Low Percentage Of Tumor Cells Research Articles

Immunological microenvironment in colon cancer patients with different phenotypes of cancer stem cells.

e15503 Background: The purpose of this study was to analyze the effect of the CD44+ and CD133+ co-expression in cancer stem cells (CSCs) on lymphocytic microenvironment of colon cancer (CC). Methods: 200 CC patients received surgery as the first stage of treatment. The percentage of CSCs with the expression of CD44+ and/or CD133+ markers was studied in the tumor homogenates by flow cytometry, as well as some indicators of local immunity (CD3+, CD4+, CD8+, T regs (CD4+CD25+CD127dim), CD19+, PD-1, PD-L1, Th0, Tm, CD16/56+ and immunophenotypic characteristics of tumor cells (PD-L1, MHC-ABC). Results: Gradation depending on the absence or presence of co-expression of CSC markers on tumor cells allowed identification of 11 statistically significant differences out of 17 studied parameters of tumor cells and their lymphocytic microenvironment. Co-expression of CSC markers was accompanied by higher percentage of T regs (7.3±0.4 versus 5.3±0.5%), together with lower levels of CD4+ cells. At the same time, a higher content of the total number of T-lymphocytes was noted due to CD8+ with an increase in the percentage of memory T cells and a decline in naive T lymphocytes within this subpopulation. In addition, the co-expression of CSC markers was accompanied by a lower content of PD-L1 (34.3±3.0 vs. 42.9±2.5%) on lymphocytes and its higher content on tumor cells (10.6±1.5 vs. 4.1±0.8%), while the PD-1 expression on lymphocytes was higher (38.4±3.7 versus 22.3±2.9%). The presence of CD44+CD133+ CSCs was also accompanied by lower percentage of tumor cells expressing MHC class I (60.4±4.9 vs. 79.3±7.6%), which characterized the inhibition of recognition processes, and increased levels of CD8+, perhaps, should be considered as compensatory. Conclusions: The lymphocytic microenvironment of CC in the presence of CSCs with the CD44+CD133+ immunophenotype seems to be more immunosuppressive, according to the increase in the local content of T regs and the decrease in MHC-ABC expression. Higher expression of PD-L1 on tumor cells and PD-1 on lymphocytes allows activation of the PD-1/PD-L1 interaction, which enhances the immunosuppressive and growth-stimulating properties of the tumor microenvironment, but at the same time, makes tumor cells adequate targets for immunotherapy with immune checkpoint inhibitors.

Construction and evaluation of dual-effect cord blood natural killer cells expressing highaffinity PD-1 and chimeric antigen CD19 receptor

To obtain novel dual-effect cord blood natural killer cells (CBNKCs) expressing high-affinity PD-1 (HAPD1) and chimeric antigen CD19 receptor (CAR) to improve the effect of CAR-based immunotherapy. A dual-effect lentiviral vector expressing both HAPD1 and CAR targeting CD19 was constructed. CBNKCs were infected with the vector to obtain HAPD1 CAR19 CBNKCs. The surface markers of the cells including CD3-/CD16+CD56+, CD3+/CD16+CD56+, CD3+/CD4+, and CD3+/CD8+ were tested during cell proliferation. The cytotoxicity of CBNKCs, CAR19 CBNKCs and HAPD1 CAR19 CBNKCs incubated with CD19-positive target cells at the effector-target ratios of 5∶1, 10∶1 and 20∶1 was tested on days 7, 9, 12, and 15 of cell culture. The cytotoxicity of the cells against the target cells was also tested in NPG mice. CBNKCs were successfully transduced with T-cell designed CAR19 and HAPD1 CAR19 with an efficiency of (18.63±1.88)%. Infection with the lentiviral vector significantly reduced the cell expansion efficiency of the CBNKCs (10.97±2.77 vs 24.84±3.17, P < 0.05) but did not significantly affect the expressions of the surface markers (P>0.05). HAPD1 CAR CBNKCs showed stronger anti-tumor effect than CAR19 CBNKCs [(68.38±8.08)% vs (49.65±13.60)% at the effector-target ratios of 5∶1 and (79.11±7.42)% vs (59.78 ± 9.32)% at 10∶1; P < 0.05]. The infected CBNKCs showed the strongest cytotoxicity at 9 and 12 days after lentivirus infection. In the mouse models, transplantation of the dual-effect cells resulted in a significantly lower percentage of tumor cells in white blood cells than transplantation CAR-CBNKCs [(19.21 ± 3.07%) vs (29.08 ± 3.15)%, P < 0.05]. We obtained a novel dual-effect CBNKC co-expressing HAPD1 and CAR. The cells show strong cytotoxicity against the target tumor cells both in vitro and in vivo, which sheds light on a new strategy of immunotherapy against tumor cells.

