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Home , Hemangioblastoma, Neuroblastoma, Parathyroid adenoma, Pheochromocytoma

Amer J Hum Genet 24:514-532, 1972

Mutation and : and ALFRED G. KNUDSON, JR.1 2 and LouISE C. STRONG' 3

A recent study of [1] led to the conclusion that the occurrence of that tumor fits a two- model. According to that model a fraction, fn, of all cases is nonhereditary and results from two somatic mutational events in one cell which is thereby transformed into a tumor cell, which, in turn, becomes a solitary primary tumor. The remaining fraction of cases, f7,, is hereditary and arises in indi- viduals who are predisposed to the tumor because they have inherited one of the mutational events, the second event occurring in one or more somatic cells; such individuals may develop one tumor, more than one, or none at all. These predisposed individuals most frequently acquire the first event as a fresh dominant mutation which has arisen in a parental germinal cell. They may present a family history of the tumor, they develop tumors earlier than do nonhereditary cases, and they may develop multiple tumors. In this report, we present an analysis of familial , multiplicity, and age at diagnosis for two sympathetic-nervous-system tumors, neuroblastoma and pheochromocytoma, which reveals that these tumors also fit the two-mutation model. ANALYSIS OF DATA Neuroblastoma A starting point for the study was a review of the 60 cases of neuroblastoma seen at M. D. Anderson Hospital and Tumor Institute (MDAH) during the period 1944-1970. Two familial cases and three multiple primary cases were observed. These data were too few for analysis, so a literature review of the accumulated experience was undertaken. Points of special interest in the review were age at diagnosis, sex, multiplicity of tumors, and family incidence. In table 1 are presented the distribution of ages at diagnosis from a large series [2] of unselected cases. The largest group of cases (26%) occurs in the first year of life, following which the incidence tapers off, with 19% of cases coming after the age of 5 years. By contrast, familial cases [3-14] are clustered even more closely

Received September 3, 1971: revised January 31, 1972. This work was supported in part by Public Health Service Training grant 5-TO-1-MH 12224 (to LCS) from the National Institute of Mental Health. 1 Graduate School of Biomedical Sciences, University of at Houston, Houston, Texas 77025. 2M. D. Anderson Hospital and Tumor Institute, University of Texas at Houston, Houston, Texas 77025. 3 Texas Research Institute of Mental Sciences, Houston, Texas 77025. o 1972 by the American Society of Human Genetics. All rights reserved. 514 NEUROBLASTOMA AND PHEOCHROMOCYTOMA 515 TABLE 1 NEUROBLASTOMA: SEX AND AGE AT DIAGNOSIS FOR UNSELECTED AND FAMILIAL CASES

UNSELECTED CASES* FAMILIAL CASESt

AGE AT DIAGNOSIS Accumulated Accumulated (YEARS) N (%) N (%)

0-1 ...... 130 26 14 56 1-2 ...... 89 43 4 72 2-3 ...... 74 58 4 88 3-4 ...... 69 72 1 92 4-5 ...... 48 81 1 96 5-6 ...... 30 87 6-7 ...... 16 90 ...... 7-8 ...... 8 92 ...... 8-9 ...... 13 95 ...... 9-10 ...... 4 96 ...... >10 ...... 23 ... 1

Total ...... 504 ... 25

* [2]; N: male, 289 (57%); female, 215 (43%). t See table 2; N: male, 11; female, 14. in the early years (table 2), with 56% occurring by the end of the first year, and 4% after 5 years. The age at diagnosis is clearly much earlier in familial cases. Another difference between unselected and familial cases is that, while multiple cases constitute approximately 5%o of all neuroblastoma ([15], three of 60 cases from MDAH), they comprise 23%o of familial cases (table 2: five of 22 cases in which data are provided). An expected concomitant is that multiple tumor cases will occur earlier in life. Unfortunately, we do not have as much evidence on this point as needed. Most reports of large series do not provide this information. Those reports which do provide it are few in number and obviously biased toward the more spectacular phenomenon of congenital neuroblastoma. However, that interest- ing phenomenon itself provides the illuminating data that in 15%'o of 40 congenital cases both adrenal glands were involved [16]. According to the two-mutation model, all multiple tumor cases are hereditary. For retinoblastoma, this is borne out by the fact that 50%o of the offspring of surviving cases are affected [1]. For neuroblastoma, there is little evidence bearing directly upon this point, although there are three instances in which there may have been an affected parent [6, 10, 14]. In the family of Zimmerman [6], the father had a mediastinal removed at the age of 10 years. In the family reported by Chatten and Voorhess [10], the mother had elevated urinary , as did the father in the family reported by Wong and his col- leagues [14]. A prediction of the model is that 50%o of the offspring of multiple tumor cases would be predisposed gene carriers. The probability that a gene carrier will develop neuroblastoma, i.e., the pene- trance, can be estimated by treatment of the solitary and multiple cases as a TABLE 2 NEUROBLASTOMA: FAMILIAL CASES

