ENCCA WP17-WP7 consensus paper on teenagers and young adults (TYA) with bone sarcomas.

Teenagers and young adults (TYA) cancer contributes substantially to morbidity and mortality in a population with much to offer society. TYA place distinct challenges upon cancer care services, many reporting feeling marginalized and their needs not being met in adult or paediatric cancer services. Bone tumours such as osteosarcoma and Ewing sarcoma, because of their age at presentation and the complexity of their care, are where challenges in managing (TYA) with cancer have often been most readily apparent. Bone sarcomas may be managed by paediatric or medical oncologists, and require fastidious attention to protocol. A lack of recent improvement in survival in TYA with bone tumours may be linked to a lack of specialist care, poor concordance with therapy in some situations and TYA-specific pharmacology. Participation in clinical trials, particularly of young adults, is low, hindering progress. All these requirements may be best met by a concerted effort to create collaborative care between adult and paediatric experts in bone sarcoma, working together to meet TYA patients' needs.

Surface proteomic analysis of osteosarcoma identifies EPHA2 as receptor for targeted drug delivery.

BACKGROUND: Osteosarcoma (OS) is the most common bone tumour in children and adolescents. Despite aggressive therapy regimens, treatment outcomes are unsatisfactory. Targeted delivery of drugs can provide higher effective doses at the site of the tumour, ultimately improving the efficacy of existing therapy. Identification of suitable receptors for drug targeting is an essential step in the design of targeted therapy for OS. METHODS: We conducted a comparative analysis of the surface proteome of human OS cells and osteoblasts using cell surface biotinylation combined with nano-liquid chromatography - tandem mass spectrometry-based proteomics to identify surface proteins specifically upregulated on OS cells. This approach generated an extensive data set from which we selected a candidate to study for its suitability as receptor for targeted treatment delivery to OS. First, surface expression of the ephrin type-A receptor 2 (EPHA2) receptor was confirmed using FACS analysis. Ephrin type-A receptor 2 expression in human tumour tissue was tested using immunohistochemistry. Receptor targeting and internalisation studies were conducted to assess intracellular uptake of targeted modalities via EPHA2. Finally, tissue micro arrays containing cores of human OS tissue were stained using immunohistochemistry and EPHA2 staining was correlated to clinical outcome measures. RESULTS: Using mass spectrometry, a total of 2841 proteins were identified of which 156 were surface proteins significantly upregulated on OS cells compared with human primary osteoblasts. Ephrin type-A receptor 2 was highly upregulated and the most abundant surface protein on OS cells. In addition, EPHA2 was expressed in a vast majority of human OS samples. Ephrin type-A receptor 2 effectively mediates internalisation of targeted adenoviral vectors into OS cells. Patients with EPHA2-positive tumours showed a trend toward inferior overall survival. CONCLUSION: The results presented here suggest that the EPHA2 receptor can be considered an attractive candidate receptor for targeted delivery of therapeutics to OS.

Instability of short tandem repeats (microsatellites) in human cancers.

The allele sizes of polymorphic microsatellite repeats in DNA from human cancers were compared to normal DNA from the same patients. In 16 out of 196 paired samples (8%), we found evidence of an extra allele of a different size in the tumour which was not present in the normal DNA. Sequence analysis confirmed that the extra allele originates from the appropriate locus and that the size change is attributable to alteration in the number of repeat units. This form of instability was more common in tri- and tetranucleotide repeats than in dinucleotide repeats. In any single tumour sample only one repeat in the set examined was abnormal, the remainder showing identical patterns in normal and tumour DNA or evidence of allele loss. The pattern of instability in diverse types of cancer differs from that reported in colorectal neoplasms.

Restoration of chemosensitivity for doxorubicin and cisplatin in chondrosarcoma in vitro: BCL-2 family members cause chemoresistance.