Napsin A is a highly sensitive marker for nephrogenic adenoma: an immunohistochemical study with a specificity test in genitourinary tumors

Nephrogenic adenomas are uncommon benign lesions that are typically cytologically bland, but degenerative and reactive changes may make it difficult to distinguish these lesions from malignant entities, such as urothelial carcinoma and prostatic adenocarcinoma. In this study, we explored whether napsin A, a sensitive marker for lung adenocarcinoma, may also have a role in distinguishing nephrogenic adenoma from other genitourinary lesions. Immunohistochemically, napsin A was expressed in all 43 nephrogenic adenomas (bladder: 38, prostatic urethra: 4, and ureter: 1; mean positive tumor cells: 72%, median: 80%, range: 15-100%) and showed regional variability in its expression pattern with a bias towardsurface architectures (flat, papillary) compared with stromal architectures (tubular/glandular, microcystic). We also compared napsin A with other markers including PAX8, GATA3, p63, and 34BE12. Although napsin A matched PAX8 in terms of its sensitivity for nephrogenic adenoma (100%), napsin A stained a lower percentage of tumor cells than PAX8 (72% vs 99%, respectively, P=1.0×10-5). P63 was negative in all nephrogenic adenomas, whereas GATA3 showed variable staining in 25 cases (58%). All 43 nephrogenic adenomas showed variable 34BE12 staining. Finally, we profiled napsin A expression among 401 genitourinary tumors on tissue microarrays (n=308) and full tissue blocks (N=93)and observed napsin A positivity in 37 tumors (9%), which included urothelial carcinomas with the glandular/microcystic component differentiation (in the glandular/microcystic component in 4/6), bladder adenocarcinomas (primary: 4/4 and metastatic: 3/3), urinary tract clear-cell carcinomas (primary: 8/9, metastatic uterine primary: 1/1), and some renal tumors (17/174). All 81 pure urothelial carcinomas and 53 prostatic acinar adenocarcinomas were negative for napsin A. Our study indicates that napsin A is a highly sensitive marker for nephrogenic adenoma and can serve as a useful addition in immunohistochemical panels seeking to distinguish it from pure urothelial carcinoma and prostatic acinar adenocarcinoma but not clear-cell carcinoma or urothelial carcinoma with glandular differentiation.

Expression of novel neuroendocrine marker insulinoma-associated protein 1 (INSM1) in genitourinary high-grade neuroendocrine carcinomas: An immunohistochemical study with specificity analysis and comparison to chromogranin, synaptophysin, and CD56

Confirmation of genitourinary high-grade neuroendocrine carcinomas (GU-HGNECs) often requires immunohistochemical staining. Here we evaluated a novel neuroendocrine marker, insulinoma-associated protein 1 (INSM1), in GU-HGNECs with comparison to chromogranin, synaptophysin and CD56. Immunohistochemical expression of INSM1, chromogranin, synaptophysin, and CD56 was evaluated in 39 GU-HGNECs using full tissue sections [4 in kidney, 28 in urinary bladder, and 7 in prostate; 31 small cell carcinomas (SmCCs), 6 large cell neuroendocrine carcinomas (LCNECs), 2 mixed SmCC-LCNECs]. In 33 SmCCs/components, INSM1 showed similar sensitivity (93.9 %) to chromogranin (87.8 %), synaptophysin (93.9 %) and CD56 (87.8 %), and stained a similar percentage of tumor cells (52 %) to chromogranin (49 %) and CD56 (52 %), but lower than synaptophysin (87 %) (p < 0.0001). In 8 LCNECs/components, INSM1 is similar to chromogranin, synaptophysin or CD56 in sensitivity (62.5 %, 62.5 %, 75 %, 62.5 %, respectively) and the mean percentage of positively stained tumor cells (21 %, 44 %, 48 %, 37 %, respectively). INSM1 is more sensitive for SmCCs than LCNECs (93.9 % vs. 62.5 %, p = 0.015). INSM1 showed 97.4 % specificity upon analyzing 273 genitourinary non-neuroendocrine tumors on tissue microarrays. Our study indicates that INSM1 is a sensitive marker for genitourinary HGNECs with high specificity. For genitourinary SmCCs, INSM1 shows similar sensitivity to chromogranin, synaptophysin and CD56 but stains a lower percentage of tumor cells than synaptophysin. For genitourinary LCNECs, INSM1 showed similar sensitivity to chromogranin, synaptophysin and CD56. INSM1 is more sensitive for genitourinary SmCCs than LCNECs. Our result and literature review indicate that whether INSM1 is more sensitive than conventional neuroendocrine markers for HGNECs depends on the tumor primary sites.

Immunohistochemical Expression of Epidermal Growth Factor Receptor in Hepatocellular Carcinoma.

