RB1 mutations and second primary malignancies after hereditary retinoblastoma.

Survivors of hereditary retinoblastoma have a high risk of second primary malignancies, but it has not been investigated whether specific RB1 germline mutations are associated with greater risk of second primary malignancies in a large cohort. We conducted a retrospective cohort study of 199 survivors of hereditary retinoblastoma with a documented RB1 germline mutation diagnosed between 1905 and 2005. In total, 44 hereditary retinoblastoma survivors developed a second primary malignancy after a median follow-up of 30.2 years (range 1.33-76.0). A significantly increased risk of second primary malignancy was observed among carriers of one of the 11 recurrent CGA>TGA nonsense RB1 mutations (hazard ratio (HR) = 3.53; [95% confidence interval (CI) = 1.82-6.84]; P = .000), and there was a significantly lower risk for subjects with a low penetrance mutation (HR = .19; [95% CI = .05-.81]; P = .025). Our findings suggest a genotype-phenotype correlation for second primary cancers of retinoblastoma survivors and may impact on long-term surveillance protocols of patients with hereditary retinoblastoma, if confirmed by future studies.

Introduction

Retinoblastoma is the most common primary intraocular malignancy of childhood [1]. Mutational inactivation of both alleles of the RB1 tumor suppressor gene in the developing retina initiates the formation of retinoblastoma [2, 3]. The RB1 gene consists of 27 exons and is located on chromosome 13q14 (GenBank accession number L11910, MIM#180200). The gene encodes a ubiquitously expressed nuclear protein, which is involved in cell cycle regulation, cellular differentiation and survival [4]. About 40% of retinoblastoma patients have a hereditary predisposition, caused by a heterozygous germline mutation in the RB1 gene and are usually bilaterally affected [5]. Over 600 different pathogenic mutations have been described. Patients with nonhereditary retinoblastoma only have one eye affected, no germline mutation in the RB1 gene and two somatic retinal RB1 mutations.

As is known from long-term follow-up studies [6-10], hereditary retinoblastoma subjects have a strongly increased risk for second primary malignancies, (including osteosarcoma, soft tissue sarcoma, melanoma and epithelial cancers) which is associated with excess mortality [11-13]. So far, it has not been examined in a large cohort of retinoblastoma patients whether specific RB1 mutations might be associated with greater risk of second malignancy.

The objective of the present study was to investigate the RB1 genotype in relation to second malignancy risk in hereditary retinoblastoma subjects.

Materials and methods

Patients

In the Netherlands we have data available of Dutch retinoblastoma subjects diagnosed from 1862 onwards. Detailed information on data collection and follow-up has been described previously [9]. Relevant data collected for the present study were family history of retinoblastoma, tumor laterality, treatment for retinoblastoma, reports on invasive cancers, and date and (underlying) cause of death. Only the first cancer after retinoblastoma was included in this study. Time at risk for a second primary cancer began at diagnosis of retinoblastoma and ended on the date of second malignancy diagnosis, emigration, the date last known to be alive, the date of death, or the closing date of the study, whichever came first.

Patients with bilateral disease, a positive family history of retinoblastoma, or a germline mutation in the RB1 gene detected by chromosomal or DNA analysis were classified as hereditary. The remaining patients, those with unilateral retinoblastoma, no family history of retinoblastoma, and no germline mutation detected in the RB1 gene, were classified as having non-hereditary retinoblastoma.

Eligible subjects for the current study included all hereditary retinoblastoma patients from the Dutch retinoblastoma cohort (1862–2005), in whom a germline RB1 mutation was documented. If a retinoblastoma patient had died before DNA-testing could be performed, but a RB1 mutation was determined in the family, the patient was considered to be a carrier of the familial mutation (n = 26). Every affected family member was handled as a single case in the analysis. Of the 1,028 retinoblastoma patients in the Dutch cohort, we identified a total of 410 (39.9%) hereditary cases. Two-hundred eleven patients were excluded because no DNA analysis could be performed (n = 180) or DNA analysis did not detect a RB1 mutation (n = 31). The remaining 199 patients were included in this study (see Fig. 1), of whom 168 were alive at the time of inclusion.