BACKGROUND: Chondrosarcomas are malignant cartilage-forming tumors notorious for their resistance to conventional chemo- and radiotherapy. Postulated explanations describe the inaccessibility due to abundant hyaline cartilaginous matrix, presence of multidrug resistance (MDR) pumps, and expression of anti-apoptotic BCL-2 family members. MATERIALS AND METHODS: We studied the sensitivity of chondrosarcoma cell lines (SW1353, CH2879, JJ012, OUMS27) and two primary cultures for doxorubicin and cisplatin. We examined the role of extracellular matrix using three-dimensional (3D) pellet models and MDR pump activity using fluorescence-activated cell sorter analysis. The role of BCL-2 family members was investigated using the BH3 mimetic ABT-737. RESULTS: Chondrosarcoma cells showed highest resistance to cisplatin. 3D cell pellets, morphologically strongly resembling chondrosarcoma in vivo, confirmed nuclear incorporation of doxorubicin. MDR pump activity was heterogeneous among cultures. Chondrosarcoma cells responded to ABT-737 and combination with doxorubicin led to complete loss of cell viability and apoptosis with cytochrome C release. CONCLUSIONS: Despite MDR pump activity and abundance of hyaline cartilaginous matrix, doxorubicin is able to accumulate in the cell nuclei. By repairing the apoptotic machinery, we were able to sensitize chondrosarcoma cells to doxorubicin and cisplatin, indicating an important role for BCL-2 family members in chemoresistance and a promising new treatment strategy for inoperable chondrosarcoma.

Profiling of high-grade central osteosarcoma and its putative progenitor cells identifies tumourigenic pathways.

BACKGROUND: Osteosarcoma is the most prevalent primary malignant bone tumour in children and young adults, with poor survival in 40% of patients. To identify the signalling pathways involved in tumourigenesis, we compared gene expression in osteosarcoma with that in its presumed normal counterparts. METHODS: Genome-wide expression profiles were generated from 25 high-grade central osteosarcoma prechemotherapy biopsies, 5 osteoblastomas, 5 mesenchymal stem cell (MSC) populations and these same MSCs differentiated into osteoblasts. Genes that were differentially expressed were analysed in the context of the pathways in which they function using the GenMAPP programme. RESULTS: MSCs, osteoblasts, osteoblastomas and osteosarcomas clustered separately and thousands of differentially expressed genes were identified. The most significantly altered pathways are involved in cell cycle regulation and DNA replication. Several upstream components of the Wnt signalling pathway are downregulated in osteosarcoma. Two genes involved in degradation of beta-catenin protein, the key effectors of Wnt signalling, Axin and GSK3-beta, show decreased expression, suggesting that Wnt signalling is no longer under the control of regular signals. Comparing benign osteoblastomas with osteosarcomas identified cell cycle regulation as the most prominently changed pathway. CONCLUSION: These results show that upregulation of the cell cycle and downregulation of Wnt signalling have an important role in osteosarcoma genesis. Gene expression differences between highly malignant osteosarcoma and benign osteoblastoma involve cell cycle regulation.

Ever since Knudson.

Many publications have documented loss of heterozygosity (LOH) on many different chromosomes in a wide variety of tumours, implicating the existence of multiple tumour suppressor genes (TSGs). Knudson's two-hit hypothesis predicts that these LOH events are the second step in the inactivation of both alleles of a TSG. However, to date the number of TSGs identified that are inactivated mainly at the somatic level in cancers and are not inherited has remained disappointingly small. Here we postulate that the accurate mapping of LOH events in a series of tumours to define a common LOH region is greatly confounded by deficient LOH detection, genetic instability and intertumour heterogeneity. Finding the TSGs in chromosomal regions of frequent LOH might require 'brute-force' genomic approaches.

Evidence for limited molecular genetic heterogeneity as defined by allelotyping and clonal analysis in nine metastatic breast carcinomas.

To investigate genetic intratumor heterogeneity, 42 samples of nine primary breast carcinomas and 29 related lymph node metastases were examined for DNA ploidy status, allelotype, and X chromosome inactivation pattern. Two primary breast carcinomas showed DNA index heterogeneity and five contained a single DNA aneuploid tumor stemline, whereas the two remaining primary tumors were solely DNA diploid. Most primary DNA tumor stemlines recurred in lymph node metastases (9 of 11). The allelotype, constructed with 31 different probes mapping to 23 different chromosome arms showed allelic imbalances on nearly all chromosome arms investigated. All tumors contained multiple allelic imbalances (range, 3-12). An allelic imbalance present in a primary tumor was consistently present in all DNA samples of that primary tumor and also in all DNA samples of related lymph node metastases, irrespective of DNA index heterogeneity. X chromosome inactivation pattern analysis with probe M27 beta (DXS255) confirmed the presence of clonal tumor cell populations in these tumors at the time of diagnosis. Densitometry of autoradiograms, which by eye showed retention of heterozygosity, revealed a narrow clustering of allelic imbalance factors between 1.0 and 1.4. In contrast, autoradiograms visually showing an allelic imbalance exhibited a marked interprobe, intertumor and intratumor variation in allelic imbalance factors. No relation between densitometry results and DNA ploidy status was found. Thus, at the time of diagnosis, an advanced primary breast carcinoma consists of a clonal tumor cell population with an established complement of allelic imbalances in all parts of the primary tumor and in the related lymph node metastases. Secondary to the establishment of allelic imbalances, intratumor heterogeneity for the copy number of involved alleles may develop, which in turn probably precedes metastasis.

