Prognostic value and functional consequences of cell cycle inhibitor p27Kip1 loss in medulloblastoma
© Hatton et al.; licensee BioMed Central Ltd. 2013
Received: 21 December 2012
Accepted: 17 February 2013
Published: 1 March 2013
The cyclin-dependent kinase inhibitor p27Kip1 functions during normal cerebellar development and has demonstrated tumor suppressor functions in mouse models of medulloblastoma. Because P27 loss is associated with increased proliferation, we assessed whether P27 absence in surgical medulloblastoma specimens correlated with response to therapy in pediatric patients enrolled in two large studies. Additionally, we examined the functional consequence of p27 Kip1 loss in the SmoA1 medulloblastoma model to distinguish whether p27Kip1 reduces tumor initiation or slows tumor progression.
Analysis of 87 well-characterized patient samples identified a threshold of P27 staining at which significant P27 loss correlated with poor patient outcome. The same criteria, applied to a second test set of tissues from 141 patients showed no difference in survival between patients with minimal P27 staining and others, suggesting that P27 levels alone are not a sufficient prognostic indicator for identifying standard-risk patients that may fail standard therapy. These findings were in contrast to prior experiments completed using a mouse medulloblastoma model. Analysis of cerebellar tumor incidence in compound mutant mice carrying the activated Smoothened (SmoA1) allele that were heterozygous or nullizygous for p27 Kip1 revealed that p27Kip1 loss did not alter the frequency of tumor initiation. Tumors haploinsufficient or nullizygous for p27 Kip1 were, however, more invasive and displayed a higher proliferative index, suggesting p27Kip1 loss may contribute to SmoA1 medulloblastoma progression.
These studies revealed P27 loss affects medulloblastoma progression rather than initiation and that this putative biomarker should not be used for stratifying children with medulloblastoma to risk-based therapeutic regimens.
KeywordsMedulloblastoma p27Kip1 Sonic hedgehog Cerebellum Mouse cancer models Prognostic factors
Granule neuron progenitor
Inner granule layer
High power field
Invasive with effacement
Non-invasive tumor with no effacement.
Approximately 20% of medulloblastoma patients classified as standard risk fail therapy and succumb to their disease . Current prognostic criteria stratify patients into standard- or high-risk groups based upon age, the extent of surgical resection and the presence or absence of disseminated disease . The intensity of radio- and chemotherapy are determined by risk group, although significant impairments induced by current strategies are well established, particularly in younger patients [2, 3]. Because of these devastating toxicities, efforts are underway to reduce therapy intensity for standard risk patients. As the pediatric neuro-oncology community begins to de-escalate standard risk therapy, it consequently becomes critically important to identify molecular markers that recognize children likely to fail therapy that would benefit from stratification to more intensive therapeutic regimens.
Studies in various mouse models have demonstrated that loss of a single p27 Kip1 [GenBank: NM_009875] allele increases tumor formation, suggesting p27 Kip1 ’s tumor suppressor function is haploinsufficient or acts in a dosage-dependent manner . In the SmoA1 mouse medulloblastoma model, loss of P27 resulted in early death compared to counterparts with wildtype P27 levels . While this work showed that P27 has tumor suppressive function in medulloblastoma, it remained to be determined whether loss of P27 caused an increase in tumor initiation or progression. This is an important distinction because molecules involved in tumor initiation are rarely good prognostic indicators, whereas a subset of molecules involved in tumor progression reliably predict response to therapy.
While p27 Kip1 mutations are generally rare, P27 abundance has provided a reliable prognostic marker for cancer progression in a subset of human cancers, such as breast, colon and prostate [6, 7]. The prognostic value of P27 expression in human medulloblastoma is currently unknown. Previous studies using 14 human medulloblastoma samples found low or absent P27 expression in highly proliferative and undifferentiated tumor regions [8, 9], but the sample size was too small to assess prognostic value.
This study first addresses the unanswered question regarding P27’s role in tumor initiation versus progression in experiments conducted in a genetically engineered mouse medulloblastoma model. This study additionally assesses P27’s potential as a prognostic indicator in children with medulloblastoma. The study evaluates whether clinical outcomes improve for children with high P27 first in a retrospective clinical trial cohort (training data set) and then in a larger clinical trial cohort (test data set).
Compound mutant mice
SmoA1 mice were crossed with heterozygous p27 wt/- mice to produce compound mutant (SmoA1, p27 wt/- and SmoA1, p27 −/− ) mice. Mice were sacrificed at 2 months to examine tumor incidence via histopathology. Transgenic mice were maintained in accordance with the NIH Guide for the Care and Use of Experimental Animals with approval from our Institutional Animal Care and Use Committee.
