Paired comparison of the analytical performance between the Oncomine™ Comprehensive Assay v3 and whole-exome sequencing of ovarian cancer tissue.

BACKGROUND: Next-generation sequencing (NGS) has been implemented in clinical oncology as a personalized medicine tool to identify targetable genetic alterations and to guide treatment decisions. However, the optimal NGS test strategy and target genes for clinical use are still being discussed. The aim was to compare the performance of the Oncomine™ Comprehensive Assay v3 (OCAv3) (targeted gene panel) and whole-exome sequencing (WES) to investigate somatic single and multiple nucleotide variants and small indels in ovarian cancer patients. METHODS AND RESULTS: Genomic DNA was isolated from fresh frozen samples of five high-grade serous (HGSC) and three clear cell ovarian (oCCC) cancer patients. Exome sequencing libraries were prepared by using the Ion AmpliSeq Exome RDY kit, whereas libraries for OCAv3 were prepared using by Ion AmpliSeq™ Library Kit Plus. Sequencing was performed using the Ion S5XL System (Thermo Fisher Scientific). When including only variants classified as pathogenic, likely pathogenic or unknown significance based on ClinVar database verdicts and comparing overlapping regions covered both by the OCAv3 assay and WES, 23 variants were detected by both assays. However, OCAv3 detected additionally two variants: ARID1A: p.Gln563Ter and TP53: p.Ser261ValfsTer84 that have not passed WES filtering criteria due to low coverage. CONCLUSIONS: With the present treatment possibilities, OCAv3 panel testing provided higher diagnostic yield due to better coverage. Our study emphasizes that WES, although offering the potential to identify novel findings in genes not covered by OCAv3, might overlook variants in genes relevant for OC.

ABCG2 Protein Levels and Association to Response to First-Line Irinotecan-Based Therapy for Patients with Metastatic Colorectal Cancer.

In this study we investigated the use of cancer cell protein expression of ABCG2 to predict efficacy of systemic first-line irinotecan containing therapy in patients with metastatic colorectal cancer (mCRC). From a Danish national cohort, we identified 119 mCRC patients treated with irinotecan containing therapy in first-line setting. Among these, 108 were eligible for analyses. Immunohistochemistry (IHC) analyses were performed on the primary tumor tissue in order to classify samples as high or low presence of ABCG2 protein. Data were then associated with patient outcome (objective response (OR), progression free survival (PFS) and overall survival (OS)). ABCG2 protein expression in the basolateral membrane was high (score 3+) in 33% of the patients. Exploratory analyses revealed a significant interaction between ABCG2 score, adjuvant treatment and OR (p = 0.041) in the 101 patients with evaluable disease. Patients with low ABCG2 (score 0-2) and no prior adjuvant therapy had a significantly higher odds ratio of 5.6 (Confidence Interval (CI) 1.68-18.7; p = 0.005) for obtaining OR. In contrast, no significant associations between ABCG2 expression and PFS or OS were found. These results suggest that measurement of the ABCG2 drug efflux pump might be used to select patients with mCRC for irinotecan treatment. However, additional studies are warranted before conclusions regarding a clinical use can be made. Moreover, patients with high ABCG2 immunoreactivity could be candidates for specific ABCG2 inhibition treatment in combination with irinotecan.

Stratification by MYC expression has prognostic impact in MYC translocated B-cell lymphoma-Identifies a subgroup of patients with poor outcome.