Evidence for a gene on 17p13.3, distal to TP53, as a target for allele loss in breast tumors without p53 mutations.

In breast cancer, loss of heterozygosity (LOH) on 17p is a frequent event and a likely target is the p53 gene on 17p13.1. However, several LOH mapping studies have indicated that, in some breast tumors, LOH affects only the most distal 17p markers, suggestive of a second tumor suppressor locus in 17p13.3. In order to distinguish which gene has most probably served as the target for LOH on 17p, we have screened 141 breast tumors for somatic mutations in the p53 gene in conjunction with detailed LOH mapping on the short arm of chromosome 17. A total of 32 mutations were detected in 31 tumors, 15 of which have never been reported in breast cancer before. The majority are point mutations leading to an amino acid change in the protein. In addition, we have stained a subset of 87 tumors for the p53 protein by immunohistochemistry. In 21 of these tumors (24%), nuclear staining was detected in over 25% of the tumor cells with the anti-p53 antibody DO7. A positive correlation was found between p53-positive staining and p53 mutation (P < 0.001). A strong association was observed between p53 mutation and LOH at the TP53 locus but not between p53 expression and LOH on 17p. In breast tumors without a detectable p53 mutation but with LOH on 17p, the 17p13.3 region is always involved and, in some cases, even exclusively involved. These results suggest that a second tumor suppressor gene, located distal to TP53, is targeted by LOH on 17p in some breast tumors and that a substantial number of breast tumors stabilize p53 through mechanisms other than mutation.

Loss of heterozygosity mapping at chromosome arm 16q in 712 breast tumors reveals factors that influence delineation of candidate regions.

Loss of heterozygosity (LOH) at the long arm of chromosome 16 occurs in at least half of all breast tumors and is considered to target one or more tumor suppressor genes. Despite extensive studies by us and by others, a clear consensus of the boundaries of the smallest region of overlap (SRO) could not be identified. To find more solid evidence for SROs, we tested a large series of 712 breast tumors for LOH at 16q using a dense map of polymorphic markers. Strict criteria for LOH and retention were applied, and results that did not meet these criteria were excluded from the analysis. We compared LOH results obtained from samples with different DNA isolation methods, ie., from microdissected tissue versus total tissue blocks. In the latter group, 16% of the cases were excluded because of noninterpretable LOH results. The selection of polymorphic markers is clearly influencing the LOH pattern because a chromosomal region seems more frequently involved in LOH when many markers from this region are used. The LOH detection method, i.e., radioactive versus fluorescence detection, has no marked effect on the results. Increasing the threshold window for retention of heterozygosity resulted in significantly more cases with complex LOH, i.e., several alternating regions of loss and retention, than seen in tumors with a small window for retention. Tumors with complex LOH do not provide evidence for clear-cut SROs that are repeatedly found in other samples. On disregarding these complex cases, we could identify three different SROs, two at band 16q24.3 and one at 16q22.1. In all three tumor series, we found cases with single LOH regions that designated the distal region at 16q24.3 and the region at 16q22.1. Comparing histological data on these tumors did not result in the identification of a particular subtype with LOH at 16q or a specific region involved in LOH. Only the rare mucinous tumors had no 16q LOH at all. Furthermore, a positive estrogen content is prevalent in tumors with 16q LOH, but not in tumors with LOH at 16q24.3 only.

Targeting survivin as a potential new treatment for chondrosarcoma of bone.