Mice were pulsed with BrdU (100 mg/kg) one hour prior to sacrifice. Tissues were paraffin embedded and cut into four micron sections. Sagittal sections were cut at multiple levels from each cerebellum analyzed and scored for the presence or absence of tumor formation. Tissue sections were stained with antibodies recognizing p27, cyclin D1 and BrdU, and five high power fields (HPF) were scored for each sample to quantify the proliferative index within cerebellar tumors. Human medulloblastoma tissues were stained with a monoclonal p27Kip1 antibody and detection carried out with 3,3’-diaminobenzidine reagent. Tissue microarrays containing tissues from 87 patient samples were previously described . P27 protein expression levels were also examined on tissue microarrays containing tissues from 141 patients enrolled on the SIOP PNET03 (1992–2000) study [11, 12]. Staining intensity was evaluated by observers blinded to patient and survival data using the Nikon Elements Imagining Software after slides were digitally scanned.
For mouse studies, sagittal sections were cut at multiple levels from each cerebellum analyzed to evaluate tumor incidence and a Fisher’s exact test was used to determine p values. A t test was used to determine p values for quantification of the proliferative index. For human patients, overall survival was calculated from diagnosis date until death or the last follow up date. Survival distribution was estimated by the Kaplan-Meier method and compared using the log-rank test (SPSS 15.0). A multivariate Cox proportional-hazards regression model, with overall survival as the dependent variable, was used to assess the impact of P27 expression levels on the three individual components of the current risk stratification model (metastasis, residual disease, age <3). Two-sided p-values of less than 0.05 using the 95% confidential interval were considered to indicate statistical significance.
Genetic alterations have been isolated from medulloblastomas within signaling pathways that normally regulate proliferation and differentiation of granule neuron progenitors (GNPs) during cerebellar development . Medulloblastomas are induced in the SmoA1 mouse model through the constitutive activation of the sonic hedgehog (Shh) [GenBank: NM_009170] pathway within GNPs, signaling normally required for GNP proliferation. p27 Kip1 loss in mice can increase tumor formation , and p27 Kip1 haploinsufficiency has been further supported by studies using the Patched1 [GenBank: NM_008957] heterozygous (Ptc1 +/−) and SmoA1 mouse medulloblastoma models [5, 14], where Shh-induced medulloblastoma incidence was accelerated by loss of one or both p27 Kip1 alleles.
An important observation can be drawn from comparing tumor incidence at two months to the survival at later time point results reported previously. Early tumor formation was relatively unchanged by the loss of a single or of both p27 Kip1 alleles in our study. In contrast, SmoA1 mice with wildtype p27 Kip1 lived on average twice as long as mice with a single copy of p27 Kip1 , and mice retaining wildtype p27 Kip1 in the heterozygous (Ptc1 +/−) background survived significantly longer than counterparts lacking p27 Kip1 , which succumbed due to an increased tumor incidence . In combination with the aggressive pathology observed in early tumors from SmoA1 mice lacking p27 Kip1 , the higher tumor incidence in Ptc1 +/− mice lacking p27 Kip1 further confirms that p27 Kip1 is haploinsufficient in Shh-mediated medulloblastomas and that its loss of function contributes to medulloblastoma progression.
Together, these data suggested that P27 absence could further stratify standard-risk patients between survivors and those that fail therapy and do not survive. Because incorrect patient stratification could result in death for children placed improperly into a lower risk group, or unnecessary toxicity in children placed improperly into a higher risk group, we sought to confirm these findings by examining tissues from a second, larger study population. Tissues from 141 patient samples from children treated on the SIOP PNET03 clinical trial in Europe were stained for P27 and analyzed as above [11, 12]. Only two patients were identified that met the <1% cutoff criteria, indicating that the threshold established in the training set was not suitable for the test data set. To determine if this threshold was too stringent, we relaxed the criteria to <10% to capture more patients with low P27 expression. No significant differences were found in overall survival (P = 0.67) or in progression free survival (P = 0.55) between patients with little to no (less than 10%, F/N) P27 cells and those with greater than 10% P27 positive cells (M) (Figure 3D). We therefore conclude that the first test set was a false positive signal, likely due to small numbers and retrospective establishment of the cutoff criteria. This discrepancy underscores the importance of evaluating potential prognostic indicators in large patient populations and using both training and test data sets when developing candidate prognostic indicators for clinical use. To our knowledge, this is the first quantitative analysis of P27 expression in a large cohort of human medulloblastoma samples.
We thank Julio Vasquez from the Fred Hutchinson Cancer Research Center (FHCRC) Scientific Imaging Core for assistance with tissue microarray imaging, the FHCRC Shared Resources Histopathology for assistance with tissue processing, Michael LeBlanc for assistance with statistical analysis and Sally Ditzler, Barbara Pullar, Keith Strand and Andrew Richards for assistance with transgenic animals.
NIH Grants CA112350-04, CA114567-03 and Children’s Hospital and Regional Medical Center Neuro-oncology Endowment Support (JMO) and a Bear Necessities Grant (BAH).
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