TSC2 mutations confer everolimus sensitivity in hepatocellular carcinomas

Hepatocellular carcinomas (HCCs) are the third leading cause of cancer deaths globally; frequently diagnosed only in the advanced stages and aggressive in nature. Although enhanced mTOR activity has a key role in HCC tumourigenesis, the EVOLVE-1 clinical trial of mTOR inhibitor everolimus found no associated improvement in overall survival (Zhu et al., 2014). However, everolimus is an effective treatment for tuberous sclerosis complex (TSC) manifestations, a rare disease associated with mutations in TSC1 and TSC2 that result in high mTOR activity. New research from Huynh et al. (2016) assessed the frequency of TSC2  loss in HCC and suggests this could predict a selective response to everolimus.

Expression of TSC2 was found to be markedly reduced in 5/9 HCC cell lines assessed (Cancer Cell line Encyclopedia collection). Huynh et al. confirmed the loss of TSC2 by immunoblot in four of these lines which showed associated increased mTOR activity and decreased AKT phosphorylation. The four TSC-null cell lines showed enhanced sensitivity, with greater inhibition of cell proliferation, to everolimus than four TSC-wildtype cell lines. Everolimus treatment also reversed the enhanced phosphorylation of S6K1, indicative of mTOR inhibition.

Huynh et al. then assessed the frequency of TSC2 mutations in patient derived xenografts. In 8/26 xenografts TSC2 was undetectable with associated increased S6K1 phosphorylation. When tumour-bearing mice where treated with 1mg/kg everolimus significant anti-tumour responses were seen in the TSC2-null tumours compared to those treated with vehicle only. In contrast the everolimus treated TSC-wildtype tumours showed little or no response indicative of selective efficacy rather than general toxicity. Treatment with everolimus also showed a dose-dependent reduction in S6K1 phosphorylation and increase in AKT activity.

Genetic sequencing of the TSC-null HCC cell lines and xenografts identified a range of mutations and deletions in 3/4 and 6/8 samples respectively. Epigenetic gene silencing was hypothesised to explain the loss of TSC2 in the remaining samples. To validate the existence of TSC2 mutations in primary tumours 13 HCC biopsies were also sequenced and three found to carry TSC2 mutations. Although sequencing can identify mutations it cannot always predict protein loss. Instead Huynh et al. developed an immunohistochemistry (IHC) assay, validated in the HCC cell lines and xenografts, where TSC2 protein levels could be quantified, and samples categorised as either TSC-null, TSC-low or TSC-wildtype. .

This IHC assay was then used to assess tumour samples from 139 patients enrolled on the EVOLVE-1 trial; eight samples were found to be TSC-null and seven to be TSC-low. Detailed assessment of overall survival in these patients determined that 6/10 treated with everolimus had an overall survival (OS) greater than the median for the whole trial: 9.53-32.72 months compared to 7.56 months. Three of the other patients receiving everolimus withdrew after 2-6 weeks treatment (OS 0.76-4.63 months). Comparatively those patients with TSC2 loss who received the placebo had a lower OS of 1.25-5.59 months. This prospective analysis supports the previous results suggesting that everolimus is more effective against tumours that lack TSC2.

Tumour heterogeneity can make treatment responses difficult to predict but understanding the molecular basis of tumourigenesis can help identify treatment targets. Everolimus has also been shown to be effective against other tumour types carrying TSC1 or TSC2 mutations including renal tumours (Voss et al., 2014), metastatic bladder cancer (Iyer et al., 2012) and thyroid tumours (Wagle et al., 2014). This highlights a need to design clinical trials, like basket and umbrella trials, that use the advances in genetic sequencing to stratify patients for a greater understanding of treatment efficacy.

This research also demonstrates how greater understanding of a protein associated with a rare inherited disease can affect the treatment of patients with a sporadic condition. Mutations associated with other cancer-predisposition conditions such as BHD and HLRCC have also been reported in sporadic tumours and could progress the development of more effective treatments based on cancer genetics rather than location.

  • Huynh H, Hao HX, Chan SL, Chen D, Ong R, Soo KC, Pochanard P, Yang D, Ruddy D, Liu M, Derti A, Balak MN, Palmer MR, Wang Y, Lee BH, Sellami D, Zhu AX, Schlegel R, & Huang A (2015). Loss of Tuberous Sclerosis Complex 2 (TSC2) Is Frequent in Hepatocellular Carcinoma and Predicts Response to mTORC1 Inhibitor Everolimus. Molecular cancer therapeutics, 14 (5), 1224-35 PMID: 25724664.
  • Iyer G, Hanrahan AJ, Milowsky MI, Al-Ahmadie H, Scott SN, Janakiraman M, Pirun M, Sander C, Socci ND, Ostrovnaya I, Viale A, Heguy A, Peng L, Chan TA, Bochner B, Bajorin DF, Berger MF, Taylor BS, Solit DB (2012). Genome sequencing identifies a basis for everolimus sensitivity. Science. 338(6104):221 PMID: 22923433.
  • Voss MH, Hakimi AA, Pham CG, Brannon AR, Chen YB, Cunha LF, Akin O, Liu H, Takeda S, Scott SN, Socci ND, Viale A, Schultz N, Sander C, Reuter VE, Russo P, Cheng EH, Motzer RJ, Berger MF, Hsieh JJ (2014). Tumor genetic analyses of patients with metastatic renal cell carcinoma and extended benefit from mTOR inhibitor therapy. Clin Cancer Res. 20(7):1955-64. PMID: 24622468.
  • Wagle N, Grabiner BC, Van Allen EM, Amin-Mansour A, Taylor-Weiner A, Rosenberg M, Gray N, Barletta JA, Guo Y, Swanson SJ, Ruan DT, Hanna GJ, Haddad RI, Getz G, Kwiatkowski DJ, Carter SL, Sabatini DM, Jänne PA, Garraway LA, Lorch JH (2014). Response and acquired resistance to everolimus in anaplastic thyroid cancer. N Engl J Med. 371(15):1426-33. PMID: 25295501.
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