Epidermal growth factor receptor (EGFR) signaling plays an important role in various cancers, including hepatocellular carcinoma (HCC). We aimed to evaluate immunoexpression of EGFR in HCC and surrounding non-tumor liver tissue and to correlate it to multiple clinicopathologic data. We analyzed 60 patients with HCC for multiple clinicopathologic characteristics and survival. Presence of the immunosignal and the percentage of positive tumor cells at the whole tumor tissue sample and adjacent cirrhotic liver tissue were semi-quantitatively determined. Nineteen patients (31.67%) were female and 41 (68.33%) were male ranging in age from 31 to 85 years, median 61.88±10.51. Mean survival time for female patients was 8.86±1.76 months, for male 13.03±1.50 months and overall survival was 11.6051±1.19 months. The most patients had: T2 status (41.67%), no enlarged lymph nodes (90%), vascular invasion (63.33%) and well differentiated (43.33%) tumors. EGFR immunoexpression was determined in range from 0% to 100% in both tumor and non-tumor tissue with mean value of 39.58% in tumor and 86.86% in cirrhotic tissue (p<0.00). Higher percent of tumor EGFR positive cells were found in cases with higher T status, higher levels of AFP and poorly differentiated carcinoma, but not significantly. Lower percent of tumor EGFR positive cells were found in patients with vascular invasion and enlarged lymph nodes, but also not significantly. EGFR expression in tumor tissue significantly influenced survival of the patients (p<0.05). The study showed that expression of EGFR in lower percentage of tumor cells was associated to favorable prognosis, making it a potential prognostic marker and therapeutic target.

Next generation sequencing-based molecular profiling of lung adenocarcinoma using pleural effusion specimens.

Molecular profiling of non-small cell lung cancer (NSCLC) is essential for therapeutic decision-making. Pleural effusion obtained by a non-invasive, repeatable procedure may provide an opportunity for molecular profiling and thereby possibly provide information enabling targeted therapy. In this study, we aimed to evaluate the diagnostic performance of pleural effusion as a specimen for molecular analysis. Thirty patients with paired malignant pleural effusion and thoracic biopsy specimens were included. Clinically actionable mutations were assessed using a validated targeted next generation sequencing assay. EGFR/KRAS/ALK mutation status in thoracic biopsy specimens was tested using ARMS PCR. The concordance rate between gene status identified by ARMS and next-generation sequencing (NGS) analysis in the thoracic biopsy and pleural effusion samples was 86.7% (26/30). Compared with the thoracic biopsy specimens, the diagnostic performance of pleural effusion showed a sensitivity of 92.3%, a specificity of 50.0%, and a positive predictive value of 92.3%. Therefore, cases with a low percentage of tumor cells (<5%) can successfully be used to detect actionable mutations in pleural effusion specimens. These results suggest that pleural effusions are suitable specimens for oncogene mutation analysis and enable targeted therapy for patients with advanced NSCLC.

Performance of next-generation sequencing on small tumor specimens and/or low tumor content samples using a commercially available platform.

BackgroundNext generation sequencing tests (NGS) are usually performed on relatively small core biopsy or fine needle aspiration (FNA) samples. Data is limited on what amount of tumor by volume or minimum number of FNA passes are needed to yield sufficient material for running NGS. We sought to identify the amount of tumor for running the PCDx NGS platform.Methods2,723 consecutive tumor tissues of all cancer types were queried and reviewed for inclusion. Information on tumor volume, success of performing NGS, and results of NGS were compiled. Assessment of sequence analysis, mutation calling and sensitivity, quality control, drug associations, and data aggregation and analysis were performed.Results6.4% of samples were rejected from all testing due to insufficient tumor quantity. The number of genes with insufficient sensitivity make definitive mutation calls increased as the percentage of tumor decreased, reaching statistical significance below 5% tumor content. The number of drug associations also decreased with a lower percentage of tumor, but this difference only became significant between 1–3%. The number of drug associations did decrease with smaller tissue size as expected. Neither specimen size or percentage of tumor affected the ability to pass mRNA quality control. A tumor area of 10 mm2 provides a good margin of error for specimens to yield adequate drug association results.ConclusionsSpecimen suitability remains a major obstacle to clinical NGS testing. We determined that PCR-based library creation methods allow the use of smaller specimens, and those with a lower percentage of tumor cells to be run on the PCDx NGS platform.

Adequately defining tumor cell proportion in tissue samples for molecular testing improves interobserver reproducibility of its assessment.