Fig. 1

Flow chart showing reasons for inclusion and exclusion of retinoblastoma patients with hereditary retinoblastoma from our cohort. In the total group of 410 hereditary retinoblastoma patients from our cohort, 99 primary tumors (SPT) have been diagnosed. In the flow chart is also depicted in which in- or excluded group these SPT’s have occurred. Percentage is calculated from the total of 99 SPT’s

This study was approved by the Medical Ethics Committees of all participating hospitals, and was conducted in accordance with the principles of the Helsinki declaration.

Mutation screening

Since the beginning of the 1990s all newly diagnosed retinoblastoma patients in the Netherlands undergo germline RB1 mutation analysis. Many patients who were diagnosed before that time underwent DNA-testing between 1990 and 2005, when they were referred to the clinical genetics department (n = 90). Rb patients diagnosed prior to 1990, in whom mutation testing had not yet been performed at the time of the study and who wanted to participate in the study, were invited to undergo DNA-testing and were offered genetic counseling (n = 23).

DNA analysis included direct sequencing of exons 1 and 15, and the RB1 promoter and Denaturing Gradient Gel Electrophoresis (DGGE) analysis of the other exons and flanking intronic sequences. To detect large deletions and duplications Multiplex Ligation-dependent Probe Amplification (MLPA) analysis was performed. If warranted, e.g. when dysmorphic features or mental retardation was noted, karyotyping was performed to detect chromosomal rearrangements. With these techniques we have been able to detect 90% of mutations in familial and/or bilateral cases.

Type of RB1 mutation

In the RB1 gene are several methylated CGA codons known to lead to 11 recurrent nonsense mutations by C>T transitions [14-16]. An important factor in the high recurrence of mutations at these sites was shown to be deamination of 5-methylcytosine [17].

For this study mutations in the promoter, exon 1, missense mutations and deletions of the complete RB1 gene were regarded as low penetrance mutations, based on previous studies [18-21]. Four out of six familial splice mutations were also regarded as low penetrance mutations, based on a diseased eye ratio (DER) of ≤1.5, defined as the total number of affected eyes per family divided by the number of mutation carriers in the family [22, 23]. To exclude possible mosaicism as a cause of milder expression the first mutation carrier in these families was excluded from the analysis.

Statistical methods

We compared the frequency of second primary cancers among hereditary retinoblastoma survivors with specific documented RB1 mutations, and tested for differences using Chi-square tests.

Multivariate Cox regression analysis was performed to quantify the effects of specific RB1 mutations on the risk of second primary malignancies. Therapy, age, laterality, sex, and familial or sporadic occurrence were taken into account as possible confounders (SPSS, Chicago, IL).

Results

Median age of all patients included in the study cohort was 30.0 years (range 1.0–75.0). Of the total of 199 participants, 111 were familial cases and 88 concerned sporadic patients. After a median follow-up time of 30.2 years (range 1.33–76.0), 44 carriers of a RB1 mutation from 31 different families developed a second primary malignancy. Table 1 shows the number of germline RB1 mutations according to type of mutation and lists the number of second primary tumors according to type of mutation. The mutations found in patients who developed a second malignancy were distributed throughout most of the RB1 gene and did not appear to cluster in one region (Fig. 2). There was no correlation between the different types of second malignancies diagnosed in these patients and the type of mutation or the region of the gene where the mutation was located. In the group of retinoblastoma survivors who developed a second malignancy, only nonsense and frameshift mutations, certain splice mutations and large rearrangements were observed.

Table 1

Number and type of second primary tumor (SPT) by mutation type

Type of RB1 mutationa	Number of carriers n (%)a	Number of cases with SPT n (%)b	Type of SPT
			Sarcomac	Melanoma	Epithelial cancer	Otherd
Nonsense/frameshift mutation	117 (58.8)	31 (26.5)	11	8	10	2
Recurrent nonsense mutation	49 (41.9)	17 (34.7)	7	7	2	1
Low penetrance mutation = exon 1	7 (6)	1 (14.3)
Splice mutation	34 (17.1)	7 (20.6)	2	1	4	0
Low penetrance mutation	11 (32.4)	0
Large rearrangements	35 (17.6)	6 (17.1)	2	2	2	0
Low penetrance mutation	21 (60)	1 (4.8)	1
Missense mutation	11 (5.5)	0	0	0	0	0
Low penetrance mutation	11 (100)	0
Promoter mutation	2 (1)	0	0	0	0	0
Low penetrance mutation	2 (100)	0
Total	199	44 (22.1)	15	11	16	2