Chondrosarcomas are malignant cartilage-forming bone tumors, which are intrinsically resistant to chemo- and radiotherapy, leaving surgical removal as the only curative treatment option. Therefore, our aim was to identify genes involved in chondrosarcoma cell survival that could serve as a target for therapy. siRNA screening for 51 apoptosis-related genes in JJ012 chondrosarcoma cells identified BIRC5, encoding survivin, as essential for chondrosarcoma survival. Using immunohistochemistry, nuclear as well as cytoplasmic survivin expression was analyzed in 207 chondrosarcomas of different subtypes. Nuclear survivin has been implicated in cell-cycle regulation while cytoplasmic localization is important for its anti-apoptotic function. RT-PCR was performed to determine expression of the most common survivin isoforms. Sensitivity to YM155, a survivin inhibitor currently in phase I/II clinical trial for other tumors, was examined in 10 chondrosarcoma cell lines using viability assay, apoptosis assay and cell-cycle analysis. Survivin expression was found in all chondrosarcoma patient samples. Higher expression of nuclear and cytoplasmic survivin was observed with increasing histological grade in central chondrosarcomas. Inhibition of survivin using YM155 showed that especially TP53 mutant cell lines were sensitive, but no caspase 3/7 or PARP cleavage was observed. Rather, YM155 treatment resulted in a block in S phase in two out of three chondrosarcoma cell lines, indicating that survivin is more involved in cell-cycle regulation than in apoptosis. Thus, survivin is important for chondrosarcoma survival and chondrosarcoma patients might benefit from survivin inhibition using YM155, for which TP53 mutational status can serve as a predictive biomarker.

E-cadherin is inactivated in a majority of invasive human lobular breast cancers by truncation mutations throughout its extracellular domain.

We have analysed a series of 49 human breast cancers for mutations in the entire coding region plus flanking intron sequences of the E-cadherin gene. The tumours included 41 infiltrating lobular carcinomas, two infiltrating ducto-lobular carcinomas and six infiltrative ductal carcinomas. In the lobular carcinomas 23 different somatic mutations were detected, of which seven were insertions, 11 deletions, two nonsense mutations and three splice site mutations. The other tumours showed no detectable E-cadherin mutations. All the frameshift and nonsense mutations are expected to generate a secreted E-cadherin fragment instead of a transmembrane protein with cell adhesion activity. The majority of the mutations (21 of 23) were found in combination with loss of heterozygosity of the wild type E-cadherin locus (16q22.1), a hallmark of classical tumour suppressor genes. The mutations were scattered over the whole coding region and no hot spots could be identified. All mutations described here were previously unreported. In conclusion, we have identified up to now E-cadherin mutations in 27 of 48 (56%) infiltrating lobular breast carcinomas and in 0 of 50 breast cancers of other histopathological subtypes. These data provide strong evidence that frequent E-cadherin mutations are involved in the particular etiology of sporadic lobular breast cancers.

The majority of 22 Dutch high-risk breast cancer families are due to either BRCA1 or BRCA2.

We have analyzed, by a combination of mutation and linkage analysis, the genetic basis of 22 breast cancer families in which at least 4 cases of either breast cancer diagnosed under the age of 60 or ovarian cancer had occurred. Chain-terminating mutations in BRCA1 were evidenced in 6 families, and posterior probabilities of > 0.90 of being linked to BRCA1 in 3. The breast versus ovarian cancer ratio in these 9 families was approximately 2:1. Among the remaining 13 families, significant linkage to markers flanking BRCA2 was established in the admixture test with a maximum multipoint lod score of 3.38, but there was no statistical evidence for genetic heterogeneity. The breast:ovarian cancer ratio in these families was 7:1, suggesting BRCA2 confers a much lower risk for ovarian cancer than does BRCA1. These results suggest that BRCA2 will explain a significant proportion of hereditary breast cancer in the Netherlands, and, together with BRCA1, account for the majority of all high-risk families.

Overexpression of the HER-2 oncogene does not play a role in high-grade osteosarcomas.