PRIMARY SITES

Adrenal AGE AT Sympa- REFERENCE DIAGNOSIS RELATION- No. thetic AND CASE No. (YEARS) SEX SHIP TUMORS Single Bilateral Chain

[3]:

1 ...... 2 / F Proband 1 + ... . *. . 2 ...... NS F Sib NS ~~~~~~...... [4]:

3 ...... 13 M Proband 1 + ... . *. . 4 ...... NS NS Sib NS ...... [5]: +

5 ...... 4 F Proband 1 ...... 6 ...... 7/12 M Sib 1 + ...... [6]:

7* ...... 29412 M Proband 3 .. - + + 8 ...... 3/12 F Half-sib >2 . .. + .. . [7]: 9 ...... 4/12 M Proband 1 + ...... 10 ...... 4/12 M Identical 1 + ...... twin [8]: 11 ...... NS NS Proband NS 12 ...... NS NS Identical NS twin [9]: 13 ...... 92 F Proband 1 14 ...... "A/12 F Identical I twin [10]:

15t ...... 1/12 M Proband 1 . . . .. 16 ...... 13/2 F Sib 3 + + .. . 17 ...... 4/12 F Sib 4 + +(2) .. . 18 ...... 5/12 F Sib 1 *.* + [11]: 19 ...... birth M Proband 1 + 20 ...... 1 M Sib 1 *. . + 21 ...... 2 F Sib NS .. . [12]: 22T ...... 42 F Proband 1 + 23 ...... birth F Sib 1 [13]: 24 ...... 13 days M Proband NS 25 ...... 1i2 M Identical NS twin

NOTE.-NS = not stated. * One parent abnormal: father had mediastinal ganglioneuroma removed at age 10 years. t One parent abnormal: mother had markedly elevated urinary catecholamines but no demonstrable tumor. $ During the course of the family study, a 15-year-old sister was discovered to have elevated urinary cate- cholamines and a calcified adrenal. 516 NEUROBLASTOMA AND PHEOCHROMOCYTOMA 517 TABLE 2 (Continued) NEUROBLASTOMA: FAMILIAL CASES

PRIMARY SITES

AGE AT Adrenal Sympa- REFERENCE DIAGNOSIS RELATION- No. thetic AND CASE No. (YEARS) SEX S}IP TUMORS Single Bilateral Chain

[14]: 26§ ...... 2 NS Proband 3 + ... ±(2) 27 ...... 52 F Sib 1 + ...... Present report: 28 ...... 2 M Proband 1 + ...... 29 ...... 3Y,2 F 2d cousin 1 + ......

§ One parent abnormal: father had significantly elevated urinary catecholamines but no demonstrable tumor.

Poisson distribution. Thus, for a Poisson mean (m) of 1.0, the fraction of unaf- fected carriers (e-m) would be 0.37, that of gene carriers with one tumor (me-rn) would be 0.37, and that of multiple tumor cases would be the remainder, 0.26. Among those with any tumor (0.63), multiple cases (0.26) would constitute 41%. However, two tumors might occur at just one site, e.g., one . If only affected sites are counted and if just two sites are available, as with paired organs, then an expectation may be calculated (table 3) in which the fraction affected

TABLE 3 EXPECTED DISTRIBUTIONS OF ADRENAL FOR A MEAN NUMBER (m) OF UNITY

EXPECTED FREQUENCY TOTAL TUMORS POISSON FRACTION BOTH ADRENALS PROBABILITY UNILATERAL (r) (mre-m/r! ) [2(1/2)1] None Unilateral Bilateral

0 ...... 0.368 ... 0.368 1 ...... 0.368 1 ... 0.368 2 ...... 0.184 M ... 0.092 0.092 3 ...... 0.061 Y4 ... 0.015 0.046 4 ...... 0.015 1/8 ... 0.002 0.013 5 ...... 0.003 146 ...... 0.003