a Subclassification of the mutation type is shown in italics, percentage as compared to total number of cases with this mutation type

b Percentage of cases with an SPT as compared to total number of cases with this mutation type

c Including soft tissue sarcoma, osteosarcoma, and chondrosarcoma

d Malignant tumor not otherwise specified and brain tumor

Fig. 2

Graphical representation of RB1 and mutations found among hereditary retinoblastoma subjects diagnosed with a second primary malignancy (n = 44). Exons 1 through 27 are not drawn to scale. Every symbol represents a retinoblastoma subject diagnosed with a second primary malignancy. Black symbols represent sporadic hereditary retinoblastoma subjects. Greyscale coloured symbols represent subjects with familial retinoblastoma. Downward-pointing symbols represent mutations in exons, and upward-pointing symbols represent mutations in introns. The horizontal lines below depict large rearrangements

Table 2 displays the mutations of all patients who developed a second primary malignancy, along with clinical details, listed according to subcategories of germline RB1 mutation type.

Table 2

Descriptions of retinoblastoma patients with a second primary malignancy according to subcategories of RB1 mutation type

Subjectno.a,b	Familyc	Number of RB patients in the family without second primary malignancy (age at inclusion)	Site	Mutation descriptiond	Protein	Laterality	First in family	Therapy	Type of second primary malignancye	Second primary malignancy (age at diagnosis)
Nonsense or frameshift mutation
1	F22	NA	Exon 1	g.2121dupC	p.Ala22GlyfsX9	Bil	Yes	RT	Epithelial	B (59)
2	F11	1 (37)	Exon 2	g.5446G>T	p.Glu54X	Bil	Yes	Enucl	Epithelial	Bl (52)
3	F12	1 (12)	Exon 2	g.5505_5506dup	p.Ala74GlufsX4	Bil	Yes	RT + CH	Other	(44)
4	F16	4 (59, 40, 14, 6)	Exon 6	g.45855delT	p.Leu199TyrfsX2	Bil	No	RT	Osteosarcoma	(5)
5a	F31	NA	Exon 8	g.59695C>T	p.Arg255X	Bil	Yes	RT	Osteosarcoma	(12)
6	F17	2 (4f, 29)	Exon 8	g.59702_59703dup	p.Asn258ArgfsX7	Ul	Yes	Enucl	Epithelial	Pa (56)
7	F17		Exon8	g.59702_59703dup	p.Asn258ArgfsX7	Bil	No	RT	Epithelial	Bl (51)
8	F17		Exon8	g.59702_59703dup	p.Asn258ArgfsX7	Bil	No	RT + CH	Epithelial	L (39)
9a	F4	0	Exon 10	g.64348C>T	p.Arg320X	Bil	Yes	Enucl	Epithelial	C (49)
10 a	F4		Exon 10	g.64348C>T	p.Arg320X	Bil	No	RT	Melanoma	(25)
11a	F5	2 (26, 1f)	Exon 11	g.65386C>T	p.Arg358X	Bil	Yes	RT + CH	Sarcoma	LMS (41)
12	F6	2 (47, 41)	Exon 12	g.70261C>T	p.Gln383X	Ul	Yes	Enucl	Epithelial	Bl (62)
13a	F8	3 (26, 11, 49)	Exon 14	g.76430C>T	p.Arg445X	Bil	Yes	Enucl	Epithelial	L (65)