Gene mutation status assessment of tumors has become standard practice in diagnostic pathology. This is done using samples comprising tumor cells but also non-tumor cells, which may dramatically dilute the proportion of tumor DNA and induce false negative results. Increasing sensitivity of molecular tests presently allows detection of a targeted mutation in a sample with a small percentage of tumor cells, but assessment of tumor cellularity remains essential to adequately interpret the results of molecular tests. Comprehensive tumor cell counting would provide the most reliable approach but is time consuming, and therefore rough global estimations are used, the reliability of which has been questioned in view of their potential clinical impact. The French association for quality assurance in pathology (AFAQAP) conducted two external quality assurance schemes, partly in partnership with the French group of oncology cytogenomics (GFCO). The purpose of the schemes was to (1) evaluate how tumor cellularity is assessed on tissue samples, (2) identify reasons for discrepancies, and (3) provide recommendations for standardization and improvement. Tumor cell percentages in tissue samples of lung and colon cancer were estimated by 40-50 participants, on 10 H&E virtual slides and 20 H&E conventional slides. The average difference between lowest and highest estimated percentage was 66. This was largely due to inadequate definition of cellularity, reflecting confusion between the percentage of tumor cells and the percentage of the area occupied by tumor in the assessed region. The widest range of interobserver variation was observed for samples with dense or scattered lymphocytic infiltrates or with mucinous stroma. Estimations were more accurate in cases with a low percentage of tumor cells. Macrodissection of the most homogeneous area in the tissue reduced inter-laboratory variation. We developed a rating system indicating potential clinical impact of a discrepancy. Fewer discrepancies were clinically relevant since the study was conducted. Although semi-quantitative estimations remain somewhat subjective, their reliability improves when tumor cellularity is adequately defined and heterogeneous tissue samples are macrodissected for molecular analysis.

Validation of the high-performance of pyrosequencing for clinical MGMT testing on a cohort of glioblastoma patients from a prospective dedicated multicentric trial.

BackgroundThe goal of this prospective multicentric trial was to validate a technique that allowed for MGMT promoter methylation analysis in routine clinical practice.MethodsThe MGMT status of 139 glioblastoma patients, whom had received standard first line treatment, was determined using pyrosequencing (PSQ) and a semi-quantitative Methylation-specific PCR (sqMS-PCR) method, using both frozen and formalin-fixed paraffin-embedded FFPE samples. Eight participating centers locally performed the analysis, including external quality controls.ResultsThere was a strong correlation between results from FFPE and frozen samples. With cut-offs of 12% and 13%, 98% and 91% of samples were identically classified with PSQ and sqMS-PCR respectively. In 12% of cases frozen samples were excluded because they had a low percentage of tumor cells. In 5-6% of cases the analysis was not feasible on FFPE samples. The optimized risk cut-offs were higher in both techniques when using FFPE samples, in comparison to frozen samples. For sqMS-PCR, we validated a cut-off between 13-15% to dichotomize patients. For PSQ, patients with a low level of methylation (<= 8%) had a median progression-free survival under 9 months, as compared with more than 15.5 months for those with a level above 12%. For intermediate values (9-12%), more discordant results between FFPE and frozen samples were observed and there was not a clear benefit of temozolomide treatment, which indicated a “grey zone”.ConclusionsMGMT status can reliably be investigated in local laboratories. PSQ is the ideal choice as proven by strong interlaboratory reproducibility, along with threshold agreements across independent studies.

Surfaceome of classical Hodgkin and non-Hodgkin lymphoma.

Classical Hodgkin lymphoma (cHL) is characterized by a low percentage of tumor cells in a background of diverse, reactive immune cells. cHL cells commonly derive from preapoptotic germinal-center B cells and are characterized by the loss of B-cell markers and the varying expression of other hematopoietic lineage markers. This phenotypic variability and the scarcity of currently available cHL-specific cell surface markers can prevent clear distinction of cHL from related lymphomas. We applied the cell surface capture technology to directly measure the pool of cell surface exposed proteins in four cHL and four non-Hodgkin lymphoma (NHL) cell lines. More than 1000 membrane proteins, including 178 cluster of differentiation annotated proteins, were identified and allowed the generation of lymphoma surfaceome maps. The functional properties of identified cell surface proteins enable, but also limit the information exchange of lymphoma cells with their microenvironment. Selected candidate proteins with potential diagnostic value were evaluated on a tissue microarray (TMA). Primary lymphoma tissues of 126 different B cell-derived lymphoma cases were included in the TMA analysis. The TMA analysis indicated gamma-glutamyltranspeptidase 1 as a potential additional marker that can be included in a panel of markers for differential diagnosis of cHL versus NHL.

The efficacy of EGFR gene mutation testing in various samples from non-small cell lung cancer patients: a multicenter retrospective study.