Totals ...... 0.999 ... 0.368 0.477 0.154 unilaterally is 0.48 and that affected bilaterally is 0.15; and, among affected in- dividuals, bilateral cases are 24%o of the total. For neuroblastoma, the fraction of multiple cases among gene carriers is difficult to estimate. No cases are reported of two primaries at one site, but this may simply reflect failure of reporting. In ad- dition, the fraction of multiple cases is probably underestimated. It may be, there- 518 KNUDSON AND STRONG fore, that the observation of 23%o multiple tumors among familial cases is compatible with a Poisson mean of unity. If so, gene carriers would be affected 63 % of the time, and, among the offspring of bilateral cases, about 32% should be affected. The fraction of hereditary cases, fh, may be estimated by assuming that all mul- tiple cases are hereditary. If 5% of all cases are multiple and if 23%'o of familial cases are multiple, 0.23 f1 - 0.05, and fh 0.22 (i.e., 22%o of all cases of neuro- occur in gene carriers). Of course, nearly all of these cases have resulted from new germinal . The incidental calculation can be made that, if 15% of congenital cases are multiple, then approximately 68%o of congenital cases are hereditary. The germinal mutation rate (jig) necessary to produce these gene carriers may be calculated from a knowledge of the incidence of neuroblastoma (i), the heredi- tary fraction (fh), the mean number of tumors (m), and the coefficient of selec- tion (s). The value of s is zero for individuals with normal survival and fertility and unity for those with no offspring; for neuroblastoma, this value is essentially unity for those affected, i.e., s - 1 - eim. As discussed later, for pheochromocy- toma s < 1 - em-"f. The relationship of these variables, as shown previously [1], is

4fh i * S 2(I1- em)(l For neuroblastoma, assuming that i 7 X 10-5, a minimal estimate from the mortality data of Miller et al. [2], and f1- 0.22, then ug - 8 X 10-6 per gen- eration. This is compatible with known mutation rates and is comparable with the rate of 5 X 10-6 per generation for retinoblastoma [1]. If a second event is necessary to produce a tumor, then, as in the case of retino- blastoma, there should be a difference between unselected and hereditary cases with respect to the fraction surviving at various times. For the hereditary cases, those not yet diagnosed at a given age should decline exponentially, since only a single remaining hit is necessary to produce tumor; on a semilogarithmic plot, this relationship should be linear. On the other hand, the unselected cases will contain a majority (presumably 78%o) of the nonhereditary form, which requires two hits and should follow a curvilinear relationship. The data on unselected and familial cases do, in fact, demonstrate this relationship, as shown in figure 1. If the above conclusions are correct, then the concordance rate in monozygotic twins may also be predicted, since concordance would be expected in a fraction (1 - e-mf) of the twins of the hereditary cases (f1). Thus,

concordance =f1 (1 - e-m), (2) which, for neuroblastoma, if f11 = 0.22 and m = 1, is 14%o. Unfortunately, report- ing has been too sparse to permit a test of this prediction. Four instances of con- cordance in monozygotic twins are recorded in table 2 (cases 10, 12, 14, and 25). A number of instances of discordance in twins have been noted, but only in five of these has monozygosity been specified [2, 17-20]. NEUROBLASTOMA AND PHEOCHROMOCYTOMA 519

100- 080-

060-

0.40-

< 020-

0.10- - 008- 0.06-

,^ 004-

NEUROBLASTOMIA cr o 504 Unselected Coses(Toble) \ 0.02 25 Foncliol Coses Tobleg)

0 2 3 4 5 6 7 AGE AT DIAGNOSIS (YEARS) FIG. 1.-Neuroblastoma: semilogarithmic plots of fractions of cases of neuroblastoma not yet diagnosed versus age in years; unselected and familial cases. Pheochromocytoma The other major tumor of the sympathetic nervous system, pheochromocytoma, may also be hereditary or nonhereditary, solitary or multiple, and is therefore sub- jected to the same analysis employed for neuroblastoma. Data on hereditary simple pheochromocytoma, on the syndrome in which pheochromocytoma is asso- ciated with medullary carcinoma of the , and on the association of pheo- chromocytoma with and with the von Hippel-Lindau syndrome will be analyzed in the light of the above model for mutation and cancer. The comprehensive reviews of Hermann and Mornex [21] and of Steiner et al. [22] provide the data recorded in table 4, which presents the age distributions for three categories of simple pheochromocytoma. The modal age at diagnosis is clearly earlier for multiple nonfamilial cases (about 20 years) and for familial cases (about 20 years) than for single nonfamilial cases (about 40 years). The difference in the number of multiple cases in the familial and nonfamilial series is striking. Of the familial cases [21-24] in table 4, 44% had bilateral adrenal tumors and 52%o had tumors at more than one adrenal or sympathetic site. While Hume [25] reported similar findings (50%o bilateral, 59%o multiple), he additionally found that only 5%o of sporadic, or nonfamilial, cases were multiple. Since familial cases are more often multiple, and, since they occur earlier, it is not surprising that 397o of childhood cases are multiple [25, 26]. Again, according to the two-mutation model, all multiple tumor cases are hereditary, which is sup- ported by the earlier age at diagnosis of familial and of multiple tumors. The simple familial pheochromocytoma gene has been shown to be inherited 520 KNUDSON AND STRONG TABLE 4 PHEOCHROMOCYTOMA: SIMPLE FORM. COMPARISON OF AGE DEPENDENCE FOR SINGLE, MULTIPLE, AND FAMiLIAL TUMORS