14 a	F8		Exon 14	g.76430C>T	p.Arg445X	Bil	No	Enucl	Melanoma	(23)
15 a	F8		Exon 14	g.76430C>T	p.Arg445X	Bil	No	RT	Melanoma	(37)
16 a	F8		Exon 14	g.76430C>T	p.Arg445X	Bil	No	RT	Melanoma	(45)
17 a	F8		Exon 14	g.76430C>T	p.Arg445X	Bil	No	RT	Melanoma	(21)
18a	F23	NA	Exon 15	g.76898C>T	p.Arg467X	Bil	No	RT + CH	Melanoma	(28)
19a	F9	1 (54)	Exon 17	g.78238C>T	p.Arg552X	Bil	Yes	RT	Sarcoma	LMS (33)
20 a	F9		Exon 17	g.78238C>T	p.Arg552X	Bil	No	RT + CH	Osteosarcoma	(12)
21a	F10	0	Exon 18	g.150037C>T	p.Arg579X	Ul	Yes	Missing	Other	(59)
22 a	F10		Exon 18	g.150037C>T	p.Arg579X	Bil	No	RT	Melanoma	(35)
23a	F24	NA	Exon 18	g.150037C>T	p.Arg579X	Bil	No	RT	Osteosarcoma	(10)
24	F25	NA	Exon 19	g.153211T>A	p.Tyr606X	Bil	No	RT	Melanoma	(35)
25	F13	3 (42f, 70, 51)	Exon 20	g.156787_156788 delGC	p.Thr687ProfsX4	Bil	No	Enucl	Epithelial	O (72)
26	F13		Exon 20	g.156787_156788 delGC	p.Thr687ProfsX4	Ul	No	Enucl	Sarcoma	Unknown location (50)
27	F14	2 (53, 50)	Exon 21	g.160832G>T	p.Glu737X	Ul	Yes	Enucl	Epithelial	O (58)
28	F14		Exon 21	g.160832G>T	p.Glu737X	Bil	No	RT	Osteosarcoma	(20)
29a	F27	NA	Exon 23	g.162237C>T	p.Arg787X	Bil	No	RT + CH	Sarcoma	LMS (32)
30a	F28	NA	Exon 23	g.162237C>T	p.Arg787X	Bil	No	RT	Sarcoma	Lipo (12)
31	F26	NA	Exon 23	g.162266delC	p.Leu797TyrfsX13	Bil	No	RT + CH	Osteosarcoma	(26)
Splice mutation
32	F7	1 (29)	Exon 13	g.73869G>A	p.Gln444Gln exon 13 skipped	Bil	Yes	RT	Epithelial	L (48)
33	F20	NA	Intron 6	g.45867G>A/ IVS6 + 1G>A	n.i.	Bil	No	RT	Epithelial	B (57)
34	F21	NA	Intron 14	g.76491G>T/ IVS14 + 5G>T	n.i.	Bil	No	RT	Melanoma	(51)
35	F19	NA	Intron 17	g.149997G>A/ IVS17 − 1G>A	n.i.	Bil	No	RT	Sarcoma	Rhab (8)
36	F1	0	Intron 20	g.160729G>C/ IVS20 − 1G>C	n.i.	Bil	Yes	RT	Epithelial	Bl (62)
37	F1		Intron 20	g.160729G>C/ IVS20 − 1G>C	n.i.	Bil	No	RT	Sarcoma	His (31)
38	F1		Intron 20	g.160729G>C/ IVS20 − 1G>C	n.i.	Bil	No	RT	Epithelial	B (43)
Large rearrangements
39	F15	1 (5)	Duplica- tion exon 3	g.39446-?_39561 + ?dup	n.i.	Ul	Yes	Enucl	Melanoma	(31)
40	F3	2 (45, 9)	Deletion RB1	c.-138-?_27841 + ?del	–	Bil	No	RT	Sarcoma	Rhab (11)
41	F2	2 (17, 15)	Deletion exon 10/11	Del exon 10 or 10 and 11	n.i.	Bil	Yes	RT	Melanoma	(31)
42	F29	NA	Deletion exon 3-17	g.39446-?_78279 + ?del	n.i.	Bil	No	RT	Sarcoma	LMS (32)
43	F30	NA	Deletion exon 6-17	g.45799-?_78279 + ?del	n.i.	Bil	No	RT	Epithelial	Seb (38)
44	F18	2 (68, 42)	Deletion exon 9–27	g.61730-?_g177078 + ?del	n.i.	Ul	Yes	Enucl	Epithelial	C (57)

RB retinoblastoma, NA not applicable (i.e. sporadic retinoblastoma patient, n.i. not investigated), Bil bilateral disease, Ul unilateral disease, RT radiation therapy, CH chemotherapy, Enucl enucleation

