Methods

STUDY POPULATION

We selected 15 consecutive patients with precancerous laryngeal conditions who were treated by microlaryngoscopy and vocal cord stripping with biopsy, but who had received no further treatment except close follow up, and experienced a progression to squamous cell carcinoma of the larynx at least one year after the first biopsy. There were 13 men and two women, with a median age at initial diagnosis of 61 years (range, 37–73). The histological aspect of each lesion was reviewed, and an assessment of the epithelial modifications was made according to the criteria of Friedmann.¹⁹ There were seven cases of simple hyperplasia, four of mild dysplasia, two of moderate dysplasia, and two of severe dysplasia. The control group consisted of 30 patients with laryngeal lesions, age, sex ratio, morphology, and risk factor exposure (smoking history and alcohol consumption) comparable with those of the study population and who had no progression of the disease during a follow up period similar to that of the first group (median, 7 years; range 1.8–11 years), after initial microlaryngoscopy and vocal cord stripping with biopsy.

IMMUNOHISTOCHEMISTRY

The immunohistochemical studies were performed on formalin fixed, paraffin wax embedded sections, using the avidin–biotin complex technique (Dako, Milan, Italy). To identify alterations in TGF-β RII expression, we used a rabbit polyclonal antibody that recognises an internal epitope of the receptor and is suitable for formalin fixed, paraffin wax embedded tissue sections (L-21; Santa Cruz Biotechnologies, Santa Cruz, California, USA). Negative controls were performed by substitution of the primary antibody with non-immune rabbit serum.

The results of the immunohistochemical staining were scored semiquantitatively, and a case was considered to show loss of TGF-β RII expression when < 20% of epithelial cells showed a positive reaction.

MICROSATELLITE ANALYSIS

Serial paraffin wax sections (12 μm thick) obtained from 18 cases (eight with progression to squamous cell carcinoma and 10 without) with enough biopsy material to allow microdissection were placed on a glass slide and individually microdissected with the use of a dissecting microscope to separate epithelial cells from stromal cells. The same procedure was applied to serial sections of the eight squamous cell carcinomas that followed the preneoplastic lesions. Samples were collected into 1.5 ml polypropylene tubes, containing 1 ml xylene, incubated at 37°C for 15 minutes and pelleted at full speed in a microcentrifuge for 15 minutes. The xylene was then removed and the pellet was washed twice in absolute ethanol (1 ml). The samples were thoroughly dried under a sterile hood and 100 ml of digestion buffer, containing 1 M Tris/HCl (pH 8.0), 0.5 M EDTA, 0.02% Tween 20, and 100 mg/ml proteinase K (Quiagen Inc, Valencia, California, USA), was added to each tube. After an incubation of three hours at 55°C, proteinase K was inactivated at 95°C for 10 minutes, and the samples were pelleted at full speed in a microcentrifuge. The supernatant was stored at −20°C until use. For each polymerase chain reaction (PCR) 1 μl of a 1/10 dilution of each sample was used. The primers used for the molecular analysis were: RII U1, 5′-AGA TGCTGCTTCTCCAAAGTGC-3′; RII D1, 5′-TTGCACTCATCAGAGCTACAGG-3′ for the poly(A) microsatellite sequence (nucleotides 709–718) and RII U2, 5′-ACTGAG TGCTGGGACCACG-3′; RII D2, 5′-AGG AATCTTCTCCTCCGAGC-3′ for the poly (GT) microsatellite sequence (nucleotides 1931–1936).²⁰

The PCRs were carried out in a volume of 30 μl and included template derived from paraffin wax sections, 1× PCR buffer, 1.5 mM MgCl₂, 250 μM of each dNTP, 0.5 U Platinum^™ Taq DNA polymerase (Life Technologies, Rockville, Maryland, USA), and 3 pmol of each primer. The thermal profile was as follows: 94°C for two minutes, 35 cycles of 94°C for one minute, 56°C for one minute, and 72°C for one minute, then a final extension of 72°C for five minutes.

Amplified PCR fragments were analysed by 8–12% polyacrylamide gel electrophoresis and run at 20 W for three to four hours. Bands were detected by silver staining as described previously.²¹

STATISTICAL ANALYSIS

Two tailed Fisher's exact test was used to analyse the correlation between TGF-β RII expression and clinicopathological features of the preneoplastic lesions.