AGE AT DIAGNOSIS (YEARS)

0-9 10-19 20-29 30-39 40-49 50-59 >59 Total

Nonfamilial*: Single ...... 11 25 81 73 61 45 24 320 (3%) (8%) (25%) (23%) (19%) (14%o) (8%o) Multiple ...... 5 17 7 8 3 6 0 46 (11%) (37%) (15%) (17%) (7%) (13%) Familialt ..... 7 17 6 6 7 3 0 46 (15%) (37%) (13%) (13%) (15%) (7%o) * [21]: from 507 cases, age at diagnosis and negative family history were ascertained for 320. t [22]: families A, D-K, M-O, T, U, Y, Al; [23]: with follow-up on family X; [24].

dominantly with a high degree of [22]. In our summary of 18 familial cases, there was only one instance of lack of penetrance [27, family F], that in which a child, her maternal aunt, and a maternal cousin were affected but in which the child's mother was not. Even that example may be spurious, since among af- fected families there have been childhood cases diagnosed earlier than their affected parents [23, 28]. As for neuroblastoma, penetrance may be estimated by comparing data on soli- tary and multiple cases with the Poisson distributions for various values of the mean (m). As shown in table 5, the observed data (44% bilateral adrenal involve- ment among hereditary cases) fall between the Poisson means of 2.0 and 3.0. Only 5%7o-137% (e-m) of gene carriers would be unaffected, indicating a high de- gree of penetrance. Since these estimates are age dependent, we specify the ap- plicable age as approximately 50 years, an inference from figure 2. For an average life-span, estimates of m and of penetrance may be more nearly 4 and 0.98, re- spectively (table 5). Those with unilateral adrenal tumors should represent 35%o-

TABLE 5 COMPARISON OF EXPECTED AND OBSERVED DISTRIBUTIONS OF ADRENAL

OBSERVED EXPECTED FREQIJENCIES FOR NVARIOUS MEAN (m) NUMBERS OF TUMORS 5yndrome with DISTRIBUTION Familial Thyroid OF TUMORS 1 2 3 4 5 Simple Type Carcinoma

None ...... 0.37 0.14 0.05 0.02 0.01 <0.10 <0.05 Unilateral ...... 0.48 0.46 0.35 0.23 0.15 0.50-0.56 0.20-0.25 Bilateral ...... 0.16 0.40 0.60 0.75 0.85 0.40-0.44 0.75-0.80 NEUROBLASTOMA AND PHEOCHROMOCYTOMA 521 46%o and those with bilateral tumors, 40%o-60%o of gene carriers. Assuming that all multiple cases are hereditary, as for retinoblastoma [1], and that multiple cases comprise 55%o of hereditary cases and 127% of an unselected series [21], the fraction of hereditary cases (fI) is 0.22 (0.55 f7, = 0.12); i.e., 22%o of all simple pheochromocytoma cases carry a dominant gene which will cause pheo- chromocytoma in approximately 90%o of the half of the offspring who inherit it. The germinal mutation rate (,ug) necessary to maintain the pheochromocytoma gene in a population may be calculated from equation [1]. The coefficient of selec- tion (s) must be estimated from data from the past, recognizing that the gene frequency will change with improved diagnostic and operative techniques which tend to decrease s. In the Hermann and Mornex series [21], children rep- resented 12.6%o of all cases and had a of 29%jo (19 deaths among 66 cases). These authors also noted that 40%o of the total patients were women of childbearing age, and, of these, one in four suffered some "disturbance" during . "Disturbance" included 29 fetal or child losses and 17 maternal deaths in 52 patients and 101 . A minimum value of s = 0.07 can be estimated from the sum of childhood and fetal mortality. (It is not known how many mater- nal deaths were associated with fetal losses.) Assuming i = 10 X 10-4, as esti- mated from files [46, 47], (1 - e-mn) = 0.9, and fI = 0.22, then 9- 9 X 10-6 per generation, which is very close to the mutation rate for neuro- blastoma. As for neuroblastoma, a test for two causative events can be made. Semilogarith- mic plots of the fraction of cases not yet diagnosed versus age in years for single,

