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 O6-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 O6-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.

Cancers evolve during tumor progression by the clonal selection of genetic alterations in tumor cells. Successive waves of clonal outgrowth overcome a multitude of selection barriers during neoplastic development. In addition, cancers face further selection pressures once they are diagnosed and treatment is initiated, and they continue to accumulate genetic alterations that confer selective growth advantage by allowing escape from the tumoricidal effects of treatment. In this regard, glioblastomas, the most frequent primary human brain tumor, are a prototypical human neoplasia: they accumulate mutations in oncogenes (e.g., EGFR) and tumor suppressor genes (e.g., PTEN; ref. 1), display clinical responsiveness to X-ray therapy (XRT) and the alkylating chemotherapeutic agent temozolomide (2), but ultimately fail these therapies and progress to fatal outcomes.

In a recent large-scale sequence analysis of malignant gliomas, somatic truncating mutations in the mismatch repair gene MSH6 were identified in two recurrent human glioblastomas (3). Both tumors had large numbers of somatic mutations within a mutational signature consistent with alkylator treatment, and both had been treated with temozolomide. Prior studies in murine embryonic stem cells (4), Chinese hamster cell lines (5), human lymphoblastoid cells (68), and human cancer cells (9, 10) have shown that MSH6 inactivation confers tolerant cell growth under cytotoxic doses of alkylating agents in vitro. Importantly, tolerant cells can be subsequently resensitized to alkylating agents by reintroduction of wild-type MSH6 (9, 10), establishing the direct role of the MSH6 gene in mediating this chemoresistance effect (11). We therefore hypothesized that MSH6 inactivation in tumor subclones may have contributed to their clonal expansion during temozolomide treatment and emergence as clinical recurrences in these two cases.

However, the overall frequency of MSH6 alterations in glioblastomas is unknown. Previous reports have identified homozygous germ line mutations of MSH6 in isolated cases of syndromic children with brain tumors (12, 13), but heterozygous germ line or somatic mutations of MSH6 have not been reported in typical adult patients with sporadic glioblastoma. Moreover, clinical phenotypic surveys of large MSH6 kindreds with familial colorectal or endometrial cancer have not identified an increased risk of glioblastoma in patients with heterozygous germ line MSH6 mutations (14, 15). Nonetheless, because heterozygous germ line mutations in the canonical mismatch repair genes MSH2 and MLH1 can cause the mixed colonic-and-glial tumor spectrum of Turcot syndrome (16), it is possible that yet undiscovered germ line or somatic MSH6 mutations could contribute to the development of a subset of glioblastomas.

In addition, this initial finding of MSH6 mutations in two tumors is counterbalanced by a substantial body of evidence that temozolomide responsiveness in glioblastomas can be predicted before treatment by determining the O6-methylguanine methyltransferase (MGMT) status (17, 18). MGMT is a well-characterized enzyme that catalyzes removal of the methyl-conjugate from O6-methylguanine, one of the nucleotide modifications that results from temozolomide treatment. High levels of MGMT activity are thought to offset alkylator modification of tumor DNA, thereby limiting temozolomide antitumor activity. Levels of MGMT have been assessed by promoter methylation analysis (17), direct enzymatic assay (18), or immunohistochemistry (19), and elevated levels correspond to more rapid tumor progression. Further complicating the understanding of these two pathways, it seems that both can be operant in the development of alkylator resistance in vitro (20), with mismatch repair deficiency serving as an alternative escape route when cancer cells have low levels of MGMT expression (21). It is thus possible that both pathways could contribute to clinical recurrences in glioblastoma patients receiving temozolomide.

We therefore sought to determine the frequency of MSH6 mutation or loss of protein expression in glioblastomas. In addition, we examined the relationship between MSH6 loss, adjuvant treatment modality, and MGMT status, and characterized the clinical consequences of MSH6 loss. To this end, we analyzed the mutational status and expression of MSH6 in both pretreatment and posttreatment glioblastoma samples, compared this pattern with the MGMT expression status of these samples, and examined the radiologic and clinical treatment course of the cohort in detail.

