represents the "independent" prognostic effect of the factor after adjusting for any prognostic effects due to stage of disease.

before t r e a t m e n t f r o m sites other than the primary (P = .0007) a n d the 33 patients with treated primaries (P = .02) t h a n it was for the 65 patients with untreated primary tumors (P = .05). No significant difference was f o u n d a m o n g the seven stage IV treated metastatic lesions, but the small n u m b e r of patients precludes a definitive evaluation of that group. Since this classification system uses different criteria d e p e n d i n g on the age of the patient, a stratified life table analysis was used within each stage, stratifying for those patients who were older and y o u n g e r than 18 m o n t h s of age at diagnosis. T h e significance levels were only slightly attenuated as compared with the previously m e n t i o n e d within stage results (stage I I I changes f r o m P = .0001 to P = .0007; stage IV changes f r o m P < .001 to P = .008). These data show that the prognostic information f r o m this classification is relatively i n d e p e n d e n t of the prognostic effect o f age alone, confirming the impression gained f r o m Fig 6. Observer variation. T h e r e were 128 stage IV tumors classified by both observers and examined for concordance. A g r e e m e n t as to favorable or unfavorable histology was f o u n d in 106 cases (83%). T h e r e were differences in detail in 44 cases. Since a given histologic discrepancy may or may not change the prognosis d e p e n d i n g on the patient's age, only 22 of the 44 discrepancies led to a change in prognostic class f r o m favorable to unfavorable histology or vice versa, with a net change of ten more unfavorable histology cases for observer no. 2. T h e major discrepancies are shown in Fig 8. T h e most c o m m o n change was low to high (16 cases) or high to low (ten cases) MKI a m o n g stroma-poor tumors. Discrepancies reg.arding stroma richness or poorness occurred six txmes, differentiation discrepancies occurred seven

I. 00

Su.r..~v:Z -Stafe

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(2)

Grade I (N:4)

o. 7s Grade II (N:B)

. (2)

Grade III (N:30)

o. 50 0.2s

I GradeIV (N=IO)

I

0.0o

3O

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GradeII (N=9)

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(2)

~. 00 FIGURE 5. Survival from diagnosis by Beck'with and Martin grading. [Parentheses at end of life table curves indicate the number of patients surviving more than 60 months.] [A) Stage III patients. [[3] Stage IV patients.

1192

NEUROBLASTOIV~HISTOLOGYAND PROGNOSIS[Chatten etal]

FAVORABLE STROMA-RICH (Well-dlff + Intermixed)

DIFFERENTIATING,

FIGURE 6. Survival by age and Shimada classification for each patient. Patients treated before tissue examination are indicated by triangles. The bold line separates favorable from unfavorable tumors. 0 + V = 3-year survival; 9 + 9

death.

=

V

LOW MKI

7

V

V

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UNFAVORABLE STRO~'~A-RICH (Nodular) age

at

diagnosis

(months):

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o

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Shimada Classification v Cytohistologic Catalog Since mitotic rate in stage III and calcification in both stages p r o v e d to be significantly related to survival, we e x a m i n e d their relationship to the Shimada classification. T h e cataloged cases were only reviewed d u r i n g the early part o f the study, with emphasis on stage III cases. Hence, only 73 t u m o r s had data on both catalog and Shimada classification, with correspondingly few cases in each group. No f o r m a l statistical study a p p e a r e d warranted, but a p p r o p r i a t e trends are shown. M i t o t i c rate v M K I . Table 5 c o m p a r e d mitotic rate in 40 stage III s t r o m a - p o o r tumors to MKI in the same tumors. T h e large n u m b e r o f intermediate mitotic r a t e t u m o r s b e s p e a k s t h e n o n q u a n t i t a t i v e m e t h o d o f estimation used. In the small n u m b e r o f cases with mitotic rates clearly less than or m o r e than 1, the survival rates are 45% and 12%, respectively. T h e rates for low and high MKI alone are not different at 50% and 12%. W h e n both values are low or

o

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e o e 7u

V

60

18

times, a n d seven diagnoses were changed f r o m favorable to unfavorable s t r o m a r i c h (ie, w e l l d i f f e r e n t i a t e d or i n t e r m i x e d to nodular).