1.00 - 080

4 040

o 020- cn

0.10- 0Q08 2 o- 006-0 PHEOCHROMOCYTOMA from ° 0.04- (Date table P/ O Non-Familil-Single

I.-C> * Non-Fomiliol-Multiple i*Foliol

AGE AT DIAGNOSIS (YEARS) F-G. 2.-Pheochromocytoma: semilogarithmic plots of fractions of cases of pheochromocytoma not yet diagnosed versus age in years; nonfamilial single, nonfamilial multiple, and familial. 522 KNUDSON AND STRONG multiple, and familial cases are shown in figure 2. Pheochromocytoma differs from retinoblastoma and neuroblastoma in that it is a nonmalignant tumor of mature, differentiated tissue; diagnosis depends upon its excessive function, which may only be apparent after a substantial asymptomatic period. However, it can be seen that familial and multiple cases follow similar linear relationships after the age of 10 years, while single tumors follow a curvilinear relation, as expected for a two-hit phenomenon. Again, in the hereditary form, one hit is inherited and one occurs in somatic cells. Concordance for monozygous twins can be predicted by equation (2) to be ap- proximately 20%. Unfortunately, there are insufficient observations to test this prediction, since we are aware of only one case in which a twin has been affected by pheochromocytoma [29]; in that case, the twins were monozygous and dis- cordant. Genetic heterogeneity of familial pheochromocytoma is revealed by the existence of the pheochromocytoma-medullary carcinoma of the thyroid syndrome [30]. This association is often familial and accounts for about 30% of familial pheochromocy- toma [22]. The very similar distributions of ages at diagnosis for familial and non- familial cases ([22, 31-45]; C. S. Hill, personal communication, 1969; table 6)

TABLE 6 PHEOCHROMOCYTOMA-MEDULLARY CARCINOMA OF THYROID SYNDROME: COMPARISON OF AGE AT DIAGNOSIS FOR FAMILIAL AND NONFAMILIAL CASES

AGE AT DIAGNOSIS OF PHEOCHROMOCYTOMA (YEARS)

CATEGORY 0-19 20-39 40-59 >59 Total

Without mucosal : Nonfamilial* ...... 0 16 (67%) 5 (2 1%) 3 (13%) 24 Familialt ...... 4 (9%) 25 (57%o) 13 (30%) 2 ( 5%) 44 With mucosal neuroma: Nonfamiialt ...... 1 (8%) 10 (83%) 1 ( 8%) 0 12 Familial§ ...... 1 (8%) 6 (50%0) 4 (33%) 1 ( 8%) 12