Tumor and DNA stocks. Tumors and peripheral blood lymphocytes for normal controls were banked under Institutional Review Board–approved protocols at the Massachusetts General Hospital. Tumor DNA was extracted from tumor specimens or early cell-culture passage after histologic confirmation (PureGene kit, Gentra Systems, Minneapolis, MN). Tumor and normal DNA for sample xT5165 and treatment information were kindly provided by Drs. Gregory Riggins and Charles Eberhart (The Johns Hopkins Hospital, Baltimore, MD). The tumors assembled for sequencing analysis were identified based on two criteria: histologically confirmed glioblastoma (WHO grade IV) and availability of appropriate quality specimen (i.e., fresh-frozen surgical resection or early cell-culture passage) for DNA sequence–based analyses.

For the panel of tumors assembled for immunohistochemical analyses, three criteria were used to identify patients: histologically confirmed glioblastoma (WHO grade IV); treatment with a chemotherapeutic regimen that included temozolomide; and availability of appropriate formalin-fixed, paraffin-embedded specimen. Five samples were analyzed by both sequencing and immunohistochemistry (three with wild-type MSH6, two with mutant MSH6). As can be seen in Tables 1 and 2, their results were concordant across both assessment modalities. As a control for adjuvant treatment modality, an additional eight glioblastomas from XRT-only–treated cases were also studied.

Table 1.

Sequence analysis of MSH6 in glioblastomas

 
 

NOTE: The top-listed samples (shaded) are derived from patients that had received treatment with temozolomide or unspecified chemotherapy. Details of treatment and mutation status are as noted. Sample ID list for the 40 pretreatment cases available on request. MSH6 status of samples xT155, xT924, xT2560, xT3017, xT3058, and xT3162 has previously been reported in ref. 3.

Abbreviations: TMZ, temozolomide; BCNU, 1,3-bis(2-chloroethyl)-1-nitrosourea; EGFR, epidermal growth factor receptor; PCV, procarbazine/1-(2-chloroethyl)-3-cyclohexyl-l-nitrosourea/vincristine.

Table 2.

MSH6 and MGMT immunohistochemical analysis

Treatment modalityPre- and post-treatment sample IDPretreatment MGMTPost–XRT + temozolomide MGMTPretreatment MSH6Post–XRT + temozolomide MSH6
XRT + temozolomide xT4981/xT5089 − − ++ 
 xT3546/xT4717 ++ ++ 
 xT4142/xT3692 − − +++ ++ 
 xT3559/xT4328 − − +++ ++ 
 xT2989/xT3296 − − ++ ++ 
 xT4107/xT3648 ++ +++ 
 xT4106/xT3307 − − +++ +++ 
 xT3506/xT3768 ++ +++ 
 xT4170/xT4987 − − ++ 
 xT4213/xT4869 
 xT4901/xT4899 ++ − 
 xT4991/xT3017 − 
 xT3495/xT4161 ++ − 
 xT3893/xT4870 n/s − 
 xT3513/xT4718 − − − 
 xT4136/xT4900 +++ − 
 xT4838/xT5065 +++ − 
XRT + temozolomide —/xT104   ++ 
 —/xT5046  n/s  − 
 —/xT4803  −  − 
 —/xT3162  −  − 
XRT-only —/xT4986  −  ++ 
 —/xT4903  −  ++ 
 —/xT4977  −  ++ 
 —/xT4975   ++ 
 —/xT2422   ++ 
 —/xT2703  −  ++ 
 —/xT3303   ++ 
 —/xT4985   ++ 
Treatment modalityPre- and post-treatment sample IDPretreatment MGMTPost–XRT + temozolomide MGMTPretreatment MSH6Post–XRT + temozolomide MSH6
XRT + temozolomide xT4981/xT5089 − − ++ 
 xT3546/xT4717 ++ ++ 
 xT4142/xT3692 − − +++ ++ 
 xT3559/xT4328 − − +++ ++ 
 xT2989/xT3296 − − ++ ++ 
 xT4107/xT3648 ++ +++ 
 xT4106/xT3307 − − +++ +++ 
 xT3506/xT3768 ++ +++ 
 xT4170/xT4987 − − ++ 
 xT4213/xT4869 
 xT4901/xT4899 ++ − 
 xT4991/xT3017 − 
 xT3495/xT4161 ++ − 
 xT3893/xT4870 n/s − 
 xT3513/xT4718 − − − 
 xT4136/xT4900 +++ − 
 xT4838/xT5065 +++ − 
XRT + temozolomide —/xT104   ++ 
 —/xT5046  n/s  − 
 —/xT4803  −  − 
 —/xT3162  −  − 
XRT-only —/xT4986  −  ++ 
 —/xT4903  −  ++ 
 —/xT4977  −  ++ 
 —/xT4975   ++ 
 —/xT2422   ++ 
 —/xT2703  −  ++ 
 —/xT3303   ++ 
 —/xT4985   ++ 

NOTE: MSH6 and MGMT status in matched samples from 17 patients' pre– and post–XRT + temozolomide surgical resections. Additionally listed are four post–XRT + temozolomide and eight post–XRT-only samples where the pretreatment sample was unavailable for study. MSH6 scored with a system of −, nil or scant staining; +, <10% of cells stained positive in the nucleus; ++, 10% to 50%; and +++, >50% positive. MGMT scored using +, >20% positive staining; −, <20% staining.

