Inactivation of tumor suppressor gene pten in early and advanced gallbladder cancer

For many years Chile has had the highest incidence and mortality due to GBC in the world, in both genders, and it represents the second cause of death by malignant tumors in women in Chile [1‐3]. Other Indo-American countries like Bolivia, Mexico and Ecuador follow in frequency, and in Asia countries such as India and Pakistan [2, 4]. GBC ranks 20th among all malignant tumors with an incidence of 2.2 × 10⁶ inhabitants, and 22nd for mortality (0.7 × 10⁶) with a prevalence at 5 years of 16.8 × 10⁶ inhabitants [2].

As with other epithelial malignant tumors, GBC is the result of multiple genetic alterations involving multiple genes from different metabolic pathways, the importance of which has not yet been clearly established [5‐9]. Numerous somatic mutations have been reported for GBC, of which only a small subgroup contributes to tumor progression. The distinction has been made only recently between mutations in the “driver” or controller genes of the multiple mutations in neutral or “passenger” genes that contribute in only a small way to cancer development [10, 11]. The most frequently mutated genes in GBC are TP53 (47.1 %), KRAS (7.8 %) and ERBB3 (11.8 %) (https://​www.​sanger.​ac.​uk/​). It has also been shown recently that the signaling pathway of ErbB, which includes EGFR, ERBB2, ERBB3 and ERBB4, is one of the most frequently altered in GBC [12, 13].

PTEN (phosphatase and tensin homolog) is a tumor suppressor gene located on chromosome 10q23. 3. PTEN encodes a protein with a phosphatase function which, as it removes phosphate groups, inactivates substrates and thus acts as a tumor suppressor gene [9, 14]. The functional loss of PTEN may be the result of somatic mutations, deletion or inactivation (LOH, methylation), abnormalities in transcriptional and post-transcriptional regulation, microRNAs, regulation of microRNAs, or due to alterations in the mechanisms that regulate the stability and degradation of the PTEN protein [15, 16]. The absence of the functional protein PTEN enables unfettered PI3k activity, which determines the uncontrolled production of PIP3, one of the most important effectors of the PI3K/AKT pathway, with mTOR stimulating protein synthesis and initiating onset of the G1 phase of the cell cycle and interacting with proteins that regulate apoptosis [17].

Germline loss of PTEN is the hallmark of Cowden syndrome [18], while sporadic alterations in PTEN have been observed in a broad spectrum of malignancies [19]. The multiple mechanisms that lead to PTEN inactivation render their study difficult; therefore, determining a gene’s protein expression is considered a good way to assess the functional status of the gene [20‐22]. The absence of expression is considered a reflection of inactivation of the gene, independent of its cause.

The aim of this work was to determine the distribution and inactivation frequency of PTEN in EC and AC of GBC and to establish the correlation with clinical and morphological parameters and survival. An additional aim was to consider indirectly the importance that PTEN inactivation may have in the deregulation of the PI3K/AKT pathway in GBC.

The group comprised 108 cases, the general characteristics of which are summarized in Table 1. Eighty-four percent of the cases were women with an average age of 65.1 years (SD ± 11.6 years) and the remaining 17 were men with an average of age of 66.1 years (SD ±10.1 years). All the cases were adenocarcinomas. Thirty-five cases (33 %) were EC (17 mucous carcinomas and 18 muscular carcinomas) and 73 (67 %) were AC (29 subserosal carcinomas, 24 serosal, and 20 cases beyond the serosa). The difference in the average ages between the early and advanced carcinomas, as well as the relation between the level of tumor infiltration of the wall and the progression in the average ages was significant (p < 0.001). 30.3 % of the tumors were well differentiated, with the great majority being moderately differentiated (51.5 %) or poorly differentiated (18.2 %).

The immunohistochemical staining in the 108 tumors and their respective internal controls are summarized in Table 2. In 96.3 % (104 cases) the non-tumor mucosa showed moderate or intense positivity (2+) and only 3.7 % (4 cases) were weak positive (1+). No negative cases were observed in this group. In the GBC, however, three cases (2.8 %) had an absence of expression or inactivation of PTEN (in the presence of positive internal controls) (Fig. 1). All the cases without PTEN expression were AC, all women (50, 56 and 62 years respectively), one subserosal and two serosal with survival of 3, 6.2 and 9.9 months respectively after diagnosis. The inactivation frequency in the AC patient group reached 4.1 % (3 of 73 cases). Unlike what was observed in non-tumor tissues, the adenocarcinomas had a similar proportion of positivity (1+) or weak compared to a moderate or intense positivity (2+), which could suggest a partial or incomplete degree of PTEN activity.

Actuarial survival of the group was 41 % at 5 years of follow-up. In the 73 patients with AC, the actuarial survival at 10 months of follow-up was 52 and 29 % at 5 years. However, the three patients who presented immunohistochemical inactivation of PTEN died before the 10 months. Although the number is small and does not permit a statistically valid conclusion, this fact is noteworthy (Fig. 2).

Our results show immunohistochemical expression of PTEN protein in all the cases used as internal controls, with this expression being moderate or intense from the immunohistochemical point of view in 96.3 % of the cases, which may reflect normal activity in this gene. No negative cases were observed, which may suggest some degree of PTEN inactivation in the non-tumor mucosa adjacent to the tumors. However, in the GBC there was a greater dispersion in the intensity of the positive staining, being weak in 54.5 % of the cases and moderate in 42.6 %, which may mean that the tumor tissues present a variable expression, possibly a reflection of differing degrees of PTEN activity or inactivation. This being a recessive gene, inactivation of an allele does not necessarily compromise its function entirely, as it can express itself partially [24]. In most tumors the loss of PTEN is monoallelic (gliomas, breast, colon, lung), with biallelic being less frequent [24‐27], as inactivation due to methylation of promoter areas is one of the inactivation mechanisms that occurs more frequently than genomic alterations [24, 28, 29]. Thus, the absence of PTEN expression in three cases of advanced GBC suggests is an infrequent phenomenon (4.1 % of all AC) and is also delayed since its inactivation could not be demonstrated through the loss of its expression in any of the 35 EC. There is little information about the inactivation of this gene in GBC measured using different molecular techniques. Some indicate inactivation frequencies ranging between 0 and 5 %, but this does not rule out that other PTEN inactivation mechanisms may be at work [16]. On the other hand, the poor prognosis of the three patients with an absence of PTEN protein expression compared to patients at similar stages must be pointed out [30]. In these patients, PTEN inactivation may have released the PI3k/AKT pathway, which through some of its effectors like mTOR is able to activate cell proliferation [31]. Although PTEN inactivation is observed in around 4 % of the advanced cancers, it is worthy of note that the other inactivation mechanisms in this proliferative pathway and which promote tumor growth and development have recently been reported in significant percentages (http://​cancer.​sanger.​ac.​uk, [32‐34]), which will open up important expectations for the use of selective blockers and inhibitors of this pathway as a targeted therapy in patients with advanced gallbladder cancers [35‐37]. Our recent work has shown activating mutations in exons 9 and 20 of the PI3k gene in 16.9 % of GBC, suggesting that this gene together with PTEN could be a therapeutic target in around 20 % of advanced gallbladder cancers (Roa et al., observations not published).