Results

IMMUNOHISTOCHEMISTRY

Strong and diffuse immunostaining for TGF-β RII was seen in normal structures present in laryngeal mucosa, such as vascular endothelial cells and mucous glands, which served as an internal positive control (fig 1). Considering the squamous epithelium, 16 lesions showed loss (< 20% of cells) of receptor expression (fig 1). Preneoplastic lesions showing moderate or severe dysplasia tended to show more frequent loss of receptor expression in comparison with those showing simple hyperplasia or mild dysplasia (66.6% v 42.8%), although the difference was not significant (p = 0.5). In the group of precancerous lesions with progression to carcinoma, 11 of 15 cases showed loss (< 20% of epithelial cells) of TGF-β RII expression (fig 1), whereas among lesions without evolution only five of 30 had similar altered expression of the receptor (p < 0.001, two tailed Fisher's exact test).

In the group of squamous cell carcinomas, 12 of 15 cases showed loss of TGF-β RII expression. Overall, there was a good correspondence with regard to loss of TGF-β RII expression between preneoplastic lesions and subsequent squamous cell carcinomas—in only one instance did we observe loss of receptor expression (< 20% of neoplastic cells) in the squamous cell carcinoma that was not evident in the corresponding preneoplastic lesion.

ANALYSIS OF TGF-β RII GENE

Of the 18 cases analysed, a mutation of the poly(A) sequence of the TGF-β RII gene was identified in one precancerous lesion; the same alteration was present in the subsequent squamous cell carcinoma (fig 2). Tissue samples from these lesions showed loss of TGF-β RII immunostaining. Conversely, the region of the GT repeat sequence was conserved in all cases examined.

Discussion

Our results confirm that the loss of TGF-β RII expression occurs frequently in squamous cell carcinoma of the larynx, being present in 80% of cases. Such findings are comparable with those reported previously in head and neck squamous cell carcinomas, which presented pronounced or complete loss of the receptor in up to 90% of cases.¹⁷ In addition, the degree of receptor expression appeared to be inversely correlated with the degree of differentiation of the tumour and with the depth of the invasion.¹⁸ A similar reduction has been noted for TGF-β RII mRNA in head and neck squamous cell carcinomas, thus indicating that the decreased protein concentrations depend upon abnormal gene transcription, and that a post-transcriptional mechanism is less likely to be responsible.^{18, 22}

To investigate whether anomalies of TGF-β receptor expression are present in the early stages of laryngeal carcinogenesis and to assess their potential importance in the development of these tumours, we examined the TGF-β RII status in laryngeal premalignant lesions coupled with malignant evolution in comparison with a control group of similar lesions without progression to cancer. According to our analysis, the frequency of loss of TGF-β RII is much higher in preneoplastic lesions with subsequent progression to cancer than in those without evolution, indicating that this event may have an important role in the development of squamous cell carcinoma of the larynx. Indeed, disregulation of the TGF-β pathway could cooperate with other genetic changes, such as alterations of p16, p53, and cyclin D1 expression in the early phases of laryngeal carcinogenesis.^{23, 24} All these events involving key regulators of the cell cycle could result in alteration of growth control of epithelial cells, finally leading to cancer promotion.

Several mechanisms might be responsible for TGF-β RII gene silencing in cancer. First, this gene contains two microsatellite loci that are frequently altered in colorectal and gastric cancers arising in patients with DNA mismatch repair deficiencies. These mutations, however, are quite rare or absent in sporadic carcinomas of the uterine cervix, pancreas, and in squamous cell carcinomas of the head and neck.^{22, 25–27} In accordance, in our series we could find only one such alteration involving the polyadenine region, which occurred in one preneoplastic lesion and in the subsequent squamous cell carcinoma.

Therefore, other genetic alterations are likely to occur in head and neck squamous cell carcinomas, which may explain the high frequency of the loss of receptor expression. Human head and neck squamous cell carcinoma cell lines have been shown to contain G : C → C : G transversions in the kinase domain of TGF-β RII, which may alter TGF-β RII activity, leading to resistance to the inhibitory control of TGF-β.⁸ Missense mutations and deletions of the serine/threonine kinase domain of TGF-β RII have also been identified in six of 28 head and neck squamous cell carcinomas in vivo.²⁷ Finally, an alternative mechanism that might lead to altered expression of the gene is hypermethylation within the promoter or other transcription regulatory sequences, which has been documented to occur in oesophageal carcinomas.²⁷ Further studies using fresh tissue samples will be necessary to determine the contribution of these mechanisms to TGF-β RII gene inactivation in laryngeal preneoplastic lesions and squamous cell carcinomas.

In conclusion, our findings indicate that precancerous laryngeal lesions frequently show altered expression of TGF-β RII, with a clear prognostic relevance for tumour progression, thus underlining the potential role of TGF-β RII immunostaining in the assessment of the risk for evolution of preneoplastic laryngeal lesions. If this is confirmed in prospective studies with larger patient populations, TGF-β RII immunostaining could be included among the biomarkers potentially useful for selecting patients for chemoprevention²⁸ or for endoscopic screening protocols.