Abbreviation: n/s, assay not successfully done.

PCR amplification and sequencing. PCR primers were derived from ref. 14, covering >98% of the MSH6 coding sequence (sequences available on request). Amplification was done using Taq-Platinum DNA polymerase (Invitrogen, Carlsbad, CA). One primer from each pair was tagged with M13F universal sequencing primer to facilitate subsequent fluorescent dye-terminator sequencing (Agencourt Biosciences, Beverly, MA). Of a total of 752 reactions, 705 were successfully amplified and sequenced, for a sequence coverage rate of >93%.

Immunohistochemistry. Monoclonal antibodies to MSH6/GTBP (clone 44, BD Biosciences, San Jose, CA) and MGMT (MT3.1, Chemicon, Temecula, CA) were used for immunohistochemistry on formalin-fixed, paraffin-embedded sections. Antigen retrieval was achieved with 20-min (MSH6) or 15-min (MGMT) incubation in 10 mmol/L sodium citrate buffer at pH 6.0. Overnight 4°C incubations with a 1:100 (MSH6) or 1:50 (MGMT) dilution were done (Vectastatin Elite avidin-biotin complex kit, Vector Laboratories, Burlingame, CA). Positive control tissue consisted of nuclear staining in colonic crypt cells and the germinal centers of lymph nodes (MSH6) or cytoplasmic and nuclear stating in colon cancer and normal brain (MGMT). Scoring was done in a blinded fashion by two reviewers (D.P.C. and D.N.L.).

Tumor growth measurements. Magnetic resonance imaging (MRI) volume calculations were done on axial imaging sections through the tumor mass using the Vitrea2 3D volumetric software (Vital Images, Inc., Minnetonka, MI). Personnel scoring tumor volume were blinded to the molecular stratification of the tumors. Tumor volume was assessed using standard criteria of T1-post-gadolinium sequence enhancing volume, correcting for acute surgical residua by exclusion of pre-gadolinium T1 sequence enhancement. Tumor growth rate was calculated on matched pairs by determining the T1-enhancing volume from the MRI obtained at the initiation of alkylator therapy (which was typically the postoperative MRI following surgical biopsy or debulking resection), and subtracting it from the T1-enhancing tumor volume on the MRI obtained immediately before cessation of alkylator therapy. This change in volume was then divided by the difference in time between the two studies to calculate an overall growth rate during alkylator therapy. Similar calculations were done for FLAIR sequences, which assess the magnitude of tumor-associated edema.

In one recurrence (corresponding to tumor specimen xT4899), a second MRI study was not obtained before cessation of therapy; therefore, we used data calculated from an MRI scan that occurred 16 days after the last dose of temozolomide because it was still within the standard treatment window of 28 days for a single cycle of temozolomide therapy. One patient (corresponding to tumor specimen xT3162) was excluded from tumor growth analyses because the follow-up MRI sequences available were obtained more than 4 months after cessation of temozolomide therapy; this patient's tumor growth data are appended to Supplementary Table S1 and can be seen as essentially comparable to the other cases, but were censored because of the length of time outside of alkylating therapy without radiologic assessment.

Statistical analysis. Pretreatment cases were defined as a patient's first surgical resection, and recurrences as the first surgical resection after failure of the specified treatment modality (XRT or XRT + temozolomide). Two-sided, 0.05 level Fisher's exact tests were used to compare two independent proportions. An exact binomial confidence interval was calculated for the frequency of negative MSH6 expression at recurrence following temozolomide treatment. An exact, two-sided, 0.05 level McNemar's test was used to compare the frequency of MSH6-negative expression at recurrence following temozolomide treatment with that in the pretreatment sample. To adjust for the length bias induced by the sampling of recurrent cases, semiparametric truncation-adjusted Kaplan-Meier estimates (22), parametric Weibull estimates, and log-rank tests were used for the analyses of age at diagnosis (right truncated by age at recurrence), length of alkylator treatment (right truncated by time from diagnosis to recurrence), time to recurrence (right truncated by time to death or end of follow-up), and time from diagnosis to death (left truncated by time to recurrence). Exact, two-sided, 0.025 level (Bonferroni adjusted) Wilcoxon tests were used to evaluate for differences in tumor growth rates under treatment, as measured by T1 post-gadolinium enhancement and FLAIR hyperintensity. For the expression of growth rate per month, the growth rate per day was multiplied by 30 days.

