Multi-center real-world comparison of the fully automated Idylla™ microsatellite instability assay with routine molecular methods and immunohistochemistry on formalin-fixed paraffin-embedded tissue of colorectal cancer.

Microsatellite instability (MSI) is present in 15-20% of primary colorectal cancers. MSI status is assessed to detect Lynch syndrome, guide adjuvant chemotherapy, determine prognosis, and use as a companion test for checkpoint blockade inhibitors. Traditionally, MSI status is determined by immunohistochemistry or molecular methods. The Idylla™ MSI Assay is a fully automated molecular method (including automated result interpretation), using seven novel MSI biomarkers (ACVR2A, BTBD7, DIDO1, MRE11, RYR3, SEC31A, SULF2) and not requiring matched normal tissue. In this real-world global study, 44 clinical centers performed Idylla™ testing on a total of 1301 archived colorectal cancer formalin-fixed, paraffin-embedded (FFPE) tissue sections and compared Idylla™ results against available results from routine diagnostic testing in those sites. MSI mutations detected with the Idylla™ MSI Assay were equally distributed over the seven biomarkers, and 84.48% of the MSI-high samples had ≥ 5 mutated biomarkers, while 98.25% of the microsatellite-stable samples had zero mutated biomarkers. The concordance level between the Idylla™ MSI Assay and immunohistochemistry was 96.39% (988/1025); 17/37 discordant samples were found to be concordant when a third method was used. Compared with routine molecular methods, the concordance level was 98.01% (789/805); third-method analysis found concordance for 8/16 discordant samples. The failure rate of the Idylla™ MSI Assay (0.23%; 3/1301) was lower than that of referenced immunohistochemistry (4.37%; 47/1075) or molecular assays (0.86%; 7/812). In conclusion, lower failure rates and high concordance levels were found between the Idylla™ MSI Assay and routine tests.

Multicenter Evaluation of the Idylla NRAS-BRAF Mutation Test in Metastatic Colorectal Cancer.

Treatment of colorectal cancer (CRC) with monoclonal antibodies against epidermal growth factor receptor requires the assessment of the mutational status of exons 2, 3, and 4 of the NRAS and KRAS oncogenes. Moreover, the mutational status of exon 15 of the BRAF oncogene is a marker of poor prognosis in CRC. The Idylla NRAS-BRAF Mutation Test is a reliable, simple (<2 minutes hands-on time), and quick (<2 hours turnaround time) sample-to-result solution, enabling the detection of clinically relevant mutations in NRAS (18 mutations) and BRAF (5 mutations). A multicenter study was conducted in 14 centers using the Idylla NRAS-BRAF Mutation Test to assess the NRAS and BRAF mutational status of 418 formalin-fixed, paraffin-embedded tissue samples from CRC patients. Results were compared with those obtained earlier by routine reference methods, including next-generation sequencing, pyrosequencing, mass spectrometry-based assays, PCR-based assays, and Sanger sequencing. In case of discordance, additional tests were performed by digital droplet PCR. Overall, after testing confirmation and excluding invalids/errors by design, concordances between the Idylla NRAS-BRAF Mutation Test and the reference test results were found in almost perfect agreement. In conclusion, the Idylla NRAS-BRAF Mutation Test enables the routine detection of all NRAS and BRAF mutations deemed clinically relevant according to the latest clinical guidelines, without necessitating molecular expertise or infrastructure.

Blood-based detection of RAS mutations to guide anti-EGFR therapy in colorectal cancer patients: concordance of results from circulating tumor DNA and tissue-based RAS testing.

An accurate blood-based RAS mutation assay to determine eligibility of metastatic colorectal cancer (mCRC) patients for anti-EGFR therapy would benefit clinical practice by better informing decisions to administer treatment independent of tissue availability. The objective of this study was to determine the level of concordance between plasma and tissue RAS mutation status in patients with mCRC to gauge whether blood-based RAS mutation testing is a viable alternative to standard-of-care RAS tumor testing. RAS testing was performed on plasma samples from newly diagnosed metastatic patients, or from recurrent mCRC patients using the highly sensitive digital PCR technology, BEAMing (beads, emulsions, amplification, and magnetics), and compared with DNA sequencing data of respective FFPE (formalin-fixed paraffin-embedded) tumor samples. Discordant tissue RAS results were re-examined by BEAMing, if possible. The prevalence of RAS mutations detected in plasma (51%) vs. tumor (53%) was similar, in accord with the known prevalence of RAS mutations observed in mCRC patient populations. The positive agreement between plasma and tumor RAS results was 90.4% (47/52), the negative agreement was 93.5% (43/46), and the overall agreement (concordance) was 91.8% (90/98). The high concordance of plasma and tissue results demonstrates that blood-based RAS mutation testing is a viable alternative to tissue-based RAS testing.

