Background: The clonoSEQ® Assay (Adaptive Biotechnologies Corporation, Seattle, USA) identifies and tracks unique disease-associated immunoglobulin (Ig) sequences by next-generation sequencing of IgH, IgK, and IgL rearrangements and IgH-BCL1/2 translocations in malignant B cells. Here, we describe studies to validate the analytical performance of the assay using patient samples and cell lines.

Abstract Background: The clonoSEQ® Assay (Adaptive Biotechnologies Corporation, Seattle, USA) identifies and tracks unique disease-associated immunoglobulin (Ig) sequences by next-generation sequencing of IgH, IgK, and IgL rearrangements and IgH-BCL1/2 translocations in malignant B cells. Here, we describe studies to validate the analytical performance of the assay using patient samples and cell lines.Methods: Sensitivity and specificity were established by defining the limit of detection (LoD), limit of quantitation (LoQ) and limit of blank (LoB) in genomic DNA (gDNA) from 66 patients with multiple myeloma (MM), acute lymphoblastic leukemia (ALL), or chronic lymphocytic leukemia (CLL), and three cell lines. Healthy donor gDNA was used as a diluent to contrive test samples with specific DNA masses and malignant-cell frequencies. Precision was validated using a range of samples contrived from patient gDNA, healthy donor gDNA, and 9 cell lines to generate numerous measurable residual disease (MRD) frequencies spanning clinically relevant thresholds. Linearity was determined using samples contrived from cell line gDNA spiked into healthy gDNA to generate 11 MRD frequencies for each DNA input, then confirmed using clinical samples. Quantitation accuracy was assessed by (1) comparing clonoSEQ and multiparametric flow cytometry (mpFC) measures of ALL and MM cell lines diluted in healthy mononuclear cells, and (2) analyzing precision study data for quantitation bias between MRD results from clonoSEQ measurements of diluted gDNA and those expected from mpFC of original, undiluted samples. Repeatability of nucleotide base calls was assessed via the assay’s ability to recover malignant clonotype sequences across several replicates, process features, and MRD levels.Results: LoD and LoQ were estimated at 1.903 cells and 2.390 malignant cells, respectively. LoB was zero in healthy donor gDNA. Precision ranged from 18% CV at higher DNA inputs to 68% CV near the LoD. Variance component analysis showed MRD results were robust, with expected laboratory process variations contributing ≤3% CV. Linearity and accuracy were demonstrated for each disease across orders of magnitude of clonal frequencies. Nucleotide sequence error rates were extremely low.Conclusions: These studies validate the analytical performance of the clonoSEQ Assay, and demonstrate its potential as a highly sensitive diagnostic tool for selected lymphoid malignancies.

Background: Here we present efficacy outcomes in ALKrearranged (ALK+) NSCLC patients with baseline (BL) brain metastases (BM) treated with the selective oral ALKi ceritinib in the ASCEND-1 (phase 1; NCT01283516) and ASCEND-3 (phase 2; NCT01685138) trials.

231-1247 leads to an increase in apoptosis, however loss of the SASH1 cleavage site and/or nuclear translocation prevents this initiation of apoptosis. Mechanistically SASH1 cleavage is required for the translocation of the transcription factor NF-kB to the nucleus. The use of the NF-kB inhibitor DHMEQ demonstrated that the effect of SASH1 on apoptosis was dependent on NF-kB, indicating a co-dependence between SASH1 and NF-kB for this process. Conclusion: We have shown that SASH1 contributes to apoptosis via a NF-kB-dependent mechanism. Agents that upregulate SASH1, such as chloropyramine or SASH1 gene therapy, are potential novel approaches to the management of NSCLC in the future.