Developing Molecular Signatures for Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is a clonal malignancy of mature B cells that displays a great clinical heterogeneity, with many patients having an indolent disease that will not require intervention for many years, while others present an aggressive and symptomatic leukemia requiring immediate treatment. Although there is no cure for CLL, the disease is treatable and current standard chemotherapy regimens have been shown to prolong survival. Recent advances in our understanding of the biology of CLL have led to the identification of numerous cellular and molecular markers with potential diagnostic, prognostic and therapeutic significance. We have used the recently developed digital multiplexed gene-expression technique (DMGE) to analyze a cohort of 30 CLL patients for the presence of specific genes with known diagnostic and prognostic potential. Starting from a set of 290 genes we were able to develop a molecular signature, based on the analysis of 13 genes, which allows distinguishing CLL from normal peripheral blood and from normal B cells, and a second signature based on 24 genes, which distinguishes mutated from unmutated cases (LymphCLL Mut). A third classifier (LymphCLL Diag), based on a 44-gene signature, distinguished CLL cases from a series of other B-cell chronic lymphoproliferative disorders (n = 51). While the methodology presented here has the potential to provide a "ready to use" classification tool in routine diagnostics and clinical trials, application to larger sample numbers are still needed and should provide further insights about its robustness and utility in clinical practice

Direct Targeting <i>KRAS</i> Mutation in Non-Small Cell Lung Cancer: Focus on Resistance

KRAS is the most frequently mutated oncogene in non-small cell lung cancers (NSCLC), with a frequency of around 30%, and encoding a GTPAse that cycles between active form (GTP-bound) to inactive form (GDP-bound). The KRAS mutations favor the active form with inhibition of GTPAse activity. KRAS mutations are often with poor response of EGFR targeted therapies. KRAS mutations are good predictive factor for immunotherapy. The lack of success with direct targeting of KRAS proteins, downstream inhibition of KRAS effector pathways, and other strategies contributed to a focus on developing mutation-specific KRAS inhibitors. KRAS p.G12C mutation is one of the most frequent KRAS mutation in NSCLC, especially in current and former smokers (over 40%), which occurs among approximately 12–14% of NSCLC tumors. The mutated cysteine resides next to a pocket (P2) of the switch II region, and P2 is present only in the inactive GDP-bound KRAS. Small molecules such as sotorasib are now the first targeted drugs for KRAS G12C mutation, preventing conversion of the mutant protein to GTP-bound active state. Little is known about primary or acquired resistance. Acquired resistance does occur and may be due to genetic alterations in the nucleotide exchange function or adaptative mechanisms in either downstream pathways or in newly expressed KRAS G12C mutation

Molecular Mechanism of EGFR-TKI Resistance in EGFR-Mutated Non-Small Cell Lung Cancer: Application to Biological Diagnostic and Monitoring

International audienceNon-small cell lung cancer (NSCLC) is the most common cancer in the world. Activating epidermal growth factor receptor (EGFR) gene mutations are a positive predictive factor for EGFR tyrosine kinase inhibitors (TKIs). For common EGFR mutations (Del19, L858R), the standard first-line treatment is actually third-generation TKI, osimertinib. In the case of first-line treatment by first (erlotinib, gefitinib)- or second-generation (afatinib) TKIs, osimertinib is approved in second-line treatment for patients with T790M EGFR mutation. Despite the excellent disease control results with EGFR TKIs, acquired resistance inevitably occurs and remains a biological challenge. This leads to the discovery of novel biomarkers and possible drug targets, which vary among the generation/line of EGFR TKIs. Besides EGFR second/third mutations, alternative mechanisms could be involved, such as gene amplification or gene fusion, which could be detected by different molecular techniques on different types of biological samples. Histological transformation is another mechanism of resistance with some biological predictive factors that needs tumor biopsy. The place of liquid biopsy also depends on the generation/line of EGFR TKIs and should be a good candidate for molecular monitoring. This article is based on the literature and proposes actual and future directions in clinical and translational research

Characterization of different types of CLL samples using PCA analysis.

<p>Samples from PB (n = 5), B cell samples (n = 4), and samples from CLL patients (n = 30) were analyzed by PCA, based on the results of the differential expression of 44 genes. Mutated CLL cases are shown in blue (n = 11), unmutated in green (n = 17), and borderline cases in red (n = 2).</p

LDOC1 expression in CLL samples.

<p>CLL samples were separated into those highly expressing LDOC1 and those with absent/low expression. Listed are those genes that fulfill the following criteria: >2-fold change in expression between LDOC1 pos samples compared to LDOC1 neg samples, with a p-value < 0.05 or >2-fold change in expression between LDOC1 neg samples compared to LDOC1 pos samples, with a p-value < 0.05.</p><p>LDOC1 expression in CLL samples.</p

Quantification of mRNA transcripts for surface proteins.

<p>Quantification of mRNA transcripts for surface proteins.</p

Analysis of the expression of 290 genes in normal PB, pure B cell and CLL samples.

<p>(A) Venn diagram of genes expressed preferentially in the different sample groups (normal PB, n = 5; pure B cells, n = 4; and CLL samples, n = 30). Genes were considered preferentially expressed by one sample group, if they showed an expression level ≥50 counts and a ≥2-fold difference in expression levels between the 2 groups, with a p-value ≤0.05. (B) Samples from PB (n = 5), B cell samples (n = 4), and samples from CLL patients (n = 30) were analyzed by PCA, based on the results of the differential expression of 290 genes. (C) PCA analysis on the same samples as in (b), but using a restricted set of 44 genes relevant for this purpose according to their differential expression in CLL, B cells and normal blood. (D) Heat map of normal PB, pure B cells, and CLL samples, analyzed with thirteen genes overexpressed homogenously (CV<5%) in all CLL samples compared to normal PB and pure B cells. Unsupervised analysis shows a perfect clustering of the CLL samples compared to the normal samples.</p

Genes expressed preferentially by B cells.

<p>Listed are genes with expression values > 50, a pure B cell/normal PB ratio > 2, with a p-value < 0.05.</p><p>Genes expressed preferentially by B cells.</p

Correlation between mRNA and protein expression.

<p><b>(A)</b> Correlation between CD38 protein expression, as measured by flow cytometry (% positive CLL B cells) and CD38 mRNA counts, as measured by the nCounter (arbitrary units). (B) Quantification of CD38 and ZAP70 mRNA counts in normal peripheral blood (PB), pure B cells, and in CLL B cells. CLL B cells were analyzed for ZAP70 and for CD38 expression by flow cytometry and then grouped for the mRNA determination, according to presence or absence of these two antigens. (C) Immunoglobulin light chain ratios in 30 CLL patient samples (rhombi) and in normal B cells (triangles: normal PB samples; circles: pure B cell samples). The mean +/- 2SD interval for ratios from polyclonal normal B cells and normal PB is shown (mean 0.89; SD = 0.22; small dots).</p

List of genes over- or under-expressed specifically by CLL B cells.

<p>(A) List of 44 genes expressed specifically by CLL B cells. Expression of mRNA was compared between the mean of 30 CLL samples and the mean of 5 normal PB and 4 pure B cell samples. Listed are those genes that fulfill the following criteria: > 2-fold change in expression between CLL samples and normal PB samples and pure B cell samples; p-value < 0.05; expression level in CLL samples > 50. (B) List of genes underexpressed by CLL samples compared to pure B cell samples. Listed are those genes that fulfill the following criteria: < 0.5-fold change in expression between CLL samples and pure B-cell samples; p-value < 0.05; expression level in pure B cell samples > 50.</p><p>List of genes over- or under-expressed specifically by CLL B cells.</p