* [22]: cases 1, 3-8, 10, 13, 17, 21-23, 29-30, 33-36, 40-41; [31-36]. t [22]: families I, B, C, L, P, R, U, W, F; [37]: followup on family C; [38-44]; C. S. Hill, personal communication, 1969: followup on case reported in [44]; [45]. i [48-531. § [48, 51, 54, 55]. and the very high incidence of bilateral adrenal tumors (75%-80%), whether familial or not, strongly suggest that all cases of this syndrome are determined prezygotically. The gene causing the syndrome is distinctly separable from that causing simple pheochromocytoma. The age at diagnosis is definitely later (mode, 30-35 years) than for hereditary simple pheochromocytoma, extra-adrenal tumors are not found, and sustained is rarely observed. The incidence of bilateral cases among familial cases is compatible with a NEUROBLASTOMA AND PHEOCHROMOCYTOMA 523 Poisson mean of 4 (table 5) and a predicted penetrance of 98%o. This level of penetrance is compatible with observed data. Presence of the medullary carcinoma indicates presence of the gene for pheochromocytoma in known familial cases of both tumors. In the familial cases reported in table 6, there were seven instances of medullary carcinoma without known pheochromocytoma. Of these, four were under 30 years, had normal catecholamines, and were living and well, although one had hypertension [22, 28, 38, 42]. Two died of medullary carcinoma with no autopsy (ages 17 and 30-40) [40], and one died at age 43 without autopsy but had symptoms of pheochromocytoma [41]. All pedigrees were compatible with dominant inheritance and a high degree of penetrance, and in no case was there an unaffected member with affected parent and child. As in simple pheochromocytoma, however, there were cases in which the offspring was diagnosed earlier than was the parent. [42]. The germinal mutation rate may be estimated for this syndrome too. If 6%o-7% of all pheochromocytomas are familial [21, 56], if 30%7o of familial pheochromocy- tomas have the pheochromocytoma-medullary-carcinoma syndrome, and if the overall incidence of pheochromocytoma is 100 X 10-5, then the minimum inci- dence for pheochromocytoma-medullary carcinoma can be estimated to be 2 X 10-5, or one in 50,000 individuals. This is comparable to the estimate of Kaye and Zak [57] who found one case of pheochromocytoma and medullary carcinoma in 20,000 , and of Sipple [30], who reported 1%o of pheochromocytomas to be associated with thyroid carcinoma. Estimation of the coefficient of selection (s) is difficult. Also, since the syndrome has only recently been recognized, it is dif- ficult to get an idea of the selection which has been operating until the present. Children are rarely symptomatic. In the family of Smits and Huizinga [58], three of seven probably affected females died during pregnancy, and there were at least two cases of fetal loss by affected mothers in 40 pregnancies. Thus, a minimal es- timate for s from fetal loss would be 0.05. Unfortunately, in many cases the ob- stetrical history is not given. Summarizing the other cases, 17%o of affected females died prior to the age of 30, and, assuming that they account for further decrease in effective fertility, s would fall near 0.10. If i = 2 X 0-5 s - 0.10, fh = 1.00, and m-=4, then f,u = I X 10-6 per generation. At present, no twins have been reported with this syndrome. However, if it is true that all cases represent germinal mutation, then concordance for monozygous twins would equal penetrance of the gene, or 98%o. We look to future reports to test this prediction. There is a variant of the pheochromocytoma-medullary-carcinoma syndrome which has been mentioned separately because of the unique expression which heralds its presence-the multiple-mucosal-neuroma syndrome. The syndrome in- cludes mucosal (plexiform neuromas) characteristically found on the lips, anterior two-thirds of the tongue, buccal mucosa, and eyelids; hypertrophied lips; medullated corneal nerves; ganglioneuromatosis of the ; intestinal hypertrophy; cafe au lait spots; peculiar, coarse, acromegalic facies; a Marfanoid habitus; and osseous deformities. Not all of these features are present 524 KNUDSON AND STRONG in every case. The oral neuromas may be present at birth or early childhood, do not appear to undergo malignant degeneration but grow with the child, and have been referred to as hamartomas [59]. The syndrome was first recognized to be distinct from neurofibromatosis as well as uniquely associated with the pheochro- mocytoma-medullary-carcinoma syndrome by Williams and Pollock [59] and was thoroughly reviewed by Gorlin et al. [48]. The age distribution of those cases as well as those of more recent reports [48-55] are in table 6. It can be seen that the age at diagnosis is similar to that in the pheochromocytoma-medullary-carcinoma syndrome. Bilaterality is again 75%o-80%. This syndrome also follows a dominant Mendelian pattern with a high penetrance [50, 55]. The incidence is difficult to determine since it has so recently been recognized and is rarely found in surveys of pheochromocytoma, but Williams and Pollock [59] found three cases in a series of 67 cases of medullary carcinoma of the thyroid. Other series have not reported such a high association [60, 61] or have not stated the association [62]. It must represent a fraction of a percentage of all cases of pheochromocytoma, but a notable one because of the early manifestations. Pheochromocytoma has been associated with neurofibromatosis, and table 7 in- cludes the age of onset of those cases reviewed by Schlegel [47] as well as more

TABLE 7

PHEOCHROMOCYTOMA WITH NEUROFIBROMATOSIS OR WITH VON HIPPEL-LINDAU DISEASE: AGE AT DIAGNOSIS

AGE AT DIAGNOSIS (YEARS)

0-19 20-39 40-59 >59 Total

Neurofibromatosis* ...... 2 ( 3%5) 24 (40%) 30 (51%) 3 (5%o) 59 Von Hippel-Lindaut ...... 4 (17%) 16 (66%o) 4 (17%) 0 24