Sequence analysis of MSH6. Our prior sequencing study analyzed six glioblastomas for MSH6 alterations and found somatic mutations in two tumors. To survey the overall spectrum of MSH6 mutation in glioblastoma, we analyzed the MSH6 coding sequence in an additional 48 glioblastomas. Thirty-seven were pretreatment tumors whereas 11 were recurrent tumors. These samples were not preselected based on their treatment regimen, and therefore the recurrences had previously received a range of alkylator, nonalkylator, or even no chemotherapy. The clinical details of these samples and sequence analysis results are listed in Table 1. No mutations of MSH6 were identified in the pretreatment tumors. Therefore, germ line or somatic MSH6 mutations do not seem to contribute significantly to the pretreatment development of glioblastoma. In the recurrent samples, one tumor (xT5165) had two sequence alterations that resulted in predicted amino acid substitutions. One was an A-to-C transversion at nucleotide 560 leading to a lysine-to-threonine substitution at amino acid position 187. The other was a T-to-G transversion at nucleotide 794 resulting in a phenylalanine-to-cysteine replacement at amino acid position 265. Neither of the alterations was found in the patient's normal DNA.

Combining data from these 48 tumors with our prior findings yielded a total of 3 MSH6-mutant cases among 14 recurrent tumors, whereas none of the 40 pretreatment tumors had mutations. Because the recurrence rate of glioblastoma is nearly 100%, random sampling of recurrences is unlikely to introduce significant selection bias with respect to prior pretreatment status. With this in mind, comparison of the frequency of mutation in our pretreatment and posttreatment samples suggests that MSH6 mutation is associated with recurrence (P = 0.015).

Immunohistochemistry for MSH6 expression. These results, in light of the known selectivity of the mismatch repair pathway in mediating alkylator resistance in vitro, raised the possibility that selection for MSH6 inactivation could be a treatment-specific phenomenon. Moreover, the treatment regimens varied among these patients, possibly accounting for the minority of recurrent cases with detectable MSH6 mutations. We therefore sought to evaluate MSH6 in a second sample set focused on glioblastomas that had undergone adjuvant treatment with the standard-of-care alkylating agent temozolomide. To maximize the assessable yield of MSH6 status, we pursued immunohistochemistry of formalin-fixed, paraffin-embedded specimens, which constitute the majority of clinically well-annotated material in glioblastoma trial settings. Most glioblastoma biopsy specimens are not sufficient to afford genomic amplification and large-scale sequence analyses; fortunately, there is extensive experience validating the usefulness of immunohistochemistry to detect MSH6 inactivation in hereditary colon and endometrial cancer (14, 15, 23, 24).

We scored both MSH6 and MGMT status in a total of 46 glioblastomas (Table 2). Within this group, 38 specimens were from 21 patients who had received treatment with XRT + temozolomide. Because our study design necessitated selection of patients who had undergone two surgeries, we examined the baseline demographic characteristics of those patients treated with XRT + temozolomide to assess for selection bias. The patient cohort was 71% male, with median age at diagnosis of 53 years. These data are indicative of a patient group similar to those reported in the recent large prospective study of XRT + temozolomide (2). Of the tumors studied, 34 were matched pairs derived from 17 patients' pre– and post–XRT + temozolomide surgical resections, providing the additional benefit of certainty about the pretreatment MSH6 status.

In the matched pretreatment specimens, every tumor examined (17 of 17) stained positively for MSH6 expression. The staining displayed variability, ranging from <10% to >50% of the total tumor cell mass in any given case. However, even accounting for this variability, every pretreatment glioblastoma evaluated was unequivocally positive for MSH6 expression (Table 2), whereas normal brain sections do not stain for MSH6 expression (Fig. 1). Thus, there seems to be an induction of MSH6 protein expression in pretreatment glioblastomas, consistent with previous immunohistochemistry studies of MSH6 in other tumors types (14, 15, 23, 24) and of other mismatch repair genes in glioblastoma (25).