Expression of telomerase genes in thyroid carcinoma.

Telomerase (T) is a ribonucleoprotein complex that includes the telomerase RNA component (hTR), telomerase associated protein (TP1) and the telomerase catalytic subunit (hTERT). Telomerase has been shown in stem cells and found to be activated in tumor tissues and immortalized cells. We wanted to test whether the expression of the telomerase complex subunits correlate with the enzyme activity in human thyroid tissue. Hence, we determined the expression of hTERT, hTR and TP1 mRNA by RT-PCR and compared the results to telomerase activity as detected by the telomeric repeat amplification protocol (TRAP) assay. Fifteen benign goiters (G), 11 follicular carcinomas (FTC) including 2 oncocytic follicular carcinomas (also called Hurthle cell carcinoma, oFTC), 12 papillary carcinomas (PTC) including 3 microcarcinomas (mPTC), and 12 undifferentiated anaplastic thyroid carcinomas (UTC) were investigated. Experienced pathologists performed histological and pTNM classification in each specimen. RT-PCR analysis revealed that TP1 was ubiquitously expressed in all G and carcinomas. hTR was expressed in 4 out of 15 G, in 2 out of 3 mPTC, in 5 out of 9 PTC, in 5 out of 9 FTC, in all oFTC and in 9 out of 12 UTC samples. Regarding all carcinomas, no statistically significant correlation was observed between hTR-expression and tumor stage, lymph node or distant metastasis. hTERT-expression was associated with malignancy and tumor stage. All mPTC and 13 out of 15 G did not express hTERT, whereas all samples of pT3-4 tumor stage of FTC, PTC, UTC and all oFTC were positive for hTERT. No telomerase activity could be detected in G. Telomerase activity in carcinoma was only measurable in tissues that expressed the catalytic subunit hTERT. Our data indicate that telomerase activity is up-regulated in neoplastic cells. In contrast to TP1 and hTR, hTERT and telomerase activity may be of help in identifying invasive tumors and may be additional markers for classification of benign goiter and malignant thyroid carcinoma.

Expression analysis of pediatric solid tumor cell lines using oligonucleotide microarrays.

We identified patterns of differentially-expressed genes in cell lines derived from several pediatric solid tumors. Affymetrix Human Cancer G110 Arrays, carrying 1,700 cancer-associated genes, were applied to a panel of 11 cell lines originating from Ewing tumors (ETs), neuroblastomas, and malignant melanoma of soft parts. Hierarchical clustering clearly differentiated these 3 entities and revealed groups of 75, 102, and 36 gene probe-sets exhibiting tumor-type specific up-regulation in these cell lines, respectively. Whereas ET lines demonstrated increased expression of microtubule-associated protein tau (MAPT), protein phosphatase 1 regulatory subunit 1A (PPP1R1A), NIMA (never in mitosis gene a)-related kinase 2 (NEK2), and cyclin D1 (CCND1), neuroblastoma samples exhibited high expression of wingless-type mouse mammary tumor virus integration site family member 11 (WNT11), Drosophila frizzled homolog 2 (FZD2), and adenomatous polyposis coli (APC) which are involved in regulating free beta-catenin levels. These genes likely maintain tumor-specific characteristics and participate in key downstream regulatory mechanisms. We also correlated the expression levels of up-regulated genes in ETs with their chromosomal localization and compared these data to the comparative genomic hybridization profiles of the cell lines. We demonstrate that gains of genetic material contribute essentially to differential gene expression.

Characterization of the malignant melanoma of soft-parts cell line GG-62 by expression analysis using DNA microarrays.