9

high, the rates change to 55% and 7%, respectively. T h e differences are small b u t the t r e n d is a p p r o p r i ate. Table 6 c o m p a r e s the Shimada classification and survival in 31 calcified a n d 60 uncalcified tumors. T h e r e is a relatively high p r o p o r t i o n o f stroma-rich and differentiated histology a m o n g calcified tumors, p e r h a p s because calcification is easier to recognize in the pale n e u r o p i l or stromal b a c k g r o u n d , and perhaps because calcification is a reflection o f t u m o r aging r a t h e r t h a n a response to necrosis. As with mitotic rates, calcification enhances the predictive value o f the Shimada classification. Alone, the latter gives survivals for favorable and u n f a v o r a b l e histology o f 66% and 24% in this sample. T h e presence or absence o f calcification alone gives the same survivals (61% and 25%, respectively). Calcified favorable histology a n d uncalcified u n f a v o r a b l e histology t u m o r s a m p l i f y those differences to 73% a n d 15%, respectively.

DISCUSSION This r e p o r t is based o n histopathologic review o f 192 patients with stage III and IV neuroblastic tu-

TABLE 4. Shimada Classification Results Prognostic Group Unfavorable Histology

Favorable Histology

Sample

n

Survival * (%)

n

Stage III Untreated 1~ Untreated lymph node

19 1

(100)

37 3

(0)

20

85

40

33

.0001

15 7 1 0 14 4 41

45 83 (0)

50 19 0 2 17 3 91

"8 7

.05

Total Stage IV Untreated 1~ Untreated lymph node Untreated Liver Untreated Bone Treated 1~ Treated lymph node Total

34 25 46

* Life table estimated survival 3 years from diagnosis. t P value for trend of survival.

1193

Survival* (%)

Pt

.0007 (0) 0 33 13

.02 .0001

HUMAN PATHOLOGY 1.00

Volume 19, No. 10 [October 1988]

Su.r,~uaZ - St.ag~ IZI

H

1

Favorable (N:20)

(6)

O. 75

O. 50

~,~iUnfavorable (N:40) s

O. g5

t4)

0.00 0

f5

~o

4~

sb ~ 8 .

1. O0

O. 75

avorable (N=41)

O. 50

(I0)

0.25

nfavorable (fl:91)

(6)

&O0

FIGURE 7. Survival from diagnosis by Shimada classification system. [Parentheses at end of life table curves indicate the number of patients surviving in excess of 60 months,] [A] Stage III patients. [B] Stage IV patients.

mors e n t e r e d in CCSG studies. T h e quality and amount of pathologic material available were entirely dependent on the referring institutional pathologists, who were given no guidelines for their submissions. It is noteworthy that only two of over 400 cases reviewed were rejected for incorrect diagnosis. This indicates that confirmatory diagnostic review is not es-

sential for monitoring therapeutic studies of neuroblastoma. However, histopathologic investigations that might contribute to the understanding of the biology of this group of tumors are expedited by this referral system, and we have therefore explored histologic analyses. Several systematic studies 13-15 evaluating individual cytologic and histologic features of the neuroblastic tumors have reported variable success. ~-5'i5-17 T h e r e is no histologic classification in general use, and even the basic distinctions between neuroblastoma, ganglioneuroblastoma, and ganglioneuroma are poorly defined. 18 Therefore, the pathologic consensus seems to be that this disease is enigmatic, unpredictable, and difficult to classify. Our catalog study results generally support this impression, with the exception of the highly significant correlation of calcification with survival. We are not aware of this correlation having been made previously, either in the pathology or radiology literature. Furthermore, it appears to contradict the negative correlation of necrosis with survival, since calcification is usually assumed to follow necrosis. This paradox requires clarification by a more detailed and quantitative study of both features. Other catalog items with weaker correlations indicate trends in appropriate directions, and these merit further study as well. Among older classifications, Stout's system did not correlate with the clinical outcome. 2 Uncertainty about the precise definition of Stout's categories and the limited number of ganglioneuroblastomas among these high stage patients restricted the use of this classification to relatively few cases. The relationship of Stout's composite tumor to Shimada's nodular tumor is discussed below. The grading system of Beckwith and Martin distinguishes four grades with three prognosis groups: excellent (grade 1), intermediate (grades 2 and 3), and poor (grade 4)? However, we found that the grouping of grades appeared different in the two stages studied (Fig 5), blurring the prognostic distinctions for practical purposes. Makinen's system successfully separated two prognosis groups only in stage III. The feature common to the Beckwith and Martin, Makinen, and Shimada classifications is an estimate of differentiation. Therefore,