Introduction Testing for the epidermal growth factor receptor (EGFR) gene mutations requires considerable multidisciplinary experience of clinicians (for appropriate patient selection), pathologists (for selection of appropriate cytological or histological material) and geneticists (for performing and reporting reliable molecular tests). We present our experience on the efficacy of routine EGFR testing in various types of tumor samples and the frequency of EGFR mutations in a large series of Polish non-small cell lung cancer (NSCLC) patients. MethodsDeletions in exon 19 and substitution L858R in exon 21 of EGFR gene were assessed using real-time PCR techniques in 1,138 small biopsies or cytological specimens and in 1,312 surgical samples. ResultsOut of 2,450 diagnostic samples (containing >10 % of tumor cells), the occurrence of EGFR gene mutations was 9 %; more frequently in women (13.9 %) and adenocarcinoma patients (10 %), particularly with accompanying expression of TTF1 (13.0 %). The frequency of EGFR gene mutations was similar in cytological and histological specimens, and in primary and metastatic lesions, and did not depend on the percentage of tumor cells and quality of isolated DNA. Cytological or small biopsy, compared to surgical specimens showed lower percentage of tumor cells, with no impact on the quality of real-time PCR assay.ConclusionCytological and small biopsy samples with low (10–20 %) content of tumor cells and specimens from metastatic lesions are a sufficient source for EGFR mutation testing in NSCLC patients. The incidence of EGFR gene mutations in examined population was similar to those reported in other Caucasian populations.

Utilization of cell-transferred cytologic smears in detection of EGFR and KRAS mutation on adenocarcinoma of lung

Cell-transfer technique has been proven useful for performing immunocytochemistry on fine-needle aspiration smears. However, its utility for EGFR and KRAS molecular testing has not been validated. Molecular testing was performed using the cell-transfer technique on both Papanicolaou-stained ethanol-fixed and Hema 3-stained air-dried smears from 32 fine-needle aspiration samples that had diagnoses of adenocarcinoma of the lung, and then was compared to the results of the corresponding formalin-fixed paraffin-embedded tissues. The molecular testing was successfully performed on 32 of 32 ethanol-fixed and 31 of 32 air-dried samples. The molecular results on ethanol-fixed and air-dried smears showed 100% agreement. There is 100% (32/32) agreement for the EGFR and 97% (31/32) agreement for the KRAS between the cell-transfer technique and formalin-fixed paraffin-embedded tissues. One discrepant case was due to low percentage of tumor cells on the smears. Cell-transfer technique is a reliable alternative method for EGFR and KRAS testing if the cell blocks lack adequate cellularity.

Tumor Necrosis on Magnetic Resonance Imaging Correlates With Aggressive Histology and Disease Progression in Clear Cell Renal Cell Carcinoma

ObjectiveThe study objective was to correlate the magnetic resonance imaging (MRI) features of clear cell renal cell carcinoma (ccRCC) with the histopathologic features and disease progression. MethodsInstitutional review board approval for this retrospective study was obtained; patient consent was not required. The initial staging MRI scans of 75 patients with histologically confirmed ccRCC were retrospectively reviewed. The imaging was assessed by 2 radiologists for the presence of tumor necrosis, cystic degeneration, intracellular fat, hemorrhage, retroperitoneal collaterals, and renal vein thrombosis. Quantitative analysis for the MRI presence of intracellular lipid within tumors was performed. MRI findings were correlated with histopathologic findings of clear cell percentage, alveolar and tubular growth pattern, and disease progression. Statistical associations were evaluated with nonparametric univariable analyses and multivariable logistic regression models. ResultsCorrelation between MRI and histopathologic features was performed in 75 patients, whereas follow-up data were available for progression analysis in 68 patients. The presence of tumor necrosis, retroperitoneal collaterals, and renal vein thrombosis on MRI was significantly associated with a low percentage of tumor cells with clear cytoplasm (P < .01) and metastatic disease at presentation or disease progression (P < .01). At multivariable analysis, necrosis remained the only feature statistically associated with disease progression (P = .03; adjusted odds ratio, 27.7; 95% confidence interval, 1.4-554.7 for reader 1 and P = .02; adjusted odds ratio, 29.3; 95% confidence interval, 1.7-520.8 for reader 2). ConclusionsNecrosis in ccRCC on MRI correlates with the histopathologic finding of lower percentage of tumor cells with clear cytoplasm and is a poor prognostic indicator irrespective of tumor size.

Evaluation of 2-Year Experience With EGFR Mutation Analysis of Small Diagnostic Samples

Mutation analysis of the epidermal growth factor receptor (EGFR) gene is an essential part of the diagnostic algorithm in patients with metastatic or recurrent non-small cell lung cancer (NSCLC). Small biopsies or cytology specimens represent >80% of the available diagnostic material. EGFR mutation analyses were realized on 835 samples (675 cytology specimens, 151 formalin-fixed paraffin-embedded blocks, 5 tumors, and 4 pleural effusions). EGFR mutation analysis was performed by high-resolution melting analysis in combination with mutant-enriched polymerase chain reaction and sequencing analysis. Because of increased risk of inaccuracy in histology diagnosis of small specimens, all subtypes of NSCLC were analyzed. EGFR mutations were detected in 83 cases (10%). EGFR mutation testing failed in 5% (42/835) and was associated with poor cellularity, low percentage of tumor cells, and bad quality of DNA. Although 281 samples were evaluated as insufficient material (poor cellularity and/or unrepresentative tumor content), mutation rates were 7%. Although only adenocarcinomas or NSCLC-not otherwise specified are recommended for EGFR mutation testing, EGFR mutations in 11% of the large cell carcinomas and 4% of the squamous cell carcinomas were observed. Our results indicate that defined algorithm for EGFR testing of small diagnostic samples is sensitive, fast, and suitable even for samples with poor cellularity. The results of this testing should be evaluated depending on tumor content and DNA quality for each sample individually. At the conclusion of our results, we recommend to realize EGFR mutation analysis of small diagnostic samples regardless of the histologic subtypes of NSCLC.