Fig. 1.

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

Fig. 1.

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

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In matched post–XRT + temozolomide tumors from the same patients, a range of MSH6 staining was also observed, with four cases showing an increase in staining and four cases showing stable intensity. Strikingly, however, staining for MSH6 was completely absent in seven of these cases (Fig. 1). This loss of MSH6 expression at recurrence within the matched pairs was significantly different from that at diagnosis (P = 0.016, based on the seven tumor pairs with discordant pretreatment and posttreatment expression). Furthermore, in four additional post–XRT + temozolomide tumors, three had absence of MSH6 protein. Therefore, in total, 10 of 21 post–XRT + temozolomide recurrent tumors displayed absence of MSH6 by immunohistochemical analysis.

To isolate the effect of temozolomide chemotherapy comparatively, we analyzed an additional eight recurrent tumors from patients who had received adjuvant treatment solely with XRT. These samples served as a treatment-modality control group; because current clinical practice does not allow withholding radiation treatment from eligible patients, it was not possible to assemble a cohort of patients who had solely received temozolomide in the absence of XRT. These post–XRT-only specimens were all positive for MSH6 expression (8 of 8 tumors). Comparing these recurrences to post–XRT + temozolomide recurrences as independent samples based on treatment modality (with the presumption of no significant selection bias among the recurrences with respect to their pretreatment status), there was a significant difference with respect to the frequency of MSH6 expression loss at recurrence between post–XRT + temozolomide and post–XRT-only glioblastomas (P = 0.012), providing evidence that MSH6 loss is specifically associated with the temozolomide alkylator component of combined adjuvant therapy.

MGMT. As noted above, decreased MGMT expression is a predictor of improved glioblastoma prognosis. It is therefore possible that acquisition of increased MGMT expression itself contributes to the emergence of resistant clones during temozolomide treatment. Given the association of MGMT status with treatment response, it is also possible that MSH6 status at recurrence is linked to pretreatment MGMT status. To determine the relationship between MGMT status, MSH6 status, and post–XRT + temozolomide recurrence, we assessed MGMT immunohistochemistry within our panel of tumors.

MGMT staining was successfully analyzed in 44 of the 46 samples, including 16 of 17 pretreatment tumors. We found a range of MGMT staining similar to prior estimates of the frequency of MGMT absence (1719), with 7 of 16 staining negatively for MGMT and 9 of 16 scoring moderately or strongly positive (Fig. 1; Table 2). Similarly consistent with prior immunohistochemistry analyses (19), absence of MGMT in the pretreatment tumor displayed a trend toward prolonged overall survival (P = 0.151). To evaluate changes in MGMT status during adjuvant therapy, we analyzed MGMT expression in the available matched post–XRT + temozolomide specimens. For any individual patient, pretreatment MGMT status was maintained in the post–XRT + temozolomide sample, including all seven glioblastomas that were MGMT negative before temozolomide treatment (Table 2). These data suggest that the induction of MGMT expression that can be seen in model systems of alkylator exposure (20) may not be frequent under the treatment dosing of temozolomide in current clinical practice. Also, we did not find a significant association between MGMT pretreatment status and MSH6 posttreatment status (P = 0.145), although our analyses suggest a possible correlative trend that might be further uncovered in larger studies.

Clinical correlation. To characterize the clinical features of patients whose tumors displayed MSH6 loss, clinical data were analyzed from patients treated with XRT + temozolomide. Medical records were abstracted for the dates of initial surgery, length of treatment with temozolomide and other alkylating agents, dates of recurrent surgery, and date of death. Comparing the two independent groups determined by MSH6-positive (n = 11) and MSH6-negative (n = 10) scoring at recurrence, we noted no significant differences in possible confounding factors such as age at diagnosis, time to initiation of alkylator treatment, length of alkylator treatment, or time to recurrent surgery (P = 0.998, P = 0.523, P = 0.820, and P = 0.698, respectively).