OBJECTIVE: In patients with large B-cell lymphoma (LBCL) according to WHO, the prognostic significance of MYC translocation is still not sufficiently clarified. We therefore aimed to investigate whether prognostication could be improved in patients with MYC translocation positive LBCL by additional stratification according to MYC and BCL2 protein expression levels or MYC translocation partner gene as well as concurrent BCL2 and/or BCL6 translocation (DH). METHODS: From an unselected consecutive cohort of >600 patients with LBCL investigated with fluorescent in situ hybridization (FISH), 64 patients were diagnosed with MYC translocation positive LBCL and included in the study. They were further investigated for supplemental translocations with FISH and MYC and BCL2 protein expression with immunohistochemistry (IHC). RESULTS: MYC expression >75% was associated with both reduced progression-free survival (PFS) and overall survival (OS) (PFS: HR 6.8 (95% CI 1.5-31), P = 0.004. OS: HR 4.3 (95% CI 0.9-21), P = 0.05). Immunoglobulin (IG) MYC translocation partner gene was related to high MYC protein expression (P = 0.047) but was not prognostic for PFS (P = 0.8) or OS (P = 0.6). DH did not confer a worse outcome compared to MYC single hit (SH). These findings were confirmed in a comparable, independent validation cohort of 28 patients with MYC translocation positive LBCL. All patients included in the survival analyses were treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) or R-CHOEP (R-CHOP + etoposide). CONCLUSION: These findings suggest that in patients with LBCL stratification by MYC protein expression level significantly improves the prognostic impact associated with MYC translocation.

Associations between primary tumor RAS, BRAF and PIK3CA mutation status and metastatic site in patients with chemo-resistant metastatic colorectal cancer.

BACKGROUND: Several studies have investigated correlations between metastatic pattern and mutation status in patients with colorectal cancer (CRC). However, most of the studies were small and heterogeneously designed and further research is needed to confirm previous results. In this study, we investigated the association between RAS (KRAS or NRAS), BRAF, PIK3CA mutations and metastatic pattern in patients with metastatic (m) CRC. MATERIAL AND METHODS: This study reviewed Danish biobank and database of patients with mCRC who received cetuximab and irinotecan, independent of RAS mutation status, after fluoropyrimidine, oxaliplatin and irinotecan treatment failure. The database contained information regarding tumor mutation status of KRAS, NRAS, BRAF and PIK3CA genes. RESULTS: Totally, 448 patients were included. On multivariate analyses, RAS mutations were significantly associated with increased odds of having lung metastases at diagnosis of mCRC (odds ratio (OR) = 2.04; 95% confidence interval (CI) = 1.32-3.17), and PIK3CA mutations with decreased odds of peritoneal metastases at diagnosis of mCRC (OR = 0.10; 95%CI = 0.01-0.79). On multivariate analyses of the hazard of developing metastases at any time during follow-up, RAS mutations were significantly associated with increased hazard of lung (hazard ratio (HR) = 1.34; 95%CI = 1.04-1.72) and ovarian metastases (HR = 3.12; 95%CI = 1.05-9.24), BRAF V600E mutation was associated with increased hazard of skin metastases (HR = 6.82; 95%CI = 1.86-25.02) and PIK3CA mutations with decreased hazard of peritoneal metastases (HR = 0.31; 95%CI = 0.11-0.86). CONCLUSIONS: This study indicated that in patients with mCRC, RAS mutations are associated with increased risk of lung and ovary metastases. BRAF V600E is associated with increased risk of skin metastases, and PIK3CA mutation with decreased risk of peritoneal metastases.

Topoisomerase I copy number alterations as biomarker for irinotecan efficacy in metastatic colorectal cancer.

BACKGROUND: No biomarker exists to guide the optimal choice of chemotherapy for patients with metastatic colorectal cancer. We examined the copy numbers (CN) of topoisomerase I (TOP1) as well as the ratios of TOP1/CEN-20 and TOP1/CEN-2 as biomarkers for irinotecan efficacy in patients with metastatic colorectal cancer. METHODS: From a national cohort, we identified 163 patients treated every third week with irinotecan 350 mg/m2 as second-line therapy. Among these 108 were eligible for analyses and thus entered the study. Primary tumors samples were collected and tissue microarray (TMA) blocks were produced. FISH analysis was performed using two probe-mixes: TOP1/CEN-20 and TOP1/CEN-2. Only samples harboring all three signals (TOP1, CEN-20 and CEN-2) using FISH were included in the analyses. RESULTS: In the TOP1/CEN-20 probe-mix the median TOP1- and CEN-20 CN were 4.46 (range: 1.5-9.5) and 2.00 (range: 0.55-4.55), respectively. The median TOP1- and CEN-2 CN in the TOP1/CEN-2 probe-mix, were 4.57 (range: 1.82-10.43) and 1.98 (range: 1.22-6.14), respectively. The median TOP1/CEN-20 ratio and TOP1/CEN-2 ratio were 1.25 (range: 0.92-2.90) and 2.05 (range: 1.00-6.00), respectively. None of the markers TOP1 CN, TOP1/CEN-20-ratio or TOP1/CEN-2-ratio were associated with progression free survival, overall survival or baseline characteristics. Yet, we observed a borderline association for a stepwise increase of the TOP1 CN in relation to objective response as hazard ratio were 1.35 (95% CI 0.96-1.90; p = 0.081). CONCLUSIONS: We verified a borderline significant association between increasing TOP1 CN and objective response as previously reported. Applying the probes representing CEN-20 and CEN-2, in order to investigate the ratios of TOP1/CEN-20 and TOP1/CEN-2 provided no further information in search of a biomarker driven patient stratification. Other biomarkers to be paired with TOP1 CN are therefore highly warranted.