KRAS and BRAF Mutation Analysis in Colorectal Adenocarcinoma Specimens with a Low Percentage of Tumor Cells

Background and ObjectiveThe rapid development of molecular biology techniques allows for the introduction of real-time polymerase chain reaction (PCR) methods with a limit of mutation detection at 1 % in a background of wild-type DNA. Analysis of KRAS mutations in codons 12, 13, and 61, together with analysis of BRAF mutations in codon 600, are predictive biomarkers for anti-epidermal growth factor receptor (EGFR) treatment in colorectal cancer. Our aim was to compare PCR methods for KRAS mutations and BRAF mutation analysis using DNA isolated from tissue samples previously evaluated for presence of tumor cells using a quantitative scale and the percentage of tumor cells (PTC) scale. We addressed the question of whether a low number of tumor cells can be qualified for somatic mutation testing.ResultsOur study showed that PTC as low as 10 % was good enough to detect KRAS G12D, G13D, and Q61L mutations in formalin-fixed paraffin-embedded (FFPE) material. Furthermore, our results indicate that up to 20 % of colorectal cancer may carry mutations in the KRAS codon 61 and BRAF codon 600, which suggests the value of these mutation analyses because patients carrying them are unlikely to respond to cetuximab or panitumumab. A low level of KRAS somatic mutation detection has not been studied in depth in the context of clinical outcomes in patients; therefore, we compared new PCR methods, (KRBR-RT 50 Entrogen; ViennaLab StripAssay) and re-evaluated KRAS and BRAF status in patients with relapse after targeted therapy.ConclusionsThe importance of molecular results was confirmed by clinical observation of a patient with relapse who had qualified for targeted therapy with KRAS WT status (but was diagnosed by less sensitive single-stranded conformation polymorphisms method). Interestingly, during anti-EGFR treatment, it came to the selection of cells with KRAS G12C mutation which were present from the beginning in the tumor but at a low level (detected by PCR methods) only and led consequently to the metastasis. Taking into consideration the limit of detection, labor time, and assay cost, the real-time PCR method seems to be very promising especially for FFPE material with the PTC below 15 %.

Abstract 36: The OncoNetwork Consortium: A European Collaborative Research study on the development of an Ion AmpliSeq gene panel targeting hot spots in colon and lung cancers.

Abstract The knowledge of molecular alterations involved in colorectal carcinoma (CRC) and non-small-cell lung carcinoma (NSCLC) has significantly increased in the past few years. Molecular subgroups of tumors carrying mutations in druggable genes have been identified in CRC and NSCLC. In this respect, the availability of approaches that are compatible with small amounts of input DNA from FFPE tissue and that allow a rapid screening of a large number of somatic mutations is definitely needed. Ion AmpliSeq™ technology introduces a groundbreaking workflow enabling the rapid sequencing of hundreds of mutations with low allele frequency using as little as 10ng of DNA per reaction. The OncoNetwork Consortium is a European collaborative effort of 8 Cancer Translational Research Institutions to evaluate a custom panel targeting hotspots mutations in 22 genes implicated in CRC and NSCLC. In particular, 7 labs will test the gene panel in 3 phases using 180 retrospective FFPE samples previously tested with orthogonal technologies, and 1 lab will perform confirmatory testing on novel SNPs. According to the established workflow, 10 ng of genomic DNA are used with a single primer pool that includes 86 pairs of primers spanning 15.7kb of targets. Individual libraries are barcoded during the ligation step, quantified and pooled 5 by 5 for amplification on spheres using the Ion OneTouch™ 200 Template Kit v2. Spheres are loaded on Ion 316 chip for sequencing with the Ion PGM™ Sequencer using the Ion PGM™ 200 Sequencing Kit. Results are analyzed using the Ion Reporter™ Software v1.2. In the first phase of the project, 7 labs received the same 5 blind, control FFPE samples. The average coverage obtained for the genes included in the panel ranged between 1.000x and 6.000x. The percentage of reads on target was 88-93% for all samples except for a sample showing a mean of 77%, with an average per base accuracy &amp;gt;98% for all the samples. Data analysis confirmed that all laboratories identified the expected mutations, when using a mapping quality value (MQV) of 4 as first filter. In the second phase of the project, each lab submitted to the consortium 10 previously tested CRC or NSCLC FFPE samples and received back 10 blind samples for sequencing. These samples were expected to contain 5 to 10 variants including point mutations and indels. The six labs that completed this phase were able to detect all the expected variants. Therefore, a 100% true positive rate was found in controls and blind study. The third phase of the project is ongoing and involves the assessment in each participating lab of 10 NSCLC and 5 CRC samples with a low percentage of tumor cells. In conclusion, the preliminary results suggest that the Onconetwork Ion AmpliSeq Colon and Lung Cancer panel is able to detect with high sensitivity and reproducibility the most frequent mutations of CRC and NSCLC by using a just 10 ng of DNA from FFPE samples. Citation Format: Nicola Normanno, Rosella Petraroli, Alain Rico, Henriette Kurth, Bastiaan Tops, Eliana Amato, Andrea Mafficini, Delphine Le Corre, Anna Maria Rachiglio, Anne Reiman, Orla Sheils, Christoph Noppen, Nora Rieber, Jonathan Mangion, Chrysanthi Ainali, Ludovic Lacroix, Ian Cree, Marjolijn Ligtenberg, Andy Felton, Aldo Scarpa, Pierre Laurent Puig. The OncoNetwork Consortium: A European Collaborative Research study on the development of an Ion AmpliSeq gene panel targeting hot spots in colon and lung cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 36. doi:10.1158/1538-7445.AM2013-36

