Loss of the mismatch repair protein MSH6 in human glioblastomas is associated with tumor progression during temozolomide treatment

Abstract

Purpose: Glioblastomas are treated by surgical resection followed by radiotherapy [X-ray therapy (XRT)] and the alkylating chemotherapeutic agent temozolomide. Recently, inactivating mutations in the mismatch repair gene MSH6 were identified in two glioblastomas recurrent post-temozolomide. Because mismatch repair pathway inactivation is a known mediator of alkylator resistance in vitro, these findings suggested that MSH6 inactivation was causally linked to these two recurrences. However, the extent of involvement of MSH6 in glioblastoma is unknown. We sought to determine the overall frequency and clinical relevance of MSH6 alterations in glioblastomas.

Experimental design: The MSH6 gene was sequenced in 54 glioblastomas. MSH6 and O(6)-methylguanine methyltransferase (MGMT) immunohistochemistry was systematically scored in a panel of 46 clinically well-characterized glioblastomas, and the corresponding patient response to treatment evaluated.

Results: MSH6 mutation was not observed in any pretreatment glioblastoma (0 of 40), whereas 3 of 14 recurrent cases had somatic mutations (P = 0.015). MSH6 protein expression was detected in all pretreatment (17 of 17) cases examined but, notably, expression was lost in 7 of 17 (41%) recurrences from matched post-XRT + temozolomide cases (P = 0.016). Loss of MSH6 was not associated with O(6)-methylguanine methyltransferase status. Measurements of in vivo tumor growth using three-dimensional reconstructed magnetic resonance imaging showed that MSH6-negative glioblastomas had a markedly increased rate of growth while under temozolomide treatment (3.17 versus 0.04 cc/mo for MSH6-positive tumors; P = 0.020).

Conclusions: Loss of MSH6 occurs in a subset of post-XRT + temozolomide glioblastoma recurrences and is associated with tumor progression during temozolomide treatment, mirroring the alkylator resistance conferred by MSH6 inactivation in vitro. MSH6 deficiency may therefore contribute to the emergence of recurrent glioblastomas during temozolomide treatment.

Figure 1

MSH6 and MGMT immunohistochemistry. Immunohistochemistry was performed with monoclonal antibodies specific for MSH6 (A – sample xT3307 and B – xT3162), with controls of normal colonic epithelium (C) and normal brain (D). The larger field of view for (C) demonstrates MSH6 expression in the crypt and transit-amplifying cells, but not in differentiated colonic epithelium. MGMT immunohistochemistry (E – sample xT3506 and F – xT4142).

Figure 2

3D MRI tumor growth calculations. A. Representative post-gadolinium enhancement T1 sequence axial sections through the tumor mass are shown, corresponding to the initiation of alkylating chemotherapy inclusive of TMZ and immediately prior to cessation of this treatment. The MSH6-positive case on the left shows recurrent tumor sample xT3692; 254 days elapsed between initial and final scan. The MSH6-negative case on the right shows recurrent glioblastoma samples xT4161; 249 days elapsed between initial and final scan. Multiple axial imaging sections through the tumor mass were summated to determine enhancing tumor volume. B. For 11 MSH6-positive tumors, volume change versus time elapsed between initial and followup MRI scan. The median rate of volume change was 0.001 cc/d. C. For 9 MSH6-negative tumors, volume change versus time, with a median rate of 0.106 cc/d. Full clinical/radiologic dataset available as Supplementary Table 1.

Figure 2

3D MRI tumor growth calculations. A. Representative post-gadolinium enhancement T1 sequence axial sections through the tumor mass are shown, corresponding to the initiation of alkylating chemotherapy inclusive of TMZ and immediately prior to cessation of this treatment. The MSH6-positive case on the left shows recurrent tumor sample xT3692; 254 days elapsed between initial and final scan. The MSH6-negative case on the right shows recurrent glioblastoma samples xT4161; 249 days elapsed between initial and final scan. Multiple axial imaging sections through the tumor mass were summated to determine enhancing tumor volume. B. For 11 MSH6-positive tumors, volume change versus time elapsed between initial and followup MRI scan. The median rate of volume change was 0.001 cc/d. C. For 9 MSH6-negative tumors, volume change versus time, with a median rate of 0.106 cc/d. Full clinical/radiologic dataset available as Supplementary Table 1.

Figure 2

3D MRI tumor growth calculations. A. Representative post-gadolinium enhancement T1 sequence axial sections through the tumor mass are shown, corresponding to the initiation of alkylating chemotherapy inclusive of TMZ and immediately prior to cessation of this treatment. The MSH6-positive case on the left shows recurrent tumor sample xT3692; 254 days elapsed between initial and final scan. The MSH6-negative case on the right shows recurrent glioblastoma samples xT4161; 249 days elapsed between initial and final scan. Multiple axial imaging sections through the tumor mass were summated to determine enhancing tumor volume. B. For 11 MSH6-positive tumors, volume change versus time elapsed between initial and followup MRI scan. The median rate of volume change was 0.001 cc/d. C. For 9 MSH6-negative tumors, volume change versus time, with a median rate of 0.106 cc/d. Full clinical/radiologic dataset available as Supplementary Table 1.