Hedgehog (Hh) signaling pathway is a valid therapeutic target in a wide range of malignancies. We focus here on glioblastoma multiforme (GBM), a lethal malignancy of the central nervous system (CNS). By analyzing RNA-sequencing based transcriptomics data on 149 clinical cases of TCGA-GBM database we show here a strong correlation (r = 0.7) between GLI1 and PTCH1 mRNA expression—as a hallmark of the canonical Hh-pathway activity in this malignancy. GLI1 mRNA expression varied in 3 orders of magnitude among the GBM patients of the same cohort showing a single continuous distribution—unlike the discrete high/low-GLI1 mRNA expressing clusters of medulloblastoma (MB). When compared with MB as a reference, the median GLI1 mRNA expression in GBM appeared 14.8 fold lower than that of the “high-Hh” cluster of MB but 5.6 fold higher than that of the “low-Hh” cluster of MB. Next, we demonstrated statistically significant up- and down-regulation of GLI1 mRNA expressions in GBM patient-derived low-passage neurospheres in vitro by sonic hedgehog ligand-enriched conditioned media (shh-CM) and by Hh-inhibitor drug vismodegib respectively. We also showed clinically achievable dose (50 μM) of vismodegib alone to be sufficient to induce apoptosis and cell cycle arrest in these low-passage GBM neurospheres in vitro. Vismodegib showed an effect on the neurospheres, both by down-regulating GLI1 mRNA expression and by inducing apoptosis/cell cycle arrest, irrespective of their relative endogenous levels of GLI1 mRNA expression. We conclude from our study that this single continuous distribution pattern of GLI1 mRNA expression technically puts almost all GBM patients in a single group rather than discrete high- or low-clusters in terms of Hh-pathway activity. That is suggestive of therapies with Hh-pathway inhibitor drugs in this malignancy without a need for further stratification of patients on the basis of relative levels of Hh-pathway activity among them.

A targeted therapy with lesser or no toxicity is an immediate need for glioblastoma multiforme (GBM). A group of Hedgehog (Hh) pathway inhibitor drugs is one among the most promising targeted therapies in a wide number of malignancies including medulloblastoma, a lethal childhood central nervous system (CNS) malignancy, about a third of which is presented with high Hh pathway activity. However, in case of GBM a great degree of ambiguity is reported in terms of this pathway activity. GBM is known to be highly heterogeneous at molecular level. It is indeed not clear whether any specific subgroup of GBM shows considerable Hh pathway activity so that could be targeted with appropriate Hh pathway inhibitory drugs. We estimated here the mRNA expression levels of 7 Hh pathway component genes — the ligand Sonic Hedgehog (SHH), trans-membrane receptors Patched (PTCH1) and Smoothened (SMO), Hh transcription factors Gli-1 (GLI1), Gli-2 (GLI2) and Gli-3 (GLI3), and also the Gli-1 target gene Snail (SNAI1), relative to the levels of β-actin expression, by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) method, in a cohort of GBM patients from the Eastern part of India. We have so far estimated the relative expression levels of these genes in 10 GBM, 8 low grade astrocytoma, 2 meningioma, 2 schwannoma, 1 medulloblastoma biopsy samples and 2 normal cerebellar tissue samples. CNS tumors with astrocytic origin were confirmed by Glial fibrillary acidic protein (GFAP) mRNA expression. We selected only the GBM cases for further analysis in the present study. We calculated the squared Pearson correlation coefficient between expression levels (2–ΔCt) of all the 7 genes and found the best correlations between three of them — SHH, GLI1 and SNAI1 respectively. High correlation of the expression of SHH with that of GLI1 (r2 = 0.9964) is suggestive of ligand-driven mechanism of this pathway activity in this disease. Although both PTCH1 and SNAI1 are bona fide targets of the Hh transcription factor GLI1, interestingly, we observed that the expression of GLI1 had a low correlation with that of PTCH1 (r2 = 0.0472) but was highly correlated with SNAI1 expression (r2 = 0.9986). Moreover, by using k-means clustering analysis three subgroups of patients were identified based on their expression patterns of all 7 Hh pathway component genes that were included in the present study. We have found detectable levels of SMO mRNA in all GBM patients included in the study so far, suggesting a potential benefit of the targeted therapy with smoothened antagonist drugs in this disease. Citation Format: Tapojyoti Das, Uttara Chatterjee, Samarendra Nath Ghosh, Sumit Deb, Suniti Kumar Saha, Puneet Gulati, Sarang Rote, Vikas Chandra, Ankur Mukherjee, Surajit Dhara. An objective clustering of GBM patients to identify clinically relevant subgroup with Hedgehog pathway activity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5587. doi:10.1158/1538-7445.AM2013-5587

In order to study the tolerability, toxicity and therapeutic efficacy of interferon-α_2b (Intron-A), 20 patients with superficial transitional cell carcinoma (TCC) of the urinary bladder (T_a, T₁) were given intravesically a low dose (9 million IU) of the drug mixed with 30 ml physiological saline for 8 weeks, starting 1 week after transurethral resection (TUR). Follow-up included urine cytology, ultrasonography of the abdomen and cystourethroscopy. Eleven patients (55%) had no signs of recurrence during a follow-up of 6–32 months (mean duration 24 months). Four patients (20%) had superficial tumor recurrence and 5 patients (25%) showed recurrence with progression to invasive tumors. Tolerability was very good, while toxicity in the form of mild flu-like symptoms was observed in only 3 patients. This pilot study showed that, at a low dose, intravesical interferon therapy can significantly lower the recurrence rate in superficial TCC of the urinary bladder with good tolerance and practically no side effects.