The aim of our study was to determine whether or not the tyrosine kinase receptor, HER2 (also known as ErbB2/Her2/neu), is overexpressed in human osteosarcomas (OS). We studied 15 biopsy and 18 resection specimens at the mRNA and protein levels. HER2 status in the OS specimens was assessed by immunohistochemistry (IHC) and quantitative Real-Time Polymerase chain reaction (PCR). In moderately immunopositive cases fluorescent in situ hybridisation (FISH) analysis was used in order to identify any possible gene amplification. 27 samples were evaluable for IHC and only 1 case showed a moderately positive membrane staining. The remaining samples showed no staining or focal cytoplasmic staining (2 samples). In the moderately positive case, FISH analysis showed no HER-2 gene amplification. There was also no overexpression of HER2 mRNA suggesting this sample was a false-positive immunostain. HER2 mRNA expression was present in all samples at a similar level to that in the breast cancer cell line, MCF7, which does not overexpress HER2 and was used as a negative control. In conclusion, this study shows that HER2 mRNA or membranous HER2 protein overexpression is absent in human OS. We noted various inconsistencies in previous published studies, with regard to methodology and the interpretation of the results based on poor methodology. We therefore conclude that the positive data with regard to HER2 overexpression reported in these previous studies is not reliable. Our results suggest that the monoclonal antibody trastuzumab (Herceptin(R)), directed against the HER2-receptor, is not likely to be an effective therapeutic agent in OS.

PCR artifacts in LOH and MSI analysis of microdissected tumor cells.

Polymerase chain reaction (PCR) analysis to study loss of heterozygosity (LOH) and microsatellite instability (MSI) in tumors is widely used. Microdissection techniques are applied to obtain tumor-specific tissue cells. By microdissection, however, the amount of template DNA extracted may vary considerably and interfere with optimal PCR amplification. To circumvent LOH and MSI misinterpretations due to low DNA input, we have assessed the critical level of DNA input for reliable PCR analysis. PCR analysis was performed by using 18 polymorphic markers (mono-, di-, tri-, and tetranucleotide) on DNA derived from both paraffin-embedded, formalin-fixed, and fresh frozen tumor specimens at template input levels ranging from 0.05 to 25.0 ng. We show a highly significant relation between DNA input and the occurrence of LOH and MSI artifacts. Furthermore, for DNA extracted from paraffin-embedded material, the percentage of LOH artifacts is significantly higher compared with DNA extracted from frozen tissue. For reliable PCR analyses using a mono-, di-, tri-, or tetranucleotide marker, a minimum of 10.0 ng DNA is required when DNA is isolated from formalin-fixed, paraffin-embedded tissue and 5.0 ng when isolated from fresh frozen tissue. HUM PATHOL 31:1414-1419.

Malignant progression in multiple enchondromatosis (Ollier's disease): an autopsy-based molecular genetic study.

Multiple enchondromatosis (Ollier's disease) is a nonhereditary disease characterized by multiple central (medullary) cartilaginous bone tumors of unknown pathogenesis. It usually involves the extremities with a unilateral predominance, and sarcomatous transformation may occur. We report an autopsy-based genetic study of a 34-year-old man presenting in early adolescence with multiple enchondromas of the extremities, predominantly left-sided, compatible with Ollier's disease. Twelve years after presentation, malignant transformation to a high grade chondrosarcoma occurred in a tibial enchondroma. The patient died after widespread metastatic disease. Loss of heterozygosity (LOH), in the tibial chondrosarcoma and its metastases, was identified exclusively on chromosome bands 13q14 and 9p21, while being absent in the femoral enchondroma analyzed. Similarly, p53 overexpression was identified immunohistochemically in the tibial chondrosarcoma and its metastases, while being absent in the femoral enchondroma; LOH at 17p13 however, was not demonstrable. It is hypothesized that inactivation of putative tumor suppressor genes at 9p21 and 13q14, and overexpression of p53, identified in the chondrosarcoma and its metastases, but absent in enchondroma, may be related to sarcomatous transformation in Ollier's disease.

Molecular evidence of field cancerization in a patient with 7 tumors of the aerodigestive tract.

Exposure of the mucosa of the upper aerodigestive tract to carcinogens can induce genetic changes resulting in various independent clones of neoplastic growth, a concept defined as "field cancerization." The risk of developing multiple tumors in this compartment of the body is well established. We studied 6 distinct tumors of the upper aerodigestive tract of a single patient for loss of heterozygosity (LOH), microsatellite instability (MSI), p53 mutations, and K-ras codon 12 point mutations. We detected a unique pattern of LOH and p53 mutations in all 6 tumors. No tumor showed a K-ras mutation or MSI. The results support the mechanism of "field cancerization" and illustrate the potential power of molecular techniques to elucidate pathogenesis.

High quality RNA isolation from tumours with low cellularity and high extracellular matrix component for cDNA microarrays: application to chondrosarcoma.