To determine the treatment consequences of MSH6 loss, we used MRI for in vivo calculations of tumor growth rates during temozolomide treatment in patients stratified for MSH6 status. For all patients analyzed by immunohistochemistry above, we identified matched pairs of MRI scans, one corresponding to the initiation of alkylating chemotherapy inclusive of temozolomide and one immediately before cessation of this treatment; appropriate matched pairs were identified for 20 of 21 patients. In aggregate, MRI data were assessed spanning 3,879 days across a total alkylator treatment time of 4,496 days, covering >85% of the clinical treatment window. To exclude the possibility of systematic confounding biases arising during the clinical care and radiologic assessment of these patients, we examined several aspects of the timing and clinical conditions of the patients' treatment assignments and MRI schedules. We found no significant differences between MSH6-positive and MSH6-negative tumors with respect to either the time between the start of alkylator treatment and the initial MRI or the time between cessation of alkylator treatment and the final assessment MRI. Furthermore, to exclude the possibility that tumors were being assessed at different stages of progression due to clinical factors, we examined radiologic proxy measures of clinical performance such as the volume of tumor, as assessed by T1-gadolinium-enhancing signal, or the volume of edema, as assessed by FLAIR signal, and found no significant differences between the two groups when comparing either the pretreatment or posttreatment scans (Table 3, with the full clinical-radiologic data set available in Supplementary Table S1).

Table 3.

Baseline characteristics of clinical and radiologic measures

MSH6-positive (n = 11), median (range)MSH6-negative (n = 9), median (range)P
Age at recurrence, y 49 (23-70) 56 (31-67) NS 
Gender (% male) 64 78  
Calculated pretreatment tumor volume, cc 11.79 (1.65-46.59) 5.12 (0-53.73) NS 
Pretreatment T1-post-gadolinium enhancement volume, cc 12.6 (1.8-54.85) 9.75 (0-53.73) NS 
Pretreatment T1 enhancement volume (i.e., pre-gadolinium), cc 1.37 (0-41.06) 0 (0-11.33) NS 
Pretreatment FLAIR volume, cc 43.16 (5-178.4) 52.75 (8.29-229.6) NS 
Recurrence T1-post-gadolinium enhancement volume, cc 9.15 (5.2-44.1) 16.17 (4.19-82.83) NS 
Recurrence FLAIR volume, cc 56.13 (8.05-185.3) 57.53 (10.92-353.2) NS 
Time elapsed between initial and recurrence scans, d 170 (71-364) 153 (57-432) NS 
Rate of FLAIR change, cc/d 0.009 (−0.645-0.406) 0.060 (−0.221-1.258) NS 
Rate of T1-post-gadolinium enhancement change, cc/d 0.001 (−0.087-0.068) 0.106 (−0.055-0.288) 0.020 
MSH6-positive (n = 11), median (range)MSH6-negative (n = 9), median (range)P
Age at recurrence, y 49 (23-70) 56 (31-67) NS 
Gender (% male) 64 78  
Calculated pretreatment tumor volume, cc 11.79 (1.65-46.59) 5.12 (0-53.73) NS 
Pretreatment T1-post-gadolinium enhancement volume, cc 12.6 (1.8-54.85) 9.75 (0-53.73) NS 
Pretreatment T1 enhancement volume (i.e., pre-gadolinium), cc 1.37 (0-41.06) 0 (0-11.33) NS 
Pretreatment FLAIR volume, cc 43.16 (5-178.4) 52.75 (8.29-229.6) NS 
Recurrence T1-post-gadolinium enhancement volume, cc 9.15 (5.2-44.1) 16.17 (4.19-82.83) NS 
Recurrence FLAIR volume, cc 56.13 (8.05-185.3) 57.53 (10.92-353.2) NS 
Time elapsed between initial and recurrence scans, d 170 (71-364) 153 (57-432) NS 
Rate of FLAIR change, cc/d 0.009 (−0.645-0.406) 0.060 (−0.221-1.258) NS 
Rate of T1-post-gadolinium enhancement change, cc/d 0.001 (−0.087-0.068) 0.106 (−0.055-0.288) 0.020 

NOTE: Radiologic assessment factors were compared between the two groups of patients with glioblastoma recurrences determined by MSH6-positive or MSH6-negative immunohistochemical staining. No significant differences were noted between potential confounding measures. Tumor growth rate, as assessed by rate of T1-post-gadolinium enhancement change, was higher in the MSH6-negative group.

Abbreviation: NS, not significant (P > 0.025).