GG-62 is a cell line previously thought to be derived from an atypical Ewing tumor (ET). Reverse-transcriptase polymerase chain reaction revealed an in-frame fusion between the Ewing sarcoma gene ( EWS) codon 325 and the activating transcription factor 1 gene ( ATF1) codon 65 which permits the production of chimeric EWS-ATF1 oncoproteins. We also identified the genomic breakpoint resulting from a reciprocal t(12;22)(q13;q12), which is the hallmark of malignant melanoma of soft parts (MMSP). We applied Affymetrix human cancer G110 arrays to compare the gene expression patterns of GG-62 and other cell lines derived from small blue round cell tumors of childhood. Hierarchical clustering of 463 differentially expressed genes distinguished GG-62 from the ETs, as well as the neuroblastomas, and revealed a cluster of 36 upregulated genes. Several of these genes are involved in signal transduction pathways that may be critical for maintaining cell transformation; some examples are avian erythroblastic leukemia viral oncogene homolog 3 ( ERBB3), neuregulin 1 ( NRG1), fibroblast growth factor 9 ( FGF9), and fibroblast growth factor receptor-1 ( FGFR1). Furthermore, genes near the chromosome-12q13 breakpoint exhibited increased expression of GG-62 including ERBB3, NR4A1 (nuclear receptor subfamily 4, group A, member 1), cyclin-dependent kinase 2 ( CDK2), and alpha 5 integrin ( ITGA5). Altogether our findings demonstrate the MMSP derivation of GG-62 and may shed light on the mechanisms of tumorigenesis in this rare disease.

Chromosomal alterations in osteosarcoma cell lines revealed by comparative genomic hybridization and multicolor karyotyping.

We characterized the chromosomal alterations in eight osteosarcoma cell lines (OST, HOS, U-2 OS, ZK-58, MG-63, SJSA-1, Saos-2, and MNNG) by comparative genomic hybridization (CGH); gains and losses of DNA sequences were defined as chromosomal regions with a fluorescence ratio, wherein all of the 95% confidence interval was above 1.25 and below 0.75, respectively. In four of 8 cell lines, multicolor karyotyping (MK) was added. CGH revealed the average number of aberrations per cell line was 20.8 (range: 10-31); the average numbers of gains and losses were 11.1 and 9.6, respectively. The frequent gains were identified on 1p21 approximately q24, 1q25-q31, 7p21, 7q31, 8q23 approximately q24, and 14q21; frequent losses were at 18q21 approximately q22, 18q12, 19p, and 3p12 approximately p14. High-level gains were observed on 8q23 approximately q24, 5p, and 1p21 approximately p22. MK revealed the most common translocations in the four cell lines were t(8;9), t(1;3), t(3;5), t(1;13), t(2;6), t(3; 17), t(1;15), t(10;20), and t(6;20). Chromosomes 1, 3, 8, 9, and 20 were most frequently involved in translocation events. The concordance rate of aberrations in CGH and translocations in MK was 76%. MK was useful to identify the chromosomal alterations and as a supplement to the CGH results in three of four chromosomes.

Comparative genomic hybridization in cartilaginous tumors.

Genetic aberrations in cartilaginous tumors have not yet been well characterized. We analyzed the molecular-chromosomal aberrations in 10 chondrosarcomas (four Grade-3 tumors, four Grade-2 tumors and two Grade-1 tumors) and in three benign cartilaginous tumors (two enchondromas and one chondromyxoid fibroma). Genomic imbalances were detected in 9 out of 10 cases of chondrosarcomas. The median number of changes was 7.0 per tumor (range 0-23) and the gain-to-loss ratio was 1:1.4. The most frequent gains involved 7q, 5p, or 21q and the most frequent losses were 17p, 13q, 16p, or 22q. The three benign cartilaginous tumors each had two (0 gains and two losses), six (one gain and five losses) and eight (one gain and seven losses) chromosomal aberrations. Both of the gains occurred on 13q21 and losses were frequently observed on chromosomes 19 and 22q in all three cases. Loss of chromosomes 16p, 17p, 22q, or 19 loss were common in both chondrosarcomas and benign cartilaginous tumors. However, aberrations from chromosomes 2 to 11, 14, 15, 18, or 21 were detected only in chondrosarcomas. Therefore, although the number of aberrations between benign and malignant cartilaginous tumors appears to be similar, these two entities may be differentiated by determining which chromosomes are affected.

Analysis of TP53 germline mutations in pediatric tumor patients using DNA microarray-based sequencing technology.