Cellular origin and diagnostic significance of high‐fluorescent cells in cerebrospinal fluid detected by the XE‐5000 hematology analyzer

The Sysmex XE-5000 is a blood and body fluid analyzer able to differentiate cells into polymorphonuclear, mononuclear, and high-fluorescent cells (HFC). The identity of HFC in cerebrospinal fluid (CSF) has been uncertain; however, compatible with their high nucleic acid content, HFC could represent intrathecal tumor cells. Here, we studied the cellular origin and the diagnostic significance of HFC in CSF. Results of CSF examinations with the XE-5000 were analyzed in 65 CSF samples with and 126 CSF samples without tumor cells, as defined by manual microscopy of CSF cytospin preparations. The XE-5000 detected HFC in 51 of 65 tumor cell-positive and in 33 of 126 tumor cell-negative CSF samples (sensitivity: 78.5%, specificity: 73.8%, positive likelihood ratio: 3.0, negative likelihood ratio: 0.29). The percentages of HFC and tumor cells in CSF samples correlated (r²=0.41, P<0.0001). Tumor cells escaped detection by the XE-5000 especially in CSF samples with a low percentage of tumor cells. While this study identifies tumor cells as the predominant correlate of HFC in CSF, it suggests that measuring HFC is not an appropriate diagnostic test for intrathecal tumor cells. However, if HFC are incidentally detected in CSF, further evaluation by CSF microscopy seems mandatory.

Molecular Biological Diagnostics of KRAS and BRAF Mutations in Patients with Colorectal Cancer – Laboratory Experience

Targeted biological therapy based on blocking growth factor receptors and inhibition of cancer-inducing signaling pathways is a new treatment facility for patients with colorectal cancer (CRC). Therapeutic agents are monoclonal antibodies targeting epidermal growth factor receptor (EGFR). Gene aberrations in the EGFR-induced pathways are negative predictors of therapeutic response. Determination of -non-mutated KRAS is a requirement for the indication of targeted anti-EGFR therapy in the present time, BRAF mutation analysis is recommended. Comparison of our results with published data and verification of routine laboratory methods in relation to diagnostic kits were the purposes of this study. In addition to routine methods based on PCR, direct sequencing as well as two diagnostic kits for KRAS (codon 12 and 13) and BRAF (codon 600) mutation analysis were used for 132 patients. KRAS mutations were detected in 45 patients (34%), V600E mutation of the BRAF gene in 9 patients (7%). Both mutations simultaneously were not detected. Tissues from primary tumor and metastases were available from 33 patients. KRAS mutation was detected in 13 cases of this group. KRAS mutations in tumor and metastasis were of the same type in 9 patients; types of mutation in both tissues were different in one case. KRAS mutation only in one tissue was detected in 3 cases. BRAF mutation in both tissues was detected in the 4 patients. A low percentage of tumor cells in 17 patients specimen did not allow performance of routine analysis and diagnostic kit was used. The frequency of KRAS and BRAF mutations in our cohort of patients corresponds to published data. The suitability of metastatic tissue analysis due to tumor heterogeneity was confirmed. KRAS analysis requires a comprehensive methodological approach with regard to reduced DNA quality and different percentage of tumor cells in tissue. For this reason, commercial diagnostic kits constitute a suitable supplement to standard methods.