Risk factors for brain metastases in patients with metastatic colorectal cancer.

BACKGROUND: Brain metastases (BM) from colorectal cancer (CRC) are rare, but the incidence is suspected to rise as treatment of metastatic (m) CRC improves. The aim of this study was to identify possible biological and clinical characteristics at initial presentation of mCRC that could predict later risk of developing BM. Furthermore, we wished to estimate the incidence of BM in long-term surviving patients. MATERIAL AND METHODS: We conducted a retrospective study on a Danish multicenter cohort of patients with mCRC who received cetuximab and irinotecan (CetIri) as third-line treatment. All patients had previously progression on 5-FU, irinotecan and oxaliplatin containing regimens and received CetIri treatment independent of RAS mutations status. We subsequently performed KRAS, NRAS, BRAF, PIK3CA, PTEN, ERBB2 and EGFR sequencing of DNA extracted from primary tumor tissue. RESULTS: Totally, 480 patients were included in our study. BM were diagnosed in 42 [8.8%; 95% confidence interval (CI) 6.4-11.6%] patients. Patients with BM had a significantly longer survival from mCRC diagnosis than non-BM patients (median = 32 versus 28 months, p = 0.001). On univariate cox regression analysis, the risk of developing BM was significantly increased in patients with rectal cancer (HR = 3.9; 95% CI = 1.2-13.3), metachronous metastatic disease (HR = 2.3; 95% CI = 1.2-4.4) and lung metastases (HR = 4.2; 95% CI = 2.2-7.9). On multivariate cox regression analysis only lung metastases were significantly associated BM (HR = 3.5; 95% CI = 1.8-6.8). None of the investigated mutations were associated with BM. CONCLUSION: The incidence of BM was 8.8% in patients with mCRC who received third-line therapy. The most important risk factor for developing BM was lung metastases. Furthermore, rectal cancer, metachronous metastatic disease and long survival were linked to BM development.

DNA alterations in ovarian adult granulosa cell tumours: A scoping review protocol.

BACKGROUND: Identifying and describing molecular alterations in tumors has become common with the development of high-throughput sequencing. However, DNA sequencing in rare tumors, such as ovarian adult granulosa cell tumor (aGCT), often lacks statistical power due to the limited number of cases in each study. Questions regarding personalized treatment or prognostic biomarkers for recurrence or other malignancies therefore still need to be elucidated. This scoping review protocol aims to systematically map the current evidence and identify knowledge gaps regarding DNA alterations, actionable variations and prognostic biomarkers in aGCT. METHODS: This scoping review will be conducted based on Arksey and O'Malley's methodological framework and later modifications by JBI Evidence Synthesis. The protocol complies with Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for scoping reviews. All original publications describing molecular alterations of aGCT will be included. The search will be performed in May 2024 in the following databases: MEDLINE (Ovid), Embase (Ovid), Web of Science Core Collection and Google Scholar (100-top ranked). DISCUSSION: This scoping review will identify knowledge and gaps in the current understanding of the molecular landscape of aGCT, clinical trials on actionable variations and priorities for future research. As aGCT are rare, a possible limitation will be the small sample sizes and heterogenic study settings. SCOPING REVIEW REGISTRATION: The review protocol is registered at Open Science Framework under https://doi.org/10.17605/OSF.IO/PX4MF.