UNFAVORABLE STROMA-RICH FAVORABLE STROMA-RICH LOW MKI, DIFF OR UNDIFF

HIGH MKI, DIFF OR UNDIFF

ZZ... !!i!i!i!:: ::::::::::

FIGURE 8. Concordance between observer no. 1 and observer no. 2 in 128 Stage IV cases. @, Agreement in histologic details. O-*, Disagreement, with observer no. 2 at arrowhead. Histologic diagnoses have been grouped for clarity. Five additional disagreements in interpretation of differentiation did not alter prognostic class and are excluded from this display.

,LTTTTTTTITTTTTIIT[[IIIII[II

llltttlllILL1JtItiiiliillil

9149149149149149 m 9149149

9 = agreement (89 cases)

t or f = disagreement (39 cases), Observer 2 at

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NEUROBLASTOM~ HISTOLOGYAND PROGNOSIS[Chatten et all TABLE 5.

Shimada MKI v Mitotic Rate, Stage III Equal to 1

Under 1

Over l

Total

Mitoses/hpf

n

(%)

n

(%)

n

(%)

n

(%)

Low MKI High MKI Total

9 2 11

(55) (0) (45)

4 8 12

(50) (25) (33)

3 14 17

(33) (7) (12)

16 24 40

(50) (12) (27)

Definitions: n, number of patients in group; %, survival at 3-year follow-up.

and have no age-related subsets. The unfavorable subtype of stroma-rich tumor is characterized by a single nodule of lesser maturity, considered to be a clonal malignant growth that has broken free from an a p p r o p r i a t e maturation sequence. On the o t h e r hand, the unfavorable stroma-poor classes may be looked on as a delay in the timing of maturation throughout the tumor. In this study, we tested the Shimada classification not only in primary but also in metastatic sites, and also tested the reliability of classifying treated material in some stage IV tumors obtained at delayed firstlook surgery. The results suggest that lymph nodes in stage III and samples from metastatic lesions in stage IV show a very significant survival advantage for favorable histology as compared with unfavorable histology patients. The fact that the evaluation of the metastasis is more predictive than that of primary seems reasonable since the metastatic lesion is expected to represent the most aggressive portion of the tumor. We noted also an absence of stroma-rich morphology in metastatic sites, suggesting that the stroma lacks metastatic potential. Since sampling of the primary tumor of stage IV cases is usually limited, leading to misinterpretation of focal finding.s, it will be useful to be able to rely on distant biopsxes. Although analysis of the after-treatment histology in stage IV tumors shows a significant difference in survival between the two prognosis groups, survival for favorable histology is quite low. Therefore, treatment (chemotherapy and/or radiotherapy) before sampling may cause tumors to appear more favorable histologically without altering their ultimate outcome. Conversely, a tumor that remains in the unfavorable histology category after treatment may be demonstrating its lack of responsiveness to such treat-