We then calculated in vivo tumor growth rates under temozolomide treatment by comparing the volume of tumor on the initial scan to the volume present at cessation of temozolomide treatment. Interestingly, we could not detect a correlation between pretreatment MGMT status and the rate of tumor growth under temozolomide treatment. Importantly however, MSH6-negative glioblastomas showed a markedly increased rate of growth while being treated with temozolomide, with a median T1-gadolinium enhancing signal change of +3.17 cc/mo (0.106 cc/d), whereas median MSH6-positive tumor growth was only +0.04 cc/mo (0.001 cc/d) under temozolomide treatment (Table 3; Fig. 2). The magnitude of this difference was in accordance with in vitro studies of MSH6 function in alkylator-tolerant growth and was significant (P = 0.020). With the caveat that there may be subtle confounding factors in these comparisons that we were unable to detect due to our cohort size, this increased growth rate suggests that MSH6 loss in vivo corresponds to decreased clinical responsiveness to temozolomide and subsequent recurrent tumor growth during treatment, mirroring the alkylator resistance conferred by MSH6 inactivation in vitro.

Fig. 2.

Three-dimensional MRI tumor growth calculations. A, representative post-gadolinium enhancement T1 sequence axial sections through the tumor mass, corresponding to the initiation of alkylating chemotherapy inclusive of temozolomide and immediately before cessation of this treatment. Left, MSH6-positive case showing recurrent tumor sample xT3692; 254 d elapsed between initial and final scan. Right, MSH6-negative case showing recurrent glioblastoma sample xT4161; 249 d 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 follow-up MRI scan. The median rate of volume change was 0.001 cc/d. C, for nine MSH6-negative tumors, volume change versus time, with a median rate of 0.106 cc/d. Full clinical/radiologic data set available in Supplementary Table S1.

Fig. 2.

Three-dimensional MRI tumor growth calculations. A, representative post-gadolinium enhancement T1 sequence axial sections through the tumor mass, corresponding to the initiation of alkylating chemotherapy inclusive of temozolomide and immediately before cessation of this treatment. Left, MSH6-positive case showing recurrent tumor sample xT3692; 254 d elapsed between initial and final scan. Right, MSH6-negative case showing recurrent glioblastoma sample xT4161; 249 d 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 follow-up MRI scan. The median rate of volume change was 0.001 cc/d. C, for nine MSH6-negative tumors, volume change versus time, with a median rate of 0.106 cc/d. Full clinical/radiologic data set available in Supplementary Table S1.

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Our findings show that loss of MSH6 expression occurs in a significant subset of post–XRT + temozolomide recurrent glioblastomas and is associated with the progressive growth of these tumors while they are under temozolomide treatment. These findings parallel in vitro studies documenting the frequent emergence of mismatch repair deficiency in cell line subclones after selection and outgrowth in alkylating agents (26, 27). Importantly, whereas many factors have been proposed to mediate therapeutic resistance based on studies of cancer cells exposed to differing doses of chemotherapeutic agents in vitro, our observations were made on patients receiving the clinical standard-of-care doses of an oral alkylating agent, indicating that MSH6 loss seems to recapitulate its known in vitro alkylator resistance role in glioblastoma patients in vivo.

This extension of in vitro mechanistic studies to an in vivo system has significant implications regarding patient-drug interactions; for example, it can be reasonably surmised that drug delivery across the blood-brain barrier and tumor vasculature is not a rate-limiting factor in temozolomide treatment failure in cases with MSH6 loss because the recurrent tumor cell mass displays the hallmarks of a selection pressure mediated by direct exposure to the drug itself. Likewise, in vitro studies indicate that there is an interactive specificity between drug and pathway in the development of resistance: mismatch repair pathway defects can mediate up to a 100-fold resistance to alkylating agents but only show ∼2-fold resistance to the intrastrand cross-linking agent cisplatin and no significant resistance to agents such as topoisomerase inhibitors or interstrand cross-linkers such as mitomycin C or 1-(2-chloroethyl)-3-cyclohexyl-l-nitrosourea (28). Our in vivo tumor growth rate measurements reflect a similar degree of difference in clinical progression between MSH6-negative and MSH6-positive glioblastomas during temozolomide treatment. However, the prediction from in vitro studies would be that chemotherapeutic agents with different mechanisms (29, 30) may still prove effective in MSH6-negative glioblastomas because the cellular resistance associated with MSH6 absence is specific for exposure to alkylators.