Somatic Variants in DNA Damage Response Genes in Ovarian Cancer Patients Using Whole-exome Sequencing.

BACKGROUND/AIM: Several clinical trials have investigated homologous recombination deficiency and BRCA1/2 status to select ovarian cancer patients for treatment with poly(ADP-ribose) polymerase-inhibitors (PARPi), but less attention has been given to other DNA-damage response (DDR) pathways. Therefore, we investigated somatic single/multiple nucleotide variants and small insertions/deletions in exonic and splice-site regions of 356 DDR genes to examine whether genes other than BRCA1/2 are altered. MATERIALS AND METHODS: Whole-exome sequencing data from eight high-grade serous adenocarcinoma (HGSC) and four clear cell carcinoma (oCCC) patients were analyzed. RESULTS: Forty-two variants (pathogenic, likely pathogenic or variants of uncertain significance) in 28 genes from DDR pathways were identified. Seven out of nine TP53 variants were previously described in The Cancer Genome Atlas Ovarian Cancer; other variants were found in 23 out of 28 unique genes, whereas no variants were reported in FAAP24, GTF2H4, POLE4, RPA3, and XRCC4. CONCLUSION: As the identified variants were not only limited to well-known TP53, BRCA1/2, and HR-associated genes, our study might contribute to the better understanding of which DDR pathways potentially influence disease progression. Moreover, they may display a potential role as biomarkers to predict platinum-based chemotherapy or PARPi treatment response or disease progression, as differences in disrupted DDR pathways were observed between patients with long and short overall survival in HGSC and oCCC groups.

Mucosal expression of PI3, ANXA1, and VDR discriminates Crohn's disease from ulcerative colitis.

Differential diagnosis of inflammatory bowel disease (IBD) to Crohn's disease (CD) or ulcerative colitis (UC) is crucial for treatment decision making. With the aim of generating a clinically applicable molecular-based tool to classify IBD patients, we assessed whole transcriptome analysis on endoscopy samples. A total of 408 patient samples were included covering both internal and external samples cohorts. Whole transcriptome analysis was performed on an internal cohort of FFPE IBD samples (CD, n = 16 and UC, n = 17). The 100 most significantly differentially expressed genes (DEG) were tested in two external cohorts. Ten of the DEG were further processed by functional enrichment analysis from which seven were found to show consistent significant performance in discriminating CD from UC: PI3, ANXA1, VDR, MTCL1, SH3PXD2A-AS1, CLCF1, and CD180. Differential expression of PI3, ANXA1, and VDR was reproduced by RT-qPCR, which was performed on an independent sample cohort of 97 patient samples (CD, n = 44 and UC, n = 53). Gene expression levels of the three-gene profile, resulted in an area under the curve of 0.84 (P = 0.02) in discriminating CD from UC, and therefore appear as an attractive molecular-based diagnostic tool for clinicians to distinguish CD from UC.

Potential Targeted Therapies in Ovarian Cancer.

BACKGROUND: We aimed to identify somatic pathogenic and likely pathogenic mutations using next-generation sequencing (NGS). The mutational findings were held against clinically well-described data to identify potential targeted therapies in Danish patients diagnosed with high-grade serous ovarian cancer (HGSC). METHODS: We characterized the mutational profile of 128 HGSC patients. Clinical data were obtained from the Danish Gynecological Database and tissue samples were collected through the Danish CancerBiobank. DNA was analyzed using NGS. RESULTS: 47 (37%) patients were platinum-sensitive, 32 (25%) partially platinum-sensitive, 35 (27%) platinum-resistant, and three (2%) platinum-refractory, while 11 (9%) patients did not receive chemotherapy. Overall, 27 (21%) had known druggable targets. Twelve (26%) platinum-sensitive patients had druggable targets for PARP inhibitors: one for tyrosine kinase inhibitors and one for immunotherapy treatment. Eight (25%) partially platinum-sensitive patients had druggable targets: seven were eligible for PARP inhibitors and one was potentially eligible for alpesilib and hormone therapy. Seven (20%) platinum-resistant patients had druggable targets: six (86%) were potentially eligible for PARP inhibitors, one for immunotherapy, and one for erdafitinib. CONCLUSIONS: PARP inhibitors are the most frequent potential targeted therapy in HGSC. However, other targeted therapies remain relevant for investigation according to our mutational findings.