the prognostic strength of each classification is at least partly dependent on the definition of differentiation used by each author, the number of categories it creates, and on the ability of each observer to apply the definition. The survival correlations found, especially for Beckwith and Martin grading, are impressive and correspond to the original authors' results. Studies of these classifications are ongoing. The Shimada classification, which became available during these reviews, proved to be useful and reproducible. It is based on a few, relatively easily defined and recognized morphologic features, and has the novel addition of patient age in the creation of prognostic subsets. The basis for this is the concept of an orderly maturation sequence for neuroblastic tissue, with any deviation from the maturation program representing an unfavorable biologic event. The sequence for neuroblastic tumors consists of the differentiation of the neuroblasts toward both ganglion cells and Schwann cells. In infancy, maturation of either element is not expected, and the only unfavorable histologic finding in that age group is therefore a high MKI. After infancy (empirically 18 months in the original study), neuronal differentiation should have begun, and undifferentiated tumors beyond that age are therefore unfavorable. The allowable frequency of nuclear abnormalities also decreases from 200/5,000 to 100/5,000, paralleling the slowing of the host's growth rate. Schwann cell development is slower to appear but is obligatory in this schema by the time the patient reaches 5 years of age. Hence, all tumors that remain stroma-poor in patients over 5 years of age are unfavorable, regardless of neuroblastic differentiation or MKI. Stroma-rich tumors, which show more advanced maturation toward ganglioneuroma, generally occur only among the older patients, TABLE 6.

Stage III Shimada classification Favorable, stroma-rich Unfavorable, stroma-rich Differentiating, low MKI Undifferentiated, low MKI Differentiating, high MKI Undifferentiated, high MKI Survival Favorable histology Unfavorable histology Total

Calcification v Shimada Classification Calcified (31)

Uncalcified (60)

14

34

7 3 6 7 1 7

4 4 13 12 4 23

11/15 (73%) 8/16 (50%) 19/31 (61%)

8/14 (57%) 7/46 (15%) 15/60 (25%)

1195

Total

19/29 (66%) 15/62 (24%)

HUMAN PATHOLOGY

Volume 19, No. 10 [October 1988)

ment, and its classification as unfavorable histology can be relied on. A practical consideration for the pathologist and surgeon is recognition of the nodular stroma-rich tumor. In 1947, Stout reported the aggressive nature of "composite" tumors containing malignant neuroblastic nodules coexisting with g a n g l i o n e u r o m a t o u s tissue. 2 T h e clinical and pathologic importance of this type of tumor was recently emphasized by Bove and McAdams? ~ The definition of "nodular" tumor in the Shimada classification is parallel to, but stricter than, that of the composite tumor by Stout. It requires a visible spatial distinction between mature and immature tumor tissue, either one within the other grossly, or one in the primary site and the other in the metastatic site. Recognition of the nodular pattern is thus highly dependent on adequate sampling of large tumors, especially sampling of hemorrhagic areas (which might previously have been avoided as areas of necrosis) and sampling of lymph nodes. The diagnosis of nodular stroma-rich tumor was a frequent difference between the two observers in this study because observer no. 1 did not apply the rule that a more primitive histology in a metastatic site, as well as in an intratumor nodule, also defines a stroma-rich nodular tumor. Another practical difficulty involves the determination of MKI by the pathologist. This was originally based on a tedious absolute count of 5,000 cells. With experience, most MKIs can be estimated without formal counting. Estimation was the only technique used by observer no. 1, accounting for some of the considerable interobserver variation in MKI in this study. Selection of different fields for evaluation also contributed to the lack of concordance, since variations were in both directions and a high MKI may be focal. When the MKI is borderline, an estimate can be made by extrapolation from a count of mitotic and karyorrhectic cells in a few cellular (neuropil free) high power fields, each of which will contain no more than 800 to 1,000 neuroblasts. Each histopathologist should probably establish his own MKI standards using archival material. A study is in progress to attempt simplification and clarification of this component of the classification. 2~ The principle of considering karyorrhectic as well as mitotic cells is more significant than the technique of counting. The variable prognostic value of classical mitotic counts in our catalog study supports that contention, as does the

APPENDIX.