KRAS mutation analysis on low percentage of colon cancer cells: the importance of quality assurance

KRAS mutation testing is mandatory for patients with metastatic colorectal cancer who are eligible for treatment with an epidermal growth factor receptor targeting agent, since tumors with a mutation are not sensitive to the drug. Several methods for mutation testing are in use and the need for external quality assurance has been demonstrated. An often little addressed but important issue in external quality assurance schemes is a low percentage of tumor cells in the test samples, where the analytical sensitivity of most tests becomes critical. Using artificial samples based on a mixture of cell lines with known mutation status of the KRAS gene, we assessed the reliability of a series of commonly used methods (Sanger sequencing, high resolution melting, pyrosequencing, and amplification refractory mutation system-polymerase chain reaction) on samples with 0, 2.5, 5, 10, and 15% mutated cells. Nine laboratories throughout Europe participated and submitted a total of ten data sets. The limit of detection of each method differed, ranging from >15-5% tumor cells. All methods showed a decreasing correct mutation call rate proportionally with decreasing percentage of tumor cells. Our findings indicate that laboratories and clinicians need to be aware of the decrease in correct mutation call rate proportionally with decreasing percentage of tumor cells and that external quality assurance schemes need to address the issue of low tumor cell percentage in the test samples.

The times have changed: molecular pathology is here to stay

Molecular pathology is now an integral part of diagnostic pathology. This has brought excitement and responsibilities to us as pathologists and molecular biologists in pathology—excitement, due to the many new possibilities we have for diagnosis, prediction of outcome, and response to therapy of disease, and responsibilities, since we need to perform the tests with high accuracy, which requires expertise. But is this different conventional histology and immunohistochemistry? At the time of Virchow, histopathology brought about enormous excitement, and as we are all aware, the responsibilities became rapidly clear. The first attempt to diagnose a tumor in 1882, on a biopsy of a Crown Prince, resulted in a failure, which led to many publications on this topic. Many of us remember the early days of immunohistochemistry, again enormous excitement, many publications (still ongoing) on its use, and obviously, great responsibilities since without immunohistochemistry, many diagnoses are impossible or, at the least, unreliable. But the pathologists looking down the microscope remained the final decision makers. And we all know that a good pathologist can make a correct diagnosis on a poor slide and/or a poor immunostained slide, but without knowledge and experience, even the best slide does not suffice. For molecular pathology, it is different. For molecular tests (excluding fluorescent in situ hybridization, which is often regarded by pathologists as a bit fancier immunostain), the tissue is ground, DNA or RNA is extracted and processed, and bands, or colors or letters, appear on a screen, blot, or paper. These need to be interpreted in the context of the histological and immunohistochemical findings in order for a final conclusion to be drawn. This needs teamwork between a molecular biologist and pathologist, and both need to understand each other's trade. For pathologists, it is important to realize that there are factors that may influence the molecular results, factors such as fixation, tissue processing, and DNA extraction to name just a few. For molecular biologists, these issues are well known, but they also need to be informed about the exact diagnosis and the absolute and relative quantity of tumor cells and other potential test confounders. Not only that, both need to be aware of the test characteristics specific to the questions asked. That this is crucial can be learned from the KRAS story in colorectal cancer. EGFR targeting agents have a modest effect on metastasized colorectal cancer. As it turned out, they are only effective on tumors with a wild-type KRAS gene, but in patients with a KRAS mutated tumor, the drug even has a negative effect. It is therefore mandatory that the test is performed with high accuracy. Therefore, the European Society of Pathology has initiated a European external quality assurance system, which is now in its fourth year [1-3]. Through this program, it has become clear not only that many different test systems can provide reliable results, but also that about 10–15% of the laboratories committed unacceptable mistakes (the good news, however, is that after feedback, these laboratories performed significantly better). It has also become clear that there are major differences in the pre-analytical phase between laboratories, that there are problems when, in a sample, the percentage of tumor cells to be tested is low, and that DNA extraction methods matter. In this issue, Lee et al. describe a novel PCR-based method for KRAS testing and compare the results with the most commonly used test, Sanger sequencing [4]. This latter method is quite robust, but may show failures in case the sample contains a low percentage of tumor cells. The TaqMelt PCR assay—the cobas® KRAS Mutation Test—designed to detect 19 mutations in codons 12, 13, and 61 was shown to be superior in samples with a low percentage of tumor cells. The paper also discusses some of the issues which are relevant in molecular pathology of today and is therefore a valuable contribution to the literature, both for pathologists and for molecular biologists. It is, however, not the only commercial test system that provides highly accurate results in such samples. It is important that experiences like those from Lee et al. are published. The results of the ESP KRAS EQA system will be published as well. In this way, a large database with knowledge on methods of DNA extraction and test platforms will be built up. Such data will enable pathologists and molecular biologists to make an argued choice for test systems for any particular question. These choices are important, since each new method that is going to be used for molecular pathology needs in-house validation, prior to its application. This implies that one cannot change too often from one test to another, even though developments in methods of molecular testing evolve extremely fast.