Pre-and Postoperative Circulating Tumour DNA in Patients With Gastrointestinal Stromal Tumour - A Methodological Assessment Study.

BACKGROUND/AIM: Gastrointestinal stromal tumours (GISTs) harbour genetic aberrations in receptor tyrosine kinase KIT (KIT) or platelet-derived growth factor receptor A (PDGFRA) in 85-90% of the patients. Circulating tumour DNA (ctDNA) is a potential biomarker in patients with GIST. Previous studies investigating ctDNA around surgery in patients with GIST presented divergent results regarding the proportion of patients with detectable ctDNA. This study aimed to 1) investigate the feasibility of detecting and monitoring ctDNA pre-and postoperative, 2) compare two different circulating free DNA (cfDNA) extraction methods, and validate results obtained by next-generation sequencing (NGS) using Real-Time PCR technology. PATIENTS AND METHODS: Eight patients planned for immediate surgery or surgery after neoadjuvant oncological treatment were included in the study, from whom blood collection was performed pre- and postoperatively for ctDNA analysis. Furthermore, blood samples from six patients with GIST harbouring a point mutation in KIT or PDGFRA in tissues from primary tumours were used for comparison and validation sub-study. RESULTS: In this explorative study, none of the patients with very low to intermediate risk GIST harboured KIT, or PDGFRA mutated ctDNA in pre-or postoperative blood samples. The methods used for cfDNA extraction gave similar output, and the two methods for ctDNA analysis gave identical results. CONCLUSION: There is no benefit in analysing ctDNA around surgery in very low to intermediate-risk GIST patients. Larger studies investigating ctDNA in patients with high-risk GIST around surgery are warranted.

Outstanding Response to Sorafenib in a Patient with Metastatic Gastrointestinal Stromal Tumour.

Gastrointestinal stromal tumour (GIST) is the most common sarcoma and can be seen in any part of the gastrointestinal tract. The effect of tyrosine kinase inhibitors varies with mutation status in receptor tyrosine kinase KIT and in platelet-derived growth factor receptor A (PDGFRA). This case presents a 61-year-old man, diagnosed with an 11-cm GIST located at the stomach with a high risk of recurrence. The patient showed intolerance to imatinib shortly after introduction and subsequently progressed on sunitinib and nilotinib. The patient started fourth-line treatment with sorafenib with an impressive response to a point at which metastases intra-abdominally and in the liver could be resected. After surgery, sorafenib was restarted. Due to toxicity, sorafenib dose was reduced over time. The dose was insufficient to control the disease since a new recurrence was detected. Mutation analyses revealed a GIST harbouring a deletion of codon p.I843_D846del, located at PDGFRA exon 18, right next to the codon D842 where mutations are known leading to imatinib resistance. In this case, the GIST was highly sensitive to sorafenib, and the response was dose related. It is mandatory to perform mutation analyses on primary tumour and at recurrence in the decision-making of the correct treatment for the patient. In March 2021, the patient had been in treatment with sorafenib for 12.5 years and was still without signs of recurrence. A multidisciplinary approach was essential for the long-term survival of the patient in this case.

Frequency and coexistence of KRAS, NRAS, BRAF and PIK3CA mutations and occurrence of MMR deficiency in Danish colorectal cancer patients.