BACKGROUND: Whereas in sporadic human malignancies mutations of the TP53 tumor-suppressor gene occur in cancers of almost every organ and histologic subtype, patients with an inborn TP53 defect are at high risk to develop, in particular, soft tissue and bone sarcomas, brain tumors, leukaemias, adrenocortical tumors, and breast cancer. To demonstrate the usefulness of microarray technology applied to TP53 sequencing in pediatric tumors, we investigated young patients suffering from tumors typical of the Li-Fraumeni context who were, therefore, candidates for harboring inborn defects in tumor-suppressor genes. PROCEDURE: Six individuals were studied, including typical Li-Fraumeni patients as well as patients without any family history of cancer. DNA samples were independently analyzed for TP53 mutations by GeneChip and standard automated laser fluorescence (ALF) sequencing technology. RESULTS: The tumor and corresponding constitutional DNA samples clearly showed identical mutations which were confirmed by ALF sequencing. All coding exons (exons 2-11) of TP53 were analyzed simultaneously. The entire sequencing procedure and data analysis was carried out within 24 hr. CONCLUSIONS: The GeneChip TP53-sequencing assay may be feasible for routine molecular genetic diagnostics in determining the TP53 status of childhood-tumor patients and in enabling a disease management based on the genetic background of the individual.

Metallothionein expression in normal, hyperplastic, and neoplastic thyroid follicular and parafollicular C cells using monoclonal antimetallothionein antibody E9.

Metallothioneins (MTs) are a set of ubiquitous low molecular proteins with a high affinity for metal ions, such as zinc, copper, and cadmium. MT overexpression can be induced by these metal ions as well as by other endogenous and exogenous factors. In this study, normal, hyperplastic, and neoplastic thyroid tissues of both follicular and C-cell origin were immuno-histochemically investigated with a monoclonal antibody against I- and II-isoforms of MTs. MT immunoreactivity was demonstrated in the follicular epithelium of 19 normal thyroid glands and in all 32 cases of Graves' disease investigated; 26 of 30 follicular adenomas and 25 of 28 follicular carcinomas showed MT immunoreactivity, whereas only 7 of 20 papillary carcinomas were MT-positive (p < 0.0001 ). In 3 of the 7 positive samples, positivity was restricted to follicular areas of differentation. No MT could be immunolocalized in normal and hyperplastic C cells and medullary thyroid carcinomas (n = 20). In mixed medullary-follicular carcinomas (n = 4), MT staining patterns resembled those seen for thyroglobulin. In anaplastic carcinomas, MTs were mainly immunolocalized in nonspindle cell areas. MT expression in thyroid tumors may reflect the different biological behavior of follicular and papillary carcinomas. Antibodies to MTs may also serve as fairly specific immunohistochemical markers of follicular cell differentiation in thyroid neoplasia.

Immunohistochemical demonstration of chromogranin A, chromogranin B, and secretoneurin in primary non-small-cell carcinomas of the lung.

Fifty-three primary non-small-cell lung carcinomas (NSCLC) were immunohistochemically investigated with antibodies against chromogranin A, chromogranin B, and ecretoneurin. All 3 peptides were focally immunolocalized in 3 of 25 adenocarcinomas and in 2 of 6 large-cell anaplastic carcinomas in more than 20% of tumor cells. Two of 15 squamous-cell carcinomas showed chromogranin B reactivity in more than 20% of tumor cells. Neuroendocrine (NE) differentiation was also demonstrated in lymphnode metastases of large-cell anaplastic carcinomas, in 1 adenocarcinoma, and in 1 squamous-cell carcinoma, with NE differentiation of the respective primary tumors. All tumors with NE differentiation exhibited (large cell) anaplastic tumor areas. We conclude that NE differentiation should be immunohistochemically proven or excluded, particularly in NSCLC with anaplastic components. Chromogranin B and secretoneurin are proposed as useful additional neuroendocrine markers for demonstration of NE differentiation in lung carcinomas.

Possible Relation of p53 and mdm-2 Oncoprotein Expression in Thyroid Carcinoma: A Molecular-Pathological and Immunohistochemical Study on Paraffin-Embedded Tissue.