AIMS: High quality RNA isolation from cartilaginous tissue is considered difficult because of relatively low cellularity and the abundance of extracellular matrix rich in glycosaminoglycans and collagens. Given the growing interest and technical possibilities to study RNA expression at a high throughput level, research on tissue with these characteristics is hampered by the lack of an efficient method for obtaining sufficient amounts of high quality RNA. METHODS: This paper presents a robust protocol combining two RNA isolation procedures, based on a combination of Trizol and RNA specific columns, which has been developed to obtain high molecular weight RNA from fresh frozen and stored tissue of normal cartilage and cartilaginous tumours. Using this method, RNA was isolated from normal cartilage, peripheral chondrosarcoma, and central chondrosarcoma. RESULTS: The yields ranged from 0.1 to 0.5 microg RNA/mg tissue. RNA isolated with this method was stable and of high molecular weight. RNA samples from normal cartilage and from two chondrosarcomas isolated using this method were applied successfully in cDNA microarray experiments. The number of genes that give interpretable results was in the range of what would be expected from microarray results obtained on chondrosarcoma cell line RNA. Signal to noise ratios were good and differential expression between tumour and normal cartilage was detectable for a large number of genes. CONCLUSION: With this newly developed isolation method, high quality RNA can be obtained from low cellular tissue with a high extracellular matrix component. These procedures can also be applied to other tumour material.

Gene expression profiling of giant cell tumor of bone reveals downregulation of extracellular matrix components decorin and lumican associated with lung metastasis.

Giant cell tumor of bone (GCTB) displays worrisome clinical features such as local recurrence and occasionally metastatic disease which are unpredictable by morphology. Additional routinely usable biomarkers do not exist. Gene expression profiles of six clinically defined groups of GCTB and one group of aneurysmal bone cyst (ABC) were determined by microarray (n = 33). The most promising differentially expressed genes were validated by Q-PCR as potential biomarkers in a larger patient group (n = 41). Corresponding protein expression was confirmed by immunohistochemistry. Unsupervised hierarchical clustering reveals a metastatic GCTB cluster, a heterogeneous, non-metastatic GCTB cluster, and a primary ABC cluster. Balanced score testing indicates that lumican (LUM) and decorin (DCN) are the most promising biomarkers as they have lower level of expression in the metastatic group. Expression of dermatopontin (DPT) was significantly lower in recurrent tumors. Validation of the results was performed by paired and unpaired t test in primary GCTB and corresponding metastases, which proved that the differential expression of LUM and DCN is tumor specific rather than location specific. Our findings show that several genes related to extracellular matrix integrity (LUM, DCN, and DPT) are differentially expressed and may serve as biomarkers for metastatic and recurrent GCTB.

Decreased EXT expression and intracellular accumulation of heparan sulphate proteoglycan in osteochondromas and peripheral chondrosarcomas.

Mutational inactivation of EXT1 or EXT2 is the cause of hereditary multiple osteochondromas. These genes function in heparan sulphate proteoglycan (HSPG) biosynthesis in the Golgi apparatus. Loss of heterozygosity of the EXT1 locus at 8q24 is frequently found in solitary osteochondromas, whereas somatic mutations are rarely found. We investigated the expression of EXT1 and EXT2 (quantitative RT-PCR) and of different HSPGs (immunohistochemistry) in solitary and hereditary osteochondromas and in cases with malignant progression to secondary peripheral chondrosarcoma, in relation to possible mutations and promoter methylation. The mutation status of patients with multiple osteochondromas correlated with decreased EXT1 or EXT2 expression found in their resected tumours. We could not show somatic point mutations or promoter hypermethylation in 17 solitary tumours; however, EXT1 expression was decreased in 15 cases, whereas EXT2 was not. Intracellular accumulation of syndecan-2 and heparan sulphate-bearing isoforms of CD44 (CD44v3) was found in most tumours, which concentrated in the Golgi apparatus as shown by confocal microscopy. This contrasted with the extracellular expression found in normal growth plates. In conclusion, mutational inactivation of either EXT1 or EXT2 leads to loss of mRNA expression of the corresponding gene. We hypothesize that loss of EXT expression disrupts the function of the EXT1/2 complex in HSPG biosynthesis, resulting in the intracellular accumulation of HSPG core proteins that we found in these tumours.