* [47, 63-74]. t [75-80]. recent cases [63-74]. The age at diagnosis is older than that of either unselected or hereditary pheochromocytomas, with a modal age of 40-45 years. It was noted by Saxena [81] that pheochromocytoma does not appear in childhood in neuro- fibromatosis. In these 59 cases, 12%o were multiple. The average age of onset for multiple cases was 60 years. There were no cases of familial pheochromocytoma, although in many cases there was a family history of neurofibromatosis. The incidence of neurofibromatosis has been estimated at 1/1,500-1/2,200 [82] and is said to contribute 5%o-8%o of unselected pheochromocytomas [21, 83]. The incidence of pheochromocytoma among patients with neurofibromatosis has been estimated at 4%o-25%o [47, 75, 81, 84]. The low incidence (one in 223) re- ported by Crowe et al. [82] can be attributed to the fact that most of his patients NEUROBLASTOMA AND PHEOCHROMOCYTOMA 525 were under 30 years of age, while the modal age of onset for pheochromocytoma in neurofibromatosis is 40-45 years. Whether the high incidence of pheochromo- cytoma results because the neurofibromatosis mutation is also a pheochromocytoma mutation analogous to that causing the dual effects seen in the medullary carci- noma of the thyroid syndrome cannot be decided with the data at hand. The other unique association of pheochromocytoma is with the von Hippel- Lindau syndrome. In table 7, 38 reported instances of this association [75-80] are presented with the distribution of age of onset. It can be seen that the modal age for diagnosis of pheochromocytoma, 30-35 years, is not different from that of unselected cases. There have been several reports in which both pheochromocytoma and the von Hippel-Lindau syndrome were present in a family, although not neces- sarily both in the same patients [75-79]. Familial cases have shown a high de- gree of penetrance for pheochromocytoma, consistent with dominant inheritance. It may be that the von Hippel-Lindau mutation occasionally includes a pheochro- mocytoma gene.

