Blanka Robešová

TaqMan based real time PCR assay targeting EML4-ALK fusion transcripts in NSCLC.

OBJECTIVES Lung cancer with the ALK rearrangement constitutes only a small fraction of patients with non-small cell lung cancer (NSCLC). However, in the era of molecular-targeted therapy, efficient patient selection is crucial for successful treatment. In this context, an effective method for EML4-ALK detection is necessary. We developed a new highly sensitive variant specific TaqMan based real time PCR assay applicable to RNA from formalin-fixed paraffin-embedded tissue (FFPE). MATERIALS AND METHODS This assay was used to analyze the EML4-ALK gene in 96 non-selected NSCLC specimens and compared with two other methods (end-point PCR and break-apart FISH). RESULTS EML4-ALK was detected in 33/96 (34%) specimens using variant specific real time PCR, whereas in only 23/96 (24%) using end-point PCR. All real time PCR positive samples were confirmed with direct sequencing. A total of 46 specimens were subsequently analyzed by all three detection methods. Using variant specific real time PCR we identified EML4-ALK transcript in 17/46 (37%) specimens, using end-point PCR in 13/46 (28%) specimens and positive ALK rearrangement by FISH was detected in 8/46 (17.4%) specimens. Moreover, using variant specific real time PCR, 5 specimens showed more than one EML4-ALK variant simultaneously (in 2 cases the variants 1+3a+3b, in 2 specimens the variants 1+3a and in 1 specimen the variant 1+3b). In one case of 96 EML4-ALK fusion gene and EGFR mutation were detected. All simultaneous genetic variants were confirmed using end-point PCR and direct sequencing. CONCLUSION Our variant specific real time PCR assay is highly sensitive, fast, financially acceptable, applicable to FFPE and seems to be a valuable tool for the rapid prescreening of NSCLC patients in clinical practice, so, that most patients able to benefit from targeted therapy could be identified.

Identification of atypical ATRNL1 insertion to EML4-ALK fusion gene in NSCLC

We herein present a rare case of an EML4-ALK positive patient. A 61-year-old man was diagnosed with locoregional non-small cell lung cancer (NSCLC). No EGFR mutations were detected, and therefore the ALK rearrangement was evaluated using immunohistochemistry (IHC), fluorescence in situ hybridization (FISH) and the reverse transcription PCR (RT-PCR) method for EML4-ALK. All methods showed a positive result and, therefore, the patient was treated with crizotinib with a good therapeutic response. However, a detailed RT-PCR analysis and sequencing revealed an unexpected 138 bp insertion of attractin-like 1 (ATRNL1) gene into the EML4-ALK fusion gene. In our case, the positive therapeutic response suggests that ATRNL1 insertion does not affect EML4-ALKs sensitivity to crizotinib. This case shows great EML4-ALK heterogeneity and also that basic detection methods (IHC, FISH) cannot fully specify ALK rearrangement but in many cases a full specification seems to be important for an effective TKI indication, and sequencing ALK variants might contribute to optimized patient selection.

Clonal heterogeneity in patients with cytogenetically normal acute myeloid leukemia with NPM1 mutations

Abstract The nucleophosmin 1 (NPM1) gene is one of the most commonly mutated genes in acute myeloid leukemia (AML), occurring in approximately 60% of adult cytogenetically normal AML (CN-AML). To date, these mutations have only been detected in cells of the myeloid lineage, whereas the potential clonal involvement of the lymphoid lineage is controversial. In our study, NPM1 mutations were analyzed using the highly sensitive real-time quantitative polymerase chain reaction (RQ-PCR) method on fluorescence-activated cell-sorted (FACS) purified different circulating mature cell populations in patients with NPM1-mutated CN-AML. As expected, NPM1 mutations were found in myeloid blood cells, including CD14+ monocytes and CD66b+ granulocytes. However, we were also able to detect NPM1 mutations in CD19+ B cells and CD3−14−16+56+ natural killer (NK) cells, albeit at lower levels. Surprisingly, mutations were also detected in CD3+ T cells from all analyzed patients. Our data demonstrate that NPM1-mutated CN-AML originates in an early stem cell with both lymphoid and myeloid differentiation potential.