Previous reports have indicated that alkylator resistance can be mediated either by MGMT overexpression or mismatch repair deficiency (20, 21, 25, 31). Importantly, our analyses of recurrent glioblastoma samples indicate that MSH6 loss occurred independently of the MGMT status of the pretreatment tumor, indicating that MSH6 loss is not merely a proxy of MGMT status. Our data illustrate the relationship between these two mechanisms in patients who have undergone temozolomide treatment, indicating that whereas pretreatment tumor MGMT levels may be linked to primary resistance, acquisition of further increased expression does not seem to be a common cause of emergent temozolomide resistance in the clinical setting. Interestingly, many current salvage chemotherapeutic regimens propose increasing the dosing schedule of temozolomide, in variations known collectively as “dose-dense” modifications (32), in an attempt to overcome increased levels of MGMT activity. However, it seems that MSH6 loss can occur during temozolomide treatment regardless of MGMT status, with subsequent clonal outgrowth leading to recurrence through an alternative route of treatment escape. Dose-dense modifications may therefore prove ineffective for patients with MSH6-negative tumors because the window of therapeutic index available for this substantially myelotoxic therapy may have closed.

Given that there may be several operative mechanisms for functional MSH6 alteration, our immunohistochemistry results may underestimate the contribution of altered MSH6 pathway function to temozolomide insensitivity. Our sequencing efforts show clear evidence of somatic mutational alteration and clonal selection in some recurrent tumors. However, at the protein level, the MSH6 protein participates in a large multiprotein complex with other mismatch repair proteins to coordinate mismatch binding, mismatch repair, and DNA damage checkpoint signaling. Recent work has shown that O6-methylguanine-thymine mismatches are specifically bound by the heterodimeric MSH2:MSH6 MutSα complex to drive activation of the ATR/Chk1 S-G2 phase checkpoint pathway (33). Posttranslational changes in the subcellular localization of the MSH2:MSH6 complex occurs in response to genotoxic agents (34), communicating repair activity signals to checkpoint machinery (35). This checkpoint signaling pathway is thought to mediate much of the tumoricidal effect of alkylating chemotherapy (11). Because study of mismatch repair checkpoint pathway signaling remains an active area of investigation, it is likely that other novel candidates for analysis in treatment-tolerant tumors will emerge.

Indeed, the existence of post–XRT + temozolomide glioblastomas that retain or even increase MSH6 expression highlights the probability that alternative genetic mechanisms do contribute to temozolomide evasion. Whereas it is plausible that alterations in other mismatch repair genes such as MSH2 or MLH1 could play a similar role to MSH6 in glioblastoma, the canonical microsatellite instability typical of MSH2 or MLH1 deficiency is rarely found in adult sporadic pretreatment or posttreatment glioblastoma specimens (36, 37). Notably, as opposed to other mismatch repair genes, MSH6 deficiency is not associated with high levels of microsatellite instability (11). Furthermore, MSH6, but not other mismatch repair genes, is preferentially targeted in vitro in unbiased chemotherapy resistance genetic screens (4). With this in mind, we speculate that temozolomide may engender a selection pressure specifically targeting for MSH6 loss instead of other mismatch repair genes, as tumor subclones negotiate a balance between the increased mutation load of mismatch repair deficiency and the selective growth advantage of temozolomide insensitivity.

Given the variability among salvage therapies within recurrent glioblastoma patient cohorts, further analyses in a more uniformly treated group of patients will be needed to confirm our findings and provide further insight into such gene-specific and treatment-specific hypotheses. Current clinical practice withholds surgical intervention in many cases of recurrence due to a perceived lack of benefit relative to the risk of surgery in rapidly declining patients. With the advent of combined XRT + temozolomide treatment, more glioblastoma patients are likely to undergo recurrent surgical intervention, affording important opportunities for well-controlled molecular pathologic studies of chemotherapeutic response and escape pathways. By better understanding the molecular basis for treatment evasion and subsequent clinical failure, we can envision the rational design of targeted combination therapeutic strategies that will limit the emergence of such treatment escape in future patients.

Grant support: Institute of Cancer Research, the Wellcome Trust, Brain Tumor Society Seth Harris Feldman Research Award, National Brain Tumor Foundation GBM grant, American Brain Tumor Association Fellowship, American Association of Neurosurgeons NREF Fellowship, and the NIH.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).

We thank the patients who participated in this research through donation of blood and tumor samples; the Massachusetts General Hospital Neuroradiology 3D Lab for use of 3D volumetric software; and Drs. Gregory Riggins and Charles Eberhart for providing tumor and normal DNA samples.

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Supplementary data