Evaluation of a worldwide EQA scheme for complex clonality analysis of clinical lymphoproliferative cases demonstrates a learning effect.

Clonality analysis of immunoglobulin (IG) or T-cell receptor (TR) gene rearrangements is routine practice to assist diagnosis of lymphoid malignancies. Participation in external quality assessment (EQA) aids laboratories in identifying systematic shortcomings. The aim of this study was to evaluate laboratories' improvement in IG/TR analysis and interpretation during five EQA rounds between 2014 and 2018. Each year, participants received a total of five cases for IG and five cases for TR testing. Paper-based cases were included for analysis of the final molecular conclusion that should be interpreted based on the integration of the individual PCR results. Wet cases were distributed for analysis of their routine protocol as well as evaluation of the final molecular conclusion. In total, 94.9% (506/533) of wet tests and 97.9% (829/847) of paper tests were correctly analyzed for IG, and 96.8% (507/524) wet tests and 93.2% (765/821) paper tests were correctly analyzed for TR. Analysis scores significantly improved when laboratories participated to more EQA rounds (p=0.001). Overall performance was significantly lower (p=0.008) for non-EuroClonality laboratories (95% for IG and 93% for TR) compared to EuroClonality laboratories (99% for IG and 97% for TR). The difference was not related to the EQA scheme year, anatomic origin of the sample, or final clinical diagnosis. This evaluation showed that repeated EQA participation helps to reduce performance differences between laboratories (EuroClonality versus non-EuroClonality) and between sample types (paper versus wet). The difficulties in interpreting oligoclonal cases highlighted the need for continued education by meetings and EQA schemes.

PCR GeneScan and Heteroduplex Analysis of Rearranged Immunoglobulin or T-Cell Receptor Genes for Clonality Diagnostics in Suspect Lymphoproliferations.

Assessment of the presence of clonal lymphoproliferations via polymerase chain reaction (PCR)-based analysis of rearranged immunoglobulin (IG) or T-cell receptor (TR) genes is a valuable method in the diagnosis of suspect lymphoproliferative disorders. Additionally, this methodology can be used for evaluating dissemination of lymphoma cells and for studying the clonal relationship between multiple (different locations) or consecutive (over time) lymphomas. Here we describe an integrated approach to assess clonality via analysis of Ig heavy chain (IGH), Ig kappa (IGK), TCR beta (TRB), and TCR gamma (TRG) gene rearrangements, based on the standardized multiplex PCRs as originally developed by the European BIOMED-2 consortium. The described protocol covers the pre-analytical phase of DNA isolation (from formalin-fixed paraffin-embedded and fresh tissues, body fluids, peripheral blood, and bone marrow), the analytical phase of PCR GeneScan and heteroduplex analysis, and the post-analytical interpretation of the obtained profiles, following established guidelines.

Standardization of Somatic Variant Classifications in Solid and Haematological Tumours by a Two-Level Approach of Biological and Clinical Classes: An Initiative of the Belgian ComPerMed Expert Panel.

In most diagnostic laboratories, targeted next-generation sequencing (NGS) is currently the default assay for the detection of somatic variants in solid as well as haematological tumours. Independent of the method, the final outcome is a list of variants that differ from the human genome reference sequence of which some may relate to the establishment of the tumour in the patient. A critical point towards a uniform patient management is the assignment of the biological contribution of each variant to the malignancy and its subsequent clinical impact in a specific malignancy. These so-called biological and clinical classifications of somatic variants are currently not standardized and are vastly dependent on the subjective analysis of each laboratory. This subjectivity can thus result in a different classification and subsequent clinical interpretation of the same variant. Therefore, the ComPerMed panel of Belgian experts in cancer diagnostics set up a working group with the goal to harmonize the biological classification and clinical interpretation of somatic variants detected by NGS. This effort resulted in the establishment of a uniform, two-level classification workflow system that should enable high consistency in diagnosis, prognosis, treatment and follow-up of cancer patients. Variants are first classified into a tumour-independent biological five class system and subsequently in a four tier ACMG clinical classification. Here, we describe the ComPerMed workflow in detail including examples for each step of the pipeline. Moreover, this workflow can be implemented in variant classification software tools enabling automatic reporting of NGS data, independent of panel, method or analysis software.

A New and Simple TRG Multiplex PCR Assay for Assessment of T-cell Clonality: A Comparative Study from the EuroClonality Consortium.