Genesis of new allele at locus D2S1360 in leukemia patient

Genotyping polymorphic short tandem repeats (STRs) is widely used in forensic DNA analysis for genetic identification of individuals in paternity disputes or mapping genetic diseases. Another use of STRs is monitoring hematopoietic chimerism in patients after allogeneic hematopoietic stem cell transplantation (HSCT). STR profiling can be used for cell line authentication. STR polymorphism consists of many alleles per locus, each containing a different number of tandemly arranged repeat units. The number of repeats can be highly variable among individuals, which is suitable for human identification purposes. According to the Mendelian inheritance, each individual inherits one allele from the mother and another from the father at every locus. In rare cases, abnormal DNA profiles can be found, such as tri-allelic pattern. This genotyping irregularity is presented as a result of four nucleotides’ mutational changes, suggesting the loss or addition of one or more tetrameric repeat units [1]. Mutations in hematologic malignancies can also cause loss of heterozygosity (LOH) or different STR length mutations. Leukemia is a type of cancer affecting the blood or bone marrow cells, thus resultant mutations and loss of alleles can be observed in both blood tissue and other tumor tissues of the same individual. Differences in STR polymorphism in healthy and malignant tissues may lead to misinterpretation of leukemia patients forensic samples. In the event of leukemia, the comparative analysis on buccal and blood samples as references should be performed [2,3]. It is also suitable to use hair follicles or semen [4]. In this article, we report a rare case of genesis of a novel allele of marker D2S1360 in a patient with acute myeloid leukemia. Changes in the profile of the patients were monitored over time and confirmed by sequencing analysis. The analyses were performed on DNA from peripheral blood (PB), bone marrow (BM) or buccal swab samples from a 47 year-old woman after HSCT. In 2001, she was diagnosed with polycytemia vera, the chronic myeloproliferative disease. In 2010, the patient’s status had evolved into post-polycytemia vera myelofibrosis, a high-risk disease, which was the cause of HSCT. Allogeneic HSCT was carried out in 2012 from HLA (human leukocyte antigen system) identical sibling (the patients sister). Complete remission was achieved after HSCT. In the time period of 16–19 months after HSCT, the value of mixed chimerism (MC) moved into the range of 30–98% of recipient. Donor lymphocyte infusions (DLIs) were given to our patient for increasing MC, the patient received DLIs in escalating doses. The first DLI dose was 5 10 CD3þ cells/kg, the second dose was 1 10 CD3þ cells/kg, and the third dose was 5 10 CD3þ cells/kg. In next 10 months, the value of MC decreased and persisted under a level of 1% in the recipient. Then (since January 2016) in a period of 5 months, the value of MC increased to 72% in the recipient and the patient’s diagnosis was changed to acute myeloid leukemia by detecting 26% myeloblasts in BM in May 2016. The patient refused a second allogeneic HSCT and now she is being treated with palliative treatment (since September 2016). Informative STR polymorphism for routine monitoring of cell chimerism was determined using a multiplex test system MentypeR ChimeraR PCR Amplification Kit by Biotype (AG, Dresden, Germany), namely D2S1360 was selected. The recipients (patients) alleles contained 21 and 25 tetranucleotide repeats; alleles 23 and 30 were found in the DNA of the donor. Monitoring of chimerism was carried out in accordance with therapeutic protocols; the PB or BM samples were analyzed by fragment analysis using ABI3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA). An MC increase over 1% of the recipient was first observed in January 2016 (26 months after HSCT). The same growth trend was evident in subsequent PB samples. The patient diagnosis was changed to acute myeloid leukemia by detecting myeloblasts in the BM (MC 14% of recipient, 27 months after HSCT). During the following period, the patients PB samples were taken at regular intervals (once a month). The proportion of recipient alleles (21–25) gradually became more significant and except these alleles, the presence of

[Effect of Erlotinib in 2nd and 3rd Line Anticancer Treatment in Patients with Squamous Cell Lung Cancer - Case Series].

BACKGROUND Squamous cell carcinoma of the lung (SCC) represents cca 30-40% of new cases of non-small cell lung cancer (NSCLC) in the Czech Republic. The tyrosine kinase inhibitor erlotinib is indicated as a 1st line treatment for patients with locally advanced and metastatic disease and activating mutations in endothelial growth factor receptor (EGFR), or as a 2nd or 3rd line treatment in EGFR-negative NSCLC patients after chemotherapeutic failure. OBSERVATION We present three case reports of patients with SCC treated with erlotinib as a 2nd or 3rd line of treatment. All patients were verified by histological analysis of tumor samples. EGFR mutation status was negative in one patient, while the other samples were not suitable for genetic screening. RESULTS The therapeutic response to erlotinib lasted for 68, 40, and 13 months, resp. The patient with the longest therapeutic response (patient no. 1) is still continuing erlotinib treatment (as of December 2016). The overall survival of the two patients who died was 50 and 43 months, resp. One patient died of an unknown cause with no signs of progression of the disease on CT scans. The other patient died of terminal progression of the oncological disease. All three patients experienced major therapeutic benefit from erlotinib treatment as shown by the long periods of progression-free survival and prolonged overall survival. CONCLUSION The three case reports demonstrate that erlotinib may be effective as a 2nd or 3rd line treatment in patients with SCC, especially in patients with limited alternative anticancer treatment options.Key words: non-small cell lung cancer - squamous cell carcinoma - erlotinib - treatment - tyrosine kinase inhibitor The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 5. 8. 2016Accepted: 14. 12. 2016.