Institution Group Operations Office, Universityof Southern California, Comprehensive Cancer Center, Los Angeles

literature. 2x T h e highest MKIs are almost always composed predominantly of abnormal rather than mitotic nuclei, and favorable infantile tumors often have appreciable numbers of mitotic figures but low MKI. In the future, more elegant methods of determining nuclear abnormalities may be readily incorporated into this system. Preliminary results with flow cytometric DNA analysis indicate that it provides an accurate growth rate evaluation which could replace t h e M K I . 22,23

The fact that differences between the two observers in the assignment of histologic details occurred in 34% of cases is disquieting. This discrepancy is dampened to 17% (83% concordance) in assignment of prognostic group (favorable or unfavorable histology) because the factor of age makes histology irrelevant in about half the discordant cases. More importantly, discordance is eliminated by experience with MKI, by recognizing the metastatic site rule for nodular tumors (see above), and by erring on the side of the more unfavorable decision in doubtful cases. The discordance indicates that if we are to assign patients to treatment protocols on the basis of histologic classification, we must be conservative in adopting such classifications, and willing to accept a certain error rate when we do. 24 This study demonstrates a role for histopathology in predicting the natural history of neuroblastic tumors, and perhaps in selecting treatment strategies. We conclude that the Shimada classification is the best predictor of outcome among the histologic variables examined in this study. The classification might better be thought of as a group of histologic classifications that apply to different age groups. This concept is particularly apt for a tumor with such wellrecognized maturational behavior, but the same approach might prove fruitful with any tumor with an infantile "oncogenic grace period. ''z5 Certain of the morphologic catalog items studied might be added to this conceptual framework when sufficient data is accrued and tested. Data from other disciplines such as neuron specific enolase, ferritin, and N-myc amplification are also being accumulated for comparison with histologic classification. We would hope that multivariate analysis may eventually create a combined molecular and histopathologic classification of superior predictive value. Acknowledgment. The authors gratefully acknowledge the support of Dr William Newton, Jr, the personnel of the

PrincipalInvestigatorsof the Childrens Cancer Study Group

Investigators Denman Hammond, MD, John Weiner, DrPH, Harland Sather, PhD, Richard Sposto, PhD, Mark Krailo, PhD, Jonathan Buckley, MBBS, PhD

Grant No. CA 13539

Institution Universityof Minnesota Health Sciences Center, Minneapolis

Investigators William Woods, MD

Grant No. CA 07306

NEUROBLASTOM~HISTOLOGYAND PROGNOSIS[Chatten et al)

APPENDIX. [Confd] Institution University of Michigan Medical Center, Ann Arbor University of California Medical Center, San Francisco University of Wisconsin Hospital, Madison Children's Orthopedic Hospital and Medical Center, Seattle Rainbow Babies and Children's Hospital, Cleveland Children's Hospital National Medical Center, Washington, DC Childrens Memorial Hospital, Chicago Children's Hospital of Los Angeles Children's Hospital of Columbus, Columbus, OH Babies Hospital, New York Children's Hospital of Pittsburgh Vanderbilt University School of Medicine, Nashville, TN Doernbecher Memorial Hospital for Children, Portland, OR

Investigators

Grant No.

Institution

Ruth Heyn, MD

CA 02971

Arthur Ablin, MD

CA 1 7 8 2 9

Nasrollah Shahidi, MD

CA 05436

Ronald Chard, MD

CA 1 0 3 8 2

Peter Coccia, MD

CA 20320

Gregory Reaman, MD

CA 03888

Children's Hospital of Philadelphia Memorial Sloan-Kettering Cancer Center, New York James Whitcomb Riley Hospital for Children, Indianapolis University of Utah Medical Center, Salt Lake City Strong Memorial Hospital, Rochester, NY Children's Hospital Medical Center, Cincinnati Harbor/UCLA & Miller Children's Medical Center, Torrance and Long Beach, CA University of California Medical Center, Los Angeles University of Iowa Hospitals and Clinic, Iowa City Children's Hospital of Denver Mayo Clinic, Rochester, MN Izaak Walton Killam Hospital for Children, Halifax, Nova Scotia

Edward Baum, MD

CA 0 7 4 3 1

Jorge Ortega, MD

CA 02649

Frederick Ruymann, MD

CA 03750

Sergio Piomelli, MD

CA 03526

Vincent Albo, MD

CA 36015

John Lukens, MD

CA 26270

Robert Neerhout, MD

CA 26044

CCSG Pathology Center, Childrens Hospital (Columbus, OH), and the generosity of many contributing pathologists.