Routinely processed tissues from a series of benign and malignant thyroid lesions were immunohistochemically investigated with antibodies against p53 and mdm-2. p53 was immunolocalized in <10% of nuclei in 2/80 nodular goiters, 2/60 follicular adenomas, 26/68 follicular carcinomas, 7/40 papillary carcinomas, 3/10 "insular" carcinomas, and 10/31 anaplastic carcinomas. More than 10% positively stained nuclei were found in 2 widely invasive follicular, 2 insular, and 15 anaplastic carcinomas. All p53-positive cases showed a concomitant immunohistochemical mdm-2 expression; an immunohistochemical colocalization on serial section was demonstrated in 12 anaplastic carcinomas. Screening by polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) analysis of these 12 cases revealed no relevant mutations in the coding regions of exons 2-11 of the p53 gene. Additionally, 1 follicular adenoma, 6 follicular carcinomas (4 minimally and 2 widely invasive), 1 papillary, and 2 poorly differentiated insular carcinomas were mdm-2 positive without immunohistochemically detectable p53 expression. These results provide evidence that wild-type p53 expression in thyroid carcinomas may be associated with mdm-2 induced formation of stable complexes. However, the role of p53 mutations and p53 protein inactivation owing to other factors (e.g., mdm-2) in the progression of thyroid carcinomas is still poorly understood.

Genetic imbalances revealed by comparative genomic hybridization in osteosarcomas.

Osteosarcomas are the most frequent bone sarcomas. The molecular chromosomal aberrations in osteosarcomas were analyzed by comparative genomic hybridization (CGH). We studied 47 frozen tumors (41 primary samples, 6 relapses) in osteosarcoma patients registered in the Cooperative Osteosarcoma Study (COSS) protocol. Genomic imbalances were detected in 40 of 41 primary tumors and 6 of 6 relapsed tumors. Gains were more frequent than losses (ratio of 1.3:1). The median number of changes was 16 and 12 in primary and relapsed osteosarcomas, respectively. The median number of aberrations in primary high-grade osteosarcomas (17.0) was significantly higher than in low- or intermediate-grade osteosarcoma subtypes (3.0) (p = 0.038). The most frequent gains included 8q, 1p21-p31 and 1q21-q24, and the most frequent losses were 10q, 5q and 13q. High-level gains were observed on 8q23-q24, 17p13 and 1q21-q24. A gain of 19p (p < 0.001) or loss of 9p (p = 0.027) was more frequent in poor responders than in good responders. Univariate analysis revealed that patients with primary metastases (p = 0.002), poor histologic responses (p = 0.005), high-level gains of 19p (p = 0.012) or losses of 13q14 (p = 0.042) had significantly lower event-free survival (EFS), whereas patients with a loss of 5q (p = 0.007) or a loss of 10q21-22 (p = 0.017) had significantly higher EFS than patients without these aberrations. Multivariate analysis demonstrated that primary metastasis, loss of 13q14 and loss of 5q were independent prognostic factors. The findings of our study seem to be useful for evaluating the prognosis of patients and may finally lead to treatment strategies based on genetic background of osteosarcoma.

Radiation-induced changes of telomerase activity in a human Ewing xenograft tumor.

AIM: The effect of ionizing irradiation on telomerase activity and further associated biological factors was evaluated in a human Ewing tumor xenograft model on nude mice. MATERIAL AND METHODS: The human Ewing tumor cell line STA-ET-1 was established in a nude mouse model. Initially, the dose-response relationship for the tumor model was established. For the radiation experiments two dose levels were chosen: 5 Gy and 30 Gy. After 5 Gy, there was no significant growth delay whereas after 30 Gy there was a marked growth delay without the induction of a complete remission. Tumors were examined 6, 12, 24, 48, 72, and 96 hours post irradiation. After irradiation with 30 Gy further time points were 6, 9, 12 and 15 days. For each dose and time group, three tumors were evaluated. RESULTS: There was a reduction of telomerase activity after 5 Gy to 50% (not statistically significant) after 3 days; however, after 30 Gy there was a reduction of telomerase activity to 23% of the initial value after 6 days (p = 0.001). Telomerase activity correlated with the expression of human telomerase reverse transcriptase (hTERT), but not with the expression of telomerase-associated protein (TP1) and human telomerase RNA (hTR). The maximal amounts of necrosis or apoptosis after 30 Gy were 19% and 6.9%, respectively. CONCLUSIONS: Ionizing radiation reduces telomerase activity and the expression of hTERT which cannot be explained by the induction of necrosis or apoptosis alone. The reduction of telomerase activity may contribute to delayed cell death after radiotherapy. The combined use of radiation and specific telomerase inhibitors may be a potentially synergistic treatment strategy.