B breast cancer, Bl bladder cancer, Pa pancreatic cancer, L lung cancer, C colon cancer, LMS leiomyosarcoma, O ovarian cancer, Lipo liposarcoma, Rhab rhabdomyosarcoma, His histiocytoma, Seb sebaceous adenocarcinoma

a Carriers of a recurrent stop mutation

b Italicized cases represent family members also affected with a second primary malignancy

c Family numbers 1–18: have more affected family members with RB; family numbers 19–31: sporadic retinoblastoma patients

d Reference sequence GenBank #L11910

e Underlined types of second primary malignancies represent tumours diagnosed inside the field of radiation; defined as tumor in the eye lids, orbits, periocular sinuses, temporal bones, or skin overlying the temporal bone region

f Died of other cause than second primary cancer

We assessed the risk of second malignancy in relation to type of mutation by multivariable Cox model analysis, adjusted for age and therapy. This showed that subjects carrying one of the recurrent nonsense mutations had a significantly elevated risk of developing second malignancies, compared to subjects carrying other mutations (hazard ratio [HR], 3.53; [95% confidence interval (CI), 1.82–6.84]; P = .000). Since 5 members of family 8 developed a second malignancy, we did the same analysis while excluding this family. This did not significantly change the outcome (HR, 3.17; [95% CI, 1.50–6.69]; P = .002). Leaving both family 8 and all low penetrance mutations out of the analysis, showed a lower but still statistically significantly increased risk for recurrent nonsense mutations as compared to other mutations (HR, 2.46; [95% CI, 1.14–5.28]; P = .02).

Table 3 shows the recurrent nonsense mutations known in the RB1 gene, and displays which of these mutations are found in our cohort in relation to the number of patients and the number of second primary cancers in these patients.

Table 3

List of the 11 recurrent RB1 nonsense mutations, the number of patients in our cohort carrying the mutation and the number of patients with this mutation who developed a second primary tumor (SPT)

Exon	Mutation	Protein	Number of patients in our cohort	Number of patients who developed a SPT
8	g.59683C>T	Arg.251X	2	0
8	g.59695C>T	Arg.255X	1	1
10	g.64348C>T	Arg.320X	5	2
11	g.65386C>T	Arg.358X	3	1
14	g.76430C>T	Arg.445X	11	5
14	g.76460C>T	Arg.455X	8	0
15	g.76898C>T	Arg.467X	1	1
17	g.78238C>T	Arg.552X	3	2
17	g.78250C>T	Arg.556X	5	0
18	g.150037C>T	Arg.579X	7	3
23	g.162237C>T	Arg.787X	3	2
Total			49	17

A statistically significantly decreased risk for a second primary malignancy was found in the 52 patients with a low penetrance mutation when compared to other mutations (HR, .19; [95% CI, .05–.81]; P = .025). Of all 34 patients carrying a splice mutation, eleven were carrier of a low penetrance mutation according to our definition, i.e. a DER ≤1.5. None of the carriers of a low penetrance splice mutation developed a second primary cancer. Out of 21 carriers of a deletion involving the whole RB1 gene, just one developed a sarcoma and one out of seven carriers of a mutation in exon 1 developed breast cancer at the age of 59. No second malignancies were observed in carriers of a RB1 promoter or missense mutation (n = 13).

Discussion

Our study is the first to examine the association between specific RB1 germline mutations and the risk of second primary malignancies in a nationwide well-documented cohort. Adjusting for age and therapy, we found a higher second primary malignancy risk for retinoblastoma subjects carrying a recurrent nonsense mutation, and a lower risk for carriers of a low penetrance mutation.

We first compared the risk of second primary malignancies for carriers of recurrent nonsense mutations to all other mutations in the RB1 gene. Because one family (F8) with many family members affected by a second malignancy may have influenced the outcome too much, we excluded this family from the analysis. This still showed a statistically significantly increased risk. It is remarkable that 4 members of this family developed a melanoma. This could be due to common genetic background, though this phenomenon does not hold true for other families. As far as we are aware, the family does not display any signs of dysplastic nevus syndrome. We further hypothesized that the increased risk of second malignancies for recurrent mutations compared to all other mutations may have been caused by a substantial contribution of the lower risk of second malignancies for low penetrance mutations, included in the comparison. Therefore we also left the low penetrance mutations out of the analysis. This still showed a significantly increased risk of second primary cancers for recurrent nonsense mutations. Three recurrent nonsense mutations did not demonstrate any second malignancies in our study cohort (Table 3). Whether these mutations do not lead to a higher second malignancy risk, needs to be clarified in future studies.

What could be the cause of the higher risk for second malignancies in carriers of recurrent nonsense mutations? Nonsense and frameshift mutations are associated with bilateral retinoblastoma and high (>90%) penetrance [19], irrespective of the location of the premature stop mutation. This is attributed to nonsense mediated mRNA decay (NMD): a mechanism of mRNA surveillance that prevents the expression of truncated proteins, by breaking down mutant mRNA containing a premature termination codon [24]. NMD can be beneficial, eliminating truncated transcripts that could lead to proteins with possible dominant negative or gain-of function effects, but may also be harmful when preventing translation of truncated protein that would otherwise still be partly functional [25, 26]. Studies have also shown that NMD efficacy may vary between tissues [27, 28]. An explanation for our findings could be that specific nonsense mutations escape NMD in certain tissues, which will result in the expression of a truncated protein. This truncated protein may either have residual activity resulting in a milder effect or may have a dominant negative effect, as has been described for other tumor-suppressor genes [25, 29]. The higher risk in recurrent nonsense mutation carriers may then be explained by a differential effect of NMD between these mutations and other truncating mutations. How such a differential effect would specifically exist between the two different types of truncating mutations remains to be determined. Alternatively, while the recurrent nonsense mutations result in the loss of a 5-methylcytosine within the gene, this may affect the chromatin structure and/or expression of the gene and thereby increasing the chance of transformation. However, in spite of a clearly elevated risk for recurrent nonsense mutations in our cohort, we cannot rule out that the elevated risk is a chance finding.

Genotype-phenotype correlations of RB1 mutations have been described for specific types of mutations: certain splice mutations, promoter, exon 1 and missense mutations lead to reduced expressivity (unilateral retinoblastoma) and incomplete penetrance (unaffected carriers) of retinoblastoma [18, 19, 21, 23, 30]. This is attributed to a reduction in the amount of normal protein that is produced or to residual activity of mutant protein [18, 19]. Reduced expressivity and incomplete penetrance have also been described for deletions of the complete RB1 gene. This is thought to be caused by co-deletion of adjacent unknown genes leading to a greater chance of apoptosis, when the wildtype allele is lost as the second hit in the tumor [20]. In line with reduced expressivity in the retina, we demonstrated a lower risk of second primary cancers for carriers of these low penetrance mutations. In our study group none of the carriers (n = 24) of a missense, promoter or low penetrance splice RB1 mutation developed a second malignancy, and just one out of 7 carriers with a mutation in exon 1 developed another cancer, i.e. breast cancer at age 59. The latter may also be attributed to the high population risk of breast cancer. A lower risk for second primary malignancies was noted for carriers of a complete deletion of the RB1 gene as well: only one out of 21 carriers developed a second malignancy (i.e. rhabdomyosarcoma at age 11).

Strength of our study is the unique large data set of genotyped survivors of retinoblastoma from a population based cohort. Very few studies on genotype-phenotype relations of RB1 mutations mention second primary cancers. Two studies on mutations in de RB1 gene stated that they could not detect an association between the mutation and manifestation of a second primary cancer or tumor type [21, 31]. These studies included only a few patients with second primary cancers, however. Some limitations of our study should be considered. First, our mutation detection rate was 90% for all familial and bilateral cases at the time of the study. Leaving 10% of hereditary patients out of the analysis may have influenced the outcome. Some mutations may have been missed, because they are present in a mosaic state; others because they might be located deep in an intron. Second, some types of mutations (e.g. promoter mutations) are relatively rare and therefore some mutation type subgroups were small, making it difficult to draw firm conclusions. A third limitation is that RB1 mutation detection has become available just 20 years ago. Although many patients from the Dutch retinoblastoma cohort have been genotyped in the past years, this study still comprises a relatively young group of retinoblastoma subjects; almost 50% of all known patients with hereditary retinoblastoma could be included. Quite a few patients diagnosed with (osteo)sarcoma had already died before it was possible to perform DNA analysis. The exclusion of these (osteo)sarcomas may have limited our ability to detect possible associations between specific mutations and sarcoma risk.

In conclusion, our results suggest a genotype-phenotype correlation for second primary malignancies of retinoblastoma survivors and may impact on long-term surveillance protocols of patients with hereditary retinoblastoma, if confirmed by future studies.