The MAPK signalling genes KRAS, NRAS and BRAF and the PIK3CA gene are routinely investigated for mutations in the diagnostic routine of colorectal cancer. Few studies have reported co-existing mutations in these genes with clinical relevance, while some have been previously regarded as mutually exclusive. We set to investigate the frequency and co-occurrent mutations in these targets, and the occurrence of mismatch repair deficiency (dMMR) in a large cohort of Danish colorectal cancers. 1000 colorectal tumours were sequenced as part of our diagnostic workflow for KRAS, NRAS, BRAF and PIK3CA mutations using next-generation sequencing (NGS) and analysed by immunohistochemistry (IHC) for loss of the MMR proteins, MLH1, PMS2, MSH2 and MSH6. Co-existing mutations in 12 patients (1.2%) occurred as multiple mutations in the same gene or spread across several genes (KRAS, NRAS and/or BRAF). The frequency of single mutations in the genes occurred with a frequency similar to previously reported, except for a higher frequency of BRAF mutations (18.0%). We found dMMR in 14.6% of the cases with a majority lacking expression of both MLH1 and PMS2. BRAF mutations were only present in dMMR cases involving MLH1 and/or PMS2. Our findings suggest that co-existing mutations occur, except for the hotspot BRAF V600E, which is mutually exclusive with KRAS/NRAS mutations. Therefore, instead of single gene alterations from the MAPK signalling, assessing co-occurrence of mutations within one or more of those genes should also be accounted. This may impact future oncological treatments and should be considered in the diagnostic workflow.

DNA sequencing of cytopathologically inconclusive EUS-FNA from solid pancreatic lesions suspicious for malignancy confirms EUS diagnosis.

BACKGROUND AND OBJECTIVES: EUS-FNA is inconclusive in up to 10%-15% of patients with solid pancreatic lesions (SPLs). We aimed to investigate whether supplementary genetic analyses with whole-exome sequencing add diagnostic value in patients with SPLs suspicious of malignancy but inconclusive EUS-FNA. PATIENTS AND METHODS: Thirty-nine patients, who underwent EUS-FNA of an SPL were retrospectively included. Three groups were defined: 16 (41.0%) had suspected malignancy on EUS confirmed by cytology (malignant), 13 (33.3%) had suspected malignancy on EUS but benign cytology (inconclusive), and 10 (25.6%) had benign EUS imaging and cytology (benign). Areas with the highest epithelial cell concentrations were macro-dissected from the FNA smears from each patient, and extracted DNA was used for whole-exome sequencing by next-generation sequencing of a selected gene panel including 19 genes commonly mutated in cancer. RESULTS: Pathogenic mutations in K-RAS, TP53, and PIK3CA differed significantly between the three groups (P < 0.001, P = 0.018, and P = 0.026, respectively). Pathogenic mutations in KRAS and TP53 were predominant in the inconclusive (54% and 31%, respectively) and malignant groups (81.3% and 50%, respectively) compared to the benign group (0%). Malignant and inconclusive diagnoses correlated strongly with poor overall survival (P < 0.001). CONCLUSION: Whole-exome sequencing of genes commonly mutated in pancreatic cancer may be an important adjunct in patients with SPLs suspicious for malignancy on EUS but with uncertain cytological diagnosis.

Overexpression of c-myc is associated with adverse clinical features and worse overall survival in multiple myeloma.

The role of c-myc in multiple myeloma (MM) is controversial. We conducted a retrospective study of 117 patients with MM diagnosed between 2004 and 2010 at Herlev Hospital. Immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) were performed on tissue microarrays (TMAs) made from diagnostic bone marrow aspirates. Clinical data were obtained from the Danish Multiple Myeloma Database (DMMD). Overexpression of c-myc was found in 40% of patients. MYC translocation was found in 10% of patients. Overexpression of c-myc was not associated with MYC translocation. Overexpression of c-myc was associated with hypercalcemia (p = 0.02) and extramedullary myeloma (p < 0.01). Overexpression of c-myc was associated with shorter overall survival (OS) by multivariable analysis of the entire patient cohort [HR 1.92 (1.06-3.45), p = 0.03] and univariable analysis of high-dose-therapy (HDT)-ineligible patients [HR 2.01 (1.05-3.86), p = 0.04]. Further studies of c-myc overexpression in larger cohorts of patients with MM are warranted.

Topoisomerase-1 and -2A gene copy numbers are elevated in mismatch repair-proficient colorectal cancers.