DISCUSSION Two important tumors, neuroblastoma and pheochromocytoma, arise in the sympathetic nervous system. For both, there is a greatly increased risk for the family members of a fraction of affected individuals. This fraction (fh) is predis- posed by a dominant mutation which is tumor specific. This fraction of predisposed individuals, when compared with that fraction (fn) of affected persons not so pre- disposed, is found to develop tumors earlier in life and may develop more than one primary tumor. For both neuroblastoma and pheochromocytoma uncomplicated by other tumors, fh is estimated to be 0.22. The mean number of tumors in the two instances is estimated to be one and two or three for neuroblastoma and pheo- chromocytoma, respectively, the corresponding estimates of gene penetrance (1- e-m) being 0.63 and 0.87-0.95. Predisposed individuals do not develop a tumor until a second event occurs in some cell. This event is evidently rare, as seen by comparing the mean number of tumors noted above with the number of predisposed cells, which is probably of the order of magnitude of 1 million. This suggests, but does not prove, that the second event may also be mutational. Some individuals will develop more than one tumor, and a majority will develop at least one. By contrast, in the nonpredisposed indi- viduals with tumors, both events have occured in a somatic cell. For neuroblastoma, the classification of patients as predisposed or not has practical significance in that another tumor may be present and that the predis- position would be inherited in a dominant fashion by any offspring of survivors, as happens with retinoblastoma. Recognition of those predisposed might include screening of parents and siblings of affected individuals for excretion, as done by Helson et al. [85], who found that five of 21 families of affected children had siblings with elevated catecholamines. Three reported pedigrees, as previously noted, suggest the pattern. In these three instances, the parent has been, or has been suspected of being, a bearer of ganglioneuroma rather than of neuro- 526 KNUDSON AND STRONG blastoma. This finding suggests that the tumor specificity of the mutant gene in- cludes both neuroblastoma and ganglioneuroma and supports the theses of other investigators that a close developmental relationship exists between the two tumors [86] and that the regularly reported excess of males in neuroblastoma (see table 1) is attributable to the more frequent maturation of neuroblastoma to ganglioneuroma in females [87]. Maturation to ganglioneuroma could result in a lower estimate of mean number of tumors per gene carrier than is actually the case and a lower estimate of pene- trance as well. However, an even greater cause for error may be spontaneous regression, the rate of which is higher for neuroblastoma than for any other malig- nant [88]. Just how high this rate may be cannot be estimated at this time. The incidental finding of in situ neuroblastomas with high frequency in infants under the age of 3 months suggested to Beckwith and Perrin [89] that a majority of neuroblastomas may regress spontaneously. Whether these are the same tumors as clinical neuroblastomas, whether they are connected pathogenetically with other causes of early infant death [90], or whether they represent a state assumed by a cell containing just one of the two necessary mutations will hopefully be answered by future research. While neuroblastoma is a highly malignant tumor of early life (related by maturation to ganglioneuroma), the other principal tumor of the sympathetic nervous system, pheochromocytoma, is usually a of adults, unrelated to either neuroblastoma or ganglioneuroma. A report such as that by Fernando et al. [91], which describes contiguous pheochromocytoma and neuroblastoma in one tumor, is discontinuous with the remainder of reported experience. From the analysis presented earlier, it is evident that pheochromocytoma may occur as the result of at least two distinct mutational patterns. Most pheochromocytomas occur in nonpredisposed individuals as the result of two somatic mutational events. About 20% of cases are predisposed by a germinal mutation, i.e., a dominant gene which produces no other effect. About 2 % of pheochromocytomas occur in persons predis- posed by a separate germinal mutation which also causes another tumor, notably medullary carcinoma of the thyroid. Since all cases represent germinal mutations, the offspring of all affected persons are at a 50% risk of receiving a dominant gene with 98%o penetrance and should be followed closely for adrenal or thyroid tumors. In fact, the latter tumor is much more dangerous in view of the usually successful treatment of pheochromocytoma. The medullary thyroid carcinoma associated with pheochromocytoma occurs at an earlier age than those of an unselected series [92], is frequently bilateral (70%-80%), and also fits a Poisson distribution with a mean number of four tumors per gene carrier. The high mean number of medullary carcinomas to be expected in this syndrome gives strong support to those who suggest total thyroidectomy for gene carriers [55, 93]. Patients with medullary carcinoma differ clinically from those with simple pheochromocytoma. As observed by Steiner et al. [22], those with pheochromo- cytoma in families with medullary carcinoma generally present with paroxysmal symptoms or hypertension, while in the unselected series of Hermann and Mornex NEUROBLASTOMA AND PHEOCHROMOCYTOMA 5217 [21], over 60% demonstrated sustained hypertension. The pheochromocytoma in the medullary carcinoma syndrome also frequently gives a negative tyramine test [22], which may be related to a unique catecholamine-secretion pattern involving a low to epinephrine ratio [21]. These characteristics of the pheochromocytoma-medullary-carcinoma syndrome may be related to the absence of extra-adrenal pheochromocytomas, which are primarily norepinephrine-secreting tumors [94]. There is one condition which has been associated with both neuroblastoma and pheochromocytoma, namely, neurofibromatosis. Since, neurofibromatosis itself is dominantly inherited, the question arises whether pheochromocytoma is a mani- festation of the mutation, as it is in the syndrome with medullary carcinoma of the thyroid. This possibility is related to the suggestion of Nicholls [95] that certain of neurofibromatosis are expressed in cells which become homozygous for an abnormal gene by from a heterozygous and that other manifestations of neurofibromatosis result from further somatic mutations. Three significant differences are found when pheochromocytoma is associated with neurofibromatosis: (1) the incidence is much lower (10%o versus 95%o-100%o); (2) the tumor is less frequently bilateral (10%o versus 70%o-80%o); and (3) it appears at a later average age. If it is a direct manifestation of the neurofibro- matosis, resulting from one of the mutational hits necessary for pheochromocytoma, then it is either a different mutation, with a much lower m value than the one asssociated with either hereditary simple pheochromocytoma or the medullary- thyroid-carcinoma syndrome, or it is only occassionally a part of the neurofibro- matosis mutation. Although the former is a distinct possibility, the latter is unlikely because there should be some families with both lesions, which has not been the case. A different kind of possibility is that the neurofibromatosis mutation results from circumstances which greatly increase the frequency of induction of pheo- chromocytoma by two somatic events. If this were the case, one would expect a low incidence, a solitary tumor, and the age of appearance seen with nonhereditary pheochromocytoma. The association of neuroblastoma, or ganglioneuroma, and neurofibromatosis is much rarer. As indicated by Bolande and Towler [96], the diagnosis of ganglio- neuroma in neurofibromatosis is complicated by the fact that ganglion cells may be captured within a and lead to an erroneous diagnosis. At the other extreme, a neuroblastoma may mature into a ganglioneuroma with many neuro- fibromatous elements, leading again to an erroneous diagnosis. Even so, the coinci- dence of neuroblastoma, ganglioneuroma, and neurofibromatosis has only been reported twice [97, 98].

SUMMARY A two-mutation model of cancer previously employed in an analysis of retino- blastoma is here applied to neuroblastoma and pheochromocytoma. The data on familial incidence, age of onset, and multiplicity are compatible with the model for neuroblastoma, and, with adjustment for delayed appearance of clinical manifesta- 528 KNUDSON AND STRONG tions, for pheochromocytoma. It is concluded that both tumors result from two events, at least one of which is mutational. In nonhereditary cases, both events occur in somatic cells, and the tumor is always solitary. In hereditary cases, one event occurs in germinal cells and is inherited, the second occurring in somatic cells. Gene carriers may develop no tumor, one tumor, or more than one tumor. Heredi- tary cases are estimated to account for 20%-25% of the total, respectively, for both neuroblastoma and pheochromocytoma. ACKNOWLEDGMENT We gratefully acknowledge the helpful suggestions of Dr. David E. 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