REFERENCES 1. Shimada H, Chatten J, Newton WA, et al: Histopathologic prognostic factors in neuroblastic tumors: Definition of subtypes of ganglioneuroblastoma and an age-linked classification of neuroblastomas. JNCI 73:405-416, 1984 2. Stout AP: Ganglioneuroma of the sympathetic nervous system. Surg Gynecol Obstet 84:101-110, 1947 3. Beckwith JB, Martin RF: Observations on the histopathology of neuroblastomas. J Pediatr Surg 3:106-110, 1968 4. Makinen J: Microscopic patterns as a guide to prognosis of neuroblastoma in childhood. Cancer 29:1637-1646, 1972 5. Evans AE, D'Angio GJ, Randolph J: A proposed staging for children with neuroblastoma. Cancer 27:374-378, 1971 6. Seeger RC, Seigel SE, Sidell N: Neuroblastoma: Clinical perspectives, monoclonal antibodies, and retinoic. Ann Intern Med 97:873-884, 1982 7. Zeltzer PM, Parma AM, Dalton A, et al: Raised neuronspecific enolase in serum of children with metastatic neuroblastoma, Lancet 2:361-363, 1983 8. Hann H-W, Evans AE, Siegel SE, et al: Prognostic importance of serum ferritin in patients with stages III and IV neuroblastoma: The Children's Cancer Study Group experience. Cancer Res 45:2843-2848, 1985 9. Seeger RC, Brodeur GM, Sather H, et al: Association of

Investigators Anna Meadows, MD Peter Steinherz, MD

Grant No. CA 11796 --

Robert Weetman, MD

CA 13809

Richard O'Brien, MD

CA 10198

Harvey Cohen, MD

CA 11174

Beatrice Lampkin, MD

CA 26126

Jerry Finklestein, MD

CA 14560

Stephen Feig, MD

CA 27678

Raymond Tannous, MD

CA 29314

David Tubergen, MD

CA 28851

Gerald Gilchrist, MD

CA 28882

Allan Pyesmany, MD

--

multiple copies of the N-myc oncogene with rapid progression of neuroblastomas. N Engl J Med 313:1111-1116, 1985 10. Beckwith JB, Palmer NF: The histopathology and prognosis Wilms' tumor: Results of the national Wilms' tumor study. Cancer 41:1937-1948, 1978 11. Kaplan E, Meier P: Nonparametric estimation from incomplete observations. J Am Stat Assoc 53:457-481, 1958 12. Mantel N: Evaluation of survival data and two new rank order statistics arising in its consideration. Cancer Chemother Rep 50:163-170, 1966 13. Peto R, Pike M, Armitage P, et ah Design and analysis of randomized clinical trials requiring prolonged observation of each patient. II. Analysis and examples. Br J Cancer 35:1-39, 1977 14. Thomas D, Breslow N, Cart J: Trend and homogeneity analyses of proportions and life table data. Comput Biomed Res 10:373-381, 1977 I5. Gitlow SE, Dziedzic LB, Strauss L, et al: Biochemical and histologic determinants in the prognosis of neuroblastoma. Cancer 32:898-905, 1973 16. Hughes M, Marsden HB, Palmer MK: Histologic patterns of neuroblastoma related to prognosis and clinical staging. Cancer 34:1706-1711, 1974 I7. Sandstedt B,Jereb B, Eklund G: Prognostic factors in neuroblastomas. Acta Pathol Microbiol Immunol Scand [A] 91:365371, 1983 18. Adam A, Hochholzer L: Ganglioneuroblastoma of the posterior mediastinum: A clinicopathologic review of 80 cases. Cancer 47:373-381, 1981 19. Bove KE, McAdams J: Composite ganglioneuroblastoma. An assessment of the significance of histological maturation in neu-

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