INTRODUCTION: Topoisomerase 1 (TOP1) and 2A (TOP2A) are potential predictive biomarkers for irinotecan and anthracycline treatment, respectively, in colorectal cancer (CRC), and we have recently reported a high frequency of gene gain of the TOP1 and TOP2A genes in CRC. Furthermore, Mismatch Repair (MMR) subtypes of CRC have been associated with benefit from adjuvant chemotherapy of primary CRC. Given the involvement of the topoisomerase enzymes in DNA replication and repair, we raised the hypothesis that an association may exist between TOP gene copy numbers and MMR proficiency/deficiency in CRC. MATERIAL AND METHODS: Test cohort: FISH analysis with an in-house TOP1/CEN20 probe mix and a commercially available TOP2A/CEN17 (Dako, Glostrup, Denmark) probe mix was performed on archival formalin fixed paraffin embedded (FFPE) tissue samples from 18 patients with proficient MMR (pMMR) CRC and 18 patients with deficient MMR (dMMR) CRC. TOP1 and TOP2A gene copy numbers and their ratios per nucleus were correlated with MMR status using the Mann-Whitney test. Validation cohort: FFPE samples from 154 patients with primary stage III CRC (originally included in the RANX05 study) were classified according to MMR status by immunohistochemical analysis using validated antibodies for MLH1, MLH2, MSH6 and PMS2, and information on TOP1, CEN20, TOP2A and CEN17 status was previously published for this cohort. RESULTS: The observed TOP1 gene copy numbers in the 36 CRC test cohort were significantly greater (p < 0.01) in the pMMR subgroup (mean: 3.84, SD: 2.03) than in the dMMR subgroup (mean: 1.50, SD: 0.12). Similarly, the TOP2A copy numbers were significantly greater (p < 0.01) in the pMMR subgroup (mean: 1.99, SD: 0.52) than in the dMMR subgroup (mean: 1.52, SD: 0.10). These findings were confirmed in the validation cohort, where in the pMMR subgroup 51% had ≥2 extra TOP1 copies per cell, while all tumors classified as dMMR had diploid TOP1 status and mean TOP2A copy numbers were 2.30 (SD: 1.36) and 1.80 (SD: 0.31) (p = 0.01) in the pMMR subgroup vs. dMMR subgroup, respectively. DISCUSSION AND CONCLUSION: Our results show that TOP1 and TOP2A gene copy numbers are increased in the pMMR subgroup. We propose that this preference may reflect a selective pressure to gain and/or maintain the gained extra copies of topoisomerase genes whose products are required to cope with high replication stress present in the pMMR tumors, thereby providing a survival advantage selectively in pMMR tumors. Future studies should test this concept and explore potential differences between pMMR and dMMR tumors in response to Top1 and Top2 inhibitors.

miR-21 Expression in Cancer Cells may Not Predict Resistance to Adjuvant Trastuzumab in Primary Breast Cancer.

Trastuzumab is established as standard care for patients with HER2-positive breast cancer both in the adjuvant and metastatic setting. However, 50% of the patients do not respond to the trastuzumab therapy, and therefore new predictive biomarkers are highly warranted. MicroRNAs (miRs) constitute a new group of biomarkers and their cellular expression can be determined in tumor samples by in situ hybridization (ISH) analysis. miR-21 is highly prevalent and up-regulated in breast cancer and has been linked to drug resistance in clinical and in vitro settings. To determine expression patterns of miR-21 in high-grade breast cancers, we examined miR-21 expression in 22 HER2-positive tumors and 15 HER2-negative high-grade tumors by ISH. The histological examination indicated that patient samples could be divided into three major expression patterns: miR-21 predominantly in tumor stroma, predominantly in cancer cells, or in both stromal and cancer cells. There was no obvious difference between the HER2-positive and HER2-negative tumors in terms of the miR-21 expression patterns and intensities. To explore the possibility that miR-21 expression levels and/or cellular localization could predict resistance to adjuvant trastuzumab in HER2-positive breast cancer patients, we analyzed additional 16 HER2-positive tumors from patients who were treated with trastuzumab in the adjuvant setting. Eight of the 16 patients showed clinical recurrence and were considered resistant. Examination of the miR-21 expression patterns and intensities revealed no association between the miR-21 scores in the cancer cell population (p = 0.69) or the stromal cells population (p = 0.13) and recurrent disease after adjuvant trastuzumab. Thus, our findings show that elevated miR-21 expression does not predict resistance to adjuvant trastuzumab.