DataSheet_2_Consequences of somatic mutations of GIRK1 detected in primary malign tumors on expression and function of G-protein activated, inwardly rectifying, K+ channels.pdf

A search in the GDC Data Portal revealed 304 documented somatic mutations of the KCNJ3 gene in primary tumors (out of 10.202 cases). Most affected tumor types were carcinomas from uterus, skin and lung, while breast cancer exerted the lowest number of somatic mutations. We focused our research on 15 missense mutations within the region between TM1 and TM2, comprising the pore helix and ion selectivity signature. Expression was measured by confocal laser scan microscopy of eGFP tagged GIRK1 subunits, expressed with and without GIRK4 in oocytes of Xenopus laevis. GIRK ion currents were activated via coexpressed m2Rs and measured by the Two Electrode Voltage Clamp technique. Magnitude of the total GIRK current, as well as the fraction of current inducible by the agonist, were measured. Ion selectivity was gauged by assessment of the PNa+/PK+ ratio, calculated by the GIRK current reversal potential in extracellular media at different Na+ and K+ concentrations. None of the tested mutations was able to form functional GIRK1 homooligomeric ion channels. One of the mutations, G145A, which locates directly to the ion selectivity signature, exerted an increased PNa+/PK+ ratio. Generally, the missense mutations studied can be categorized into three groups: (i) normal/reduced expression accompanied by reduced/absent function (S132Y, F136L, E139K, G145A, R149Q, R149P, G178D, S185Y, Q186R), (ii) normal/increased expression as well as increased function (E140M, A142T, M184I) and (iii) miniscule expression but increased function relative to expression levels (I151N, G158S). We conclude, that gain of function mutations, identical or similar to categories (ii) and (iii), may potentially be involved in genesis and progression of malignancies in tissues that exert a high rate of occurrence of somatic mutations of KCNJ3.</p

DataSheet_1_Consequences of somatic mutations of GIRK1 detected in primary malign tumors on expression and function of G-protein activated, inwardly rectifying, K+ channels.pdf

A search in the GDC Data Portal revealed 304 documented somatic mutations of the KCNJ3 gene in primary tumors (out of 10.202 cases). Most affected tumor types were carcinomas from uterus, skin and lung, while breast cancer exerted the lowest number of somatic mutations. We focused our research on 15 missense mutations within the region between TM1 and TM2, comprising the pore helix and ion selectivity signature. Expression was measured by confocal laