MATERIALS AND METHODS

Patients

Blood samples and/or paraffin embedded tumour samples were obtained for DNA solation from available subjects that had developed a carcinoma, to determine their p16-Leiden mutation status. Unavailable subjects with p16-Leiden positive offspring were classified as “obligate carriers”. Informed consent was given by family members themselves or by their relatives, in case of deceased subjects. Tumours were pathologically verified whenever possible.

Tumour analysis

Paraffin embedded tumour tissues were obtained, and revision of histology was performed. Areas of highest tumour density were selected for further molecular analysis. Serial sections were produced for immunohistochemical analysis.

DNA isolation

Genomic DNA of normal and tumour tissue was isolated from formalin fixed paraffin embedded material, resuspended in 96 μl of PK-1 lysis buffer (50 mmol/l KCL, 10 mmol/l Tris pH 8.3, 2.5 mmol/l MgCl₂, 0.45% NP40, 0.45% Tween 20, 0.1 mg/ml gelatine) containing 5% Chelex beads (Biorad, Hercules, CA, USA) and 5 μl proteinase K (10 mg/ml), and incubated for 12 h at 56°C. The suspension was incubated for 10 minutes at 100°C, centrifuged, and the supernatant carefully decanted.

Polymerase chain reaction amplification

The p16-Leiden deletion comprises 19 bp and removes nucleotides 225–243 of exon 2.¹⁰ Genomic DNA from tumour and normal tissue was subjected to PCR amplification using labelled primers containing the 225–243 region; p16-forward-TET M1 (tumour) or FAM M1 (normal), sequence 5′-ATGATGGGCAGCGCCCGAGT-3′ and p16-reverse A2, sequence 5′-ACCAGCGTGTCCAGGAAG-3′ (Life Technologies). The total volume per reaction was 12 μl including 5 μmol of each primer (stock forward and reverse primer), a mix of 0.25 μl dNTP (10 mmol/l), 1.2 μl magnesium chloride (20 mmol/l), 1.2 μl bovine serum albumin (1 mg/ml), 1.2 μl AmpliTaq Gold buffer (without MgCl₂) and 0.25 μl AmpliTaq Gold DNA polymerase, 10 ng of normal or tumour DNA, and H₂O. The following conditions were used: 33 cycles of 1 minute at 96°C, 2 minutes at 55°C, 1 minute at 72°C, and a delayed extension step of 7 minutes at 72°C in a GeneAmp 9700 thermocycler (Applied Biosystems, Foster City, CA, USA). Mixtures of 24 μl dionised formamide, 1 μl TAMRA 500 (Applied Biosystems) and 1.2 μl of PCR product were run on a ABI 310 Genetic analyser (Applied Biosystems) for 20 minutes with run profile GS STR POP4 (1.0 ml) C and analysed with Genescan 3.1 computer software (Perkin-Elmer Corp).

Loss of heterozygosity analysis

Owing to the 19 bp deletion, we could specifically analyse the fate of the wild type allele in terms of LOH. Analysis of LOH was possible when both normal and tumour tissue was available. LOH was scored when there was loss of intensity of one allele in the tumour sample with respect to the matched wild type allele from normal tissue. The quotient of the peak height ratios from normal and tumour DNA served as the allelic imbalance factor (AIF)—that is, the ratio of the peak height at 101 bp of the deleted allele and the peak height at 120 bp of the wild type allele. The threshold for allelic imbalance was defined as 40% reduction of one allele, agreeing with an AIF of ⩽0.59 or >1.3. The threshold for retention was defined to range from 0.76 to 1.3 as previously empirically determined.¹¹ AIFs of 0.60–0.75 and 1.3–1.69 were considered to belong to a so-called grey area, for which no definitive decision has been made.

Immunohistochemical testing for p16

Tissue sections (4 μm) were prepared on APES coated slides, and dried overnight in a 37°C oven. Sections were deparaffinised in xylene (3×5 minutes). Endogenous peroxidase was blocked by incubation in methanol/H₂O₂ 0.3% for 20 minutes and sections were rehydrated with ethanol and distilled water. Antigen retrieval for p16 immunostaining was performed by microwaving in boiling 0.01 mol/l sodium citrate buffer (pH 6.0) for 10 minutes. After cooling for 2 hours and washing (2×5 minutes) in PBS, the sections were incubated overnight at room temperature with mouse anti-human p16 (1:500, clone JC8; Neomarkers Fremont, CA, USA) with tonsil tissue as positive control. Sections were subsequently washed (3×5 minutes in PBS) and incubated (30 minutes) with biotinylated secondary antibody in PBS/BSA 1%, washed (3×5 minutes in PBS) and incubated (30 minutes) with a horseradish peroxidase/streptavidin complex (SABC). Diaminobenzidine-tetrahydrochloride (DAB) was used as a chromogen, followed by counterstaining with haematoxylin. As a negative control, the primary antibody was omitted. Expression was scored by microscopic examination. Loss of p16 expression was scored when nuclei of tumour cells stained negative and nuclei of normal (stromal) cells stained positive (internal positive control).

BRCA1 and BRCA2 mutation screening

As described above, we were able to obtain tumour material of five p16-Leiden carriers with breast cancer. Three (NFDHT 1–3, table 1) had no first or second degree relative with breast cancer. The other two (EMC 13769 No 50 and LUMC 152, table 1) had several relatives with breast cancer diagnosed before the age of 60 years. Complete BRCA1 and BRCA2 mutation analysis was performed in the suspect families (EMC 13769 and LUMC 152) and found to be negative. We screened for germline mutations frequently detected in the Dutch population. Protein truncation tests¹² were also performed for PCR fragments of exon 11, and denaturating gradient gel electrophoresis was performed for the remaining exons and exon/intron junctions of BRCA1 and BRCA2. Additionally we screened for the deletions of exon 13 (3.8 kb) and exon 22 (510 bp) of BRCA1.¹³

Microsatellite instability

Microsatellite instability was analysed in a diagnostic setting as previously described using markers D2S123, D5S346, D17S250, BAT25, BAT26, and BAT40,¹⁴ and immunohistochemical testing for MLH1, MSH2, and MSH6 was performed.¹⁵

RESULTS

Lung cancer

We analysed four different p16-Leiden families (table 1, fig 1) with one or more cases of lung cancer. Family EMC13769 (fig 1) harbours four cases of lung cancer. One subject was a proven carrier of a germline p16-Leiden mutation (subject 51), two subjects are obligate carriers, and the p16-Leiden carrier status remains unknown for one (subject 38). The p16 immunohistochemistry analysis in the tumour of subject 51, a smoker, tested negative, and LOH of the wild type allele was found. The three other (NFDHT) families harbour 4 p16-Leiden mutation carriers with documented lung cancers. The immunohistochemistry analysis for p16 was negative in three analysed lung tumours. LOH of the wild type allele was ambiguous in one tumour, and in one tumour a (carcinoid) retention was found (table 1). In the other two tumours no normal tissue was available to perform the analysis.

Oral squamous cell carcinoma (OSCC)

Two subjects of family EMC13769 had a tumour originating in the oral cavity—that is, one tongue carcinoma (subject 36 at 65 years of age) and one subject with three primary OSCCs (subject 48 at 49 years). Immunohistochemical analysis of the tongue carcinoma was negative for p16 but lacked an internal positive control, and LOH analysis was not possible. Immunohistochemical analysis of the one of the three OSCCs from subject 48 (fig 1) tested negative for p16, and LOH of the wild type allele in this tumour was found (table 1).

Breast cancer

We analysed five families with breast cancer. Family EMC 13769 shows five cases of breast cancer. Only one was carrying the p16-Leiden mutation (subject 50). Germline mutations in BRCA1 & BRCA2 were excluded for subjects no 41, 50 and 67. The p16 protein in the tumour from EMC13769 subject 50 stained positive and no LOH was found (table 1). Of the four additional typed breast carcinomas from p16 mutation carriers from the families LUMC 152 and NFDHT 1–3 (table 1), only one showed expression loss of the p16 protein with LOH of the wild type allele, although in two of four other analysed breast carcinomas only a few tumour nuclei stained positive (with the retention of the p16 wild type allele in one, the other not tested).

Digestive tract

Family EMC13769 harbours six cases of carcinomas of the digestive tract. Of the two tumours analysed (both patients had a germline p16-Leiden mutation), one tumour stained positive and one negative. Neither showed LOH (table 1), nor microsatellite instability (microsatellite stable phenotype of the tumours with normal expression of MLH1, MSH2, and MSH6).

DISCUSSION

All lung and oral cavity tumours studied developed (most likely) in p16-Leiden mutation carriers. For two persons we cannot rule out the possibility that the p16-Leiden germline mutation in their offspring came from the non-bloodline spouses. However, as this family does not come from the “Dutch region” where multiple p16-Leiden mutation carriers have been identified, we think that they are most probably obligate carriers of the same p16-Leiden mutation. The age of onset in most patients is unusually young and abrogation of p16 seems present in all analysed cases (4/4), a ratio that seems higher than that encountered in sporadic lung cancer (36–45%).¹⁶ The p16-Leiden mutation might therefore indeed predispose carriers to an increased risk of lung and oral cavity carcinomas. With respect to lung cancer, this is supported by two other important observations. Firstly, an increased cumulative risk of developing lung cancer in male p16-Leiden mutation carriers was found compared with the general Dutch population (14.3% v 8.9%).⁴ Secondly, Cdkn2a is the most likely candidate for the lung tumour susceptibility locus pulmonary adenoma progression gene 1 (Papg1) in mice.^17,18Papg1 has been mapped to a 1.5 cM segment on chromosome 4, which contains the Cdkn2a gene that encodes p16^INK4a. Cdkn2a is polymorphic between the lung tumour resistant mouse strain BALB/cJ and the lung tumour susceptible A/J strain, and the resistant allele is preferentially lost in lung tumours of p16^INK4a heterozygous mice. Additionally, germline deletion of the gene in mice leads to increased tumour size and notable histological signs of malignant progression.¹⁷

Sufficient information on the smoking habits of most subjects in our study was lacking. However, smoking may have contributed to the unusually early age of onset of three tumours, although one of the tumours is classified as an adenocarcinoma, a type not typically associated with smoking.

Our study does not provide evidence for a dominant role of p16-Leiden in the development of breast cancer. Breast cancer seems also statistically not increased in our cohort studied⁴ However, in view of the early onset of breast cancer in our p16-Leiden positive cases, we cannot rule out a role for the gene in tumour progression, either due to haploinsufficiency or total abrogation of p16 as seen in one of our cases (LUMC152). Recently, it has been postulated for other genes that mutation or loss of a single allele may be sufficient to play an important role in progression towards cancer.¹⁹ Furthermore, an interaction with as yet unidentified modifying factors (genetic and/or environmental) has yet to be elucidated.

Both analysed tumours from the digestive tract showed no LOH; however, one stained negative. In this case methylation might have inactivated the wild type allele, which is a frequent event in sporadic colon cancer.²⁰ The role of the p16-Leiden germline mutation in the development of colon cancer needs further research.

In conclusion, the p16-Leiden mutation not only seems to predispose to melanoma and pancreatic tumours but also to head and neck tumours^5–7, and tumours of the lung in some families. Promoter methylation⁵ or loss of the wild type allele seems to be the mechanism for the “second genetic hit”. Clinical criteria for p16 germline mutation screening should be adapted accordingly.

ELECTRONIC-DATABASE INFORMATION

Online Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm.nih.gov/Omim/ (for FAMMM (OMIM 155601) and p16-Leiden (OMIM 600160.0003).