T-cell Receptor Gamma (TRG) rearrangements are commonly used to detect clonal lymphoproliferations in hematopathology, since they are rearranged in virtually all T lymphocytes and have a relatively limited recombinatorial repertoire, which reduces the risk of false negative results, at the cost of potential false positivity. We developed an initial one-tube, 2-fluorochrome EuroClonality TRG PCR multiplex (TRG-1T-2F) which was compared to the original 2-tube, 2-fluorochrome EuroClonality/BIOMED-2 TRG PCR (TRG-2T-2F) and a commercial Invivoscribe one-tube, one-fluorochrome kit (IVS-1T-1F) on a series of 239 samples, including both T-cell malignancies and reactive cases. This initial assay yielded discrepant results between the 10 participating EuroClonality laboratories when using 2 fluorochromes, leading to adoption of a final single color EuroClonality strategy (TRG-1T-1F). Compared to TRG-2T-2F, both TRG-1T-1F and IVS-1T-1F demonstrated easier interpretation and a lower risk of false positive from minor peaks in dispersed repertoires. Both generate smaller fragments and as such are likely to be better adapted to analysis of formalin-fixed paraffin-embedded (FFPE) tissue samples. Their differential performance was mainly explained by (i) superposition of biallelic rearrangements with IVS-1T-1F, due to more extensive overlapping of the repertoires and (ii) intentional omission of the TRGJP primer in TRG-1T-1F, in order to avoid the potential risk of confusion of consensus TRG V9-JP normal rearrangements with a pathological clone.

Attenuation of mitogen- and stress-activated protein kinase-1-driven nuclear factor-kappaB gene expression by soy isoflavones does not require estrogenic activity.

We have analyzed in molecular detail how soy isoflavones (genistein, daidzein, and biochanin A) suppress nuclear factor-kappaB (NF-kappaB)-driven interleukin-6 (IL6) expression. In addition to its physiologic immune function as an acute stress cytokine, sustained elevated expression levels of IL6 promote chronic inflammatory disorders, aging frailty, and tumorigenesis. Our results in estrogen-unresponsive fibroblasts, mitogen- and stress-activated protein kinase (MSK) knockout cells, and estrogen receptor (ER)-deficient breast tumor cells show that phytoestrogenic isoflavones can selectively block nuclear NF-kappaB transactivation of specific target genes (in particular IL6), independently of their estrogenic activity. This occurs via attenuation of mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK) and ERK activity, which further down-regulates MSK-dependent NF-kappaB p65 and histone H3 phosphorylation. As constitutive NF-kappaB and MSK activity are hallmarks of aggressive metastatic ER-deficient breast cancer, the MSK signaling pathway may become an attractive target for chemotherapy.

Screening of indigenous Palestinian medicinal plants for potential anti-inflammatory and cytotoxic activity.

Organic extracts of 24 selected plant species, used by Palestinian traditional healers to treat different illnesses and diseases, were tested for their anti-inflammatory and anti-tumoral activities. The plant selection was based on existing ethnobotanic information and interviews with local healers. The extracts of the plants under investigation were tested for their potential anti-tumor (cytotoxic) effect on the murine fibrosarcoma L929sA cells, and on the human breast cancer cells MDA-MB231 and MCF7. Cytotoxicity screening models provide important preliminary data to select plant extracts with potential antineoplastic properties. MTT (Tetrazolium blue) colorimetric assay was used to evaluate the reduction of viability of cell cultures in the presence or absence of the extracts. The extract from Withania somnifera, L. Dunal (Solanaceae) presented an IC(50) value at 24h of 150 and 60 microg/ml, on L929sA and MCF7 cells, respectively, while the extract from Psidium guajava L. (Myrtaceae) presented an IC(50) value at 24h of 55 microg/ml on MCF7 cells. Other extracts examined, like Laurus nobilis L. (Lauraceae) and Salvia fruticosa M. (Labiatae), also displayed a remarkable activity. Additionally, as the nuclear transcription factor NFkappaB regulates the expression of various genes that play critical roles in apoptosis and immunomodulation, we further investigated the effect of nine promising plant extracts, withheld from the first cell viability screening on NFkappaB activation. The extracts showed variable degrees of NFkappaB-inhibitory activity. Whereas Withania somnifera extract demonstrated the strongest NFkappaB-inhibitory activity, other extracts derived from Laurus nobilis, Psidium guajava and Foeniculum vulgare M. (Umbiliferrae) also revealed immunomodulatory NFkappaB activities. These species are good candidates for further activity-monitored fractionation to identify active constituents.

Antitumoral activity of tyrosine kinase inhibitors in patients with non-small cell lung cancer harbouring rare epidermal growth factor receptor mutations.

INTRODUCTION: Mutations in the epidermal growth factor receptor (EGFR) have been reported as predictive markers of tumour response to tyrosine kinase inhibitors (TKIs) in patients with non-small cell lung cancer (NSCLC). Although the "common" EGFR mutations have been associated with response to EGFR-TKIs, the correlation with response to treatment for many other rare mutations is still unclear. The aim of this study was to investigate the clinical significance of rare and complex mutations, and the efficacy of EGFR-TKIs in this selected group of patients. METHODS: Three hundred and thirty patients with stage IIIB/IV NSCLC (106 females aged 62.5 ± 1.1 years; 224 males aged 68.0 ± 0.6 years) were enrolled in the study. Formalin fixed paraffin embedded tissue samples were screened for mutations using a high resolution melting technique, followed by Sanger sequencing of exons 18-21 of the EGFR-gene. Mutation status was also tested using the Roche Cobas(®) EGFR mutation test. RESULTS: EGFR mutations were detected in 31 tumours (9.4 %). Eleven cases carried novel mutations, six of these patients were treated with erlotinib or gefitinib. A response rate (RR) of 50.0 % was obtained in the group with rare EGFR mutations, the PFS was 3.0 months [standard deviation (STD) = 5.4 months]. The RR to EGFR-TKIs in patients with conventional EGFR mutations was 85 % with a median PFS of 10.5 months (STD = 3.6 months). CONCLUSION: We reported six patients with rare EGFR mutations of unknown clinical significance and their association with EGFR-TKIs. Report of cases harbouring rare mutations can support the decision making progress in this subset of patients.

PCR-based analysis of rearranged immunoglobulin or T-cell receptor genes by GeneScan analysis or heteroduplex analysis for clonality assessment in lymphoma diagnostics.

The assessment of the presence of clonal lymphoproliferations via polymerase chain reaction (PCR)-based analysis of rearranged immunoglobulin (Ig) or T-cell receptor (TCR) genes is a valuable technique in the diagnosis of suspect lymphoproliferative disorders. Furthermore this technique is more and more used to evaluate dissemination of non-Hodgkin lymphoma and/or the presence of (minimal) residual disease. In this chapter we describe an integrated approach to assess clonality via analysis of Ig heavy chain (IGH), Ig kappa (IGK), TCR beta (TCRB), and TCR gamma (TCRG) gene rearrangements. The described PCR protocol is based on the standardized multiplex PCRs as developed by the European BIOMED-2 collaborative study (Concerted Action BMH4-CT98-3936). Furthermore it also includes the pre-analytical DNA isolation step from various tissues (formalin fixed paraffin-embedded tissue, fresh tissues, body fluids, peripheral blood and bone marrow), GeneScan analysis of labeled PCR products on a genetic analyzer, heteroduplex analysis of unlabeled PCR products, and post-analytical guidelines for the interpretation of the obtained "molecular morphology" patterns.

Guideline on the requirements of external quality assessment programs in molecular pathology.

Molecular pathology is an integral part of daily diagnostic pathology and used for classification of tumors, for prediction of prognosis and response to therapy, and to support treatment decisions. For these reasons, analyses in molecular pathology must be highly reliable and hence external quality assessment (EQA) programs are called for. Several EQA programs exist to which laboratories can subscribe, but they vary in scope, number of subscribers, and execution. The guideline presented in this paper has been developed with the purpose to harmonize EQA in molecular pathology. It presents recommendations on how an EQA program should be organized, provides criteria for a reference laboratory, proposes requirements for EQA test samples, and defines the number of samples needed for an EQA program. Furthermore, a system for scoring of the results is proposed as well as measures to be taken for poorly performing laboratories. Proposals are made regarding the content requirements of an EQA report and how its results should be communicated. Finally, the need for an EQA database and a participant manual are elaborated. It is the intention of this guideline to improve EQA for molecular pathology in order to provide more reliable molecular analyses as well as optimal information regarding patient selection for treatment.

A case of Candida lambica fungemia misidentified as Candida krusei in an intravenous drug abuser.

Only a handful of cases of human Candida lambica infections have been published up to now. We report a Candida lambica fungemia in a young intravenous drug abuser. Using a popular chromogenic agar and a commercial phenotyping gallery, the fungus was initially misidentified as Candida krusei. Key tests to distinguish these closely related species are maximum growth temperature and assimilation of certain substrates present in more elaborate phenotyping assays. Definite confirmation is possible using molecular techniques. Susceptibility testing of the isolate demonstrated amphotericin B (MIC 0.125 microg/ml) susceptible, flucytosine (MIC 2 microg/ml) susceptible, itraconazole (MIC 0.064 microg/ml) susceptible, voriconazole (MIC 1 microg/ml) susceptible, and fluconazole (MIC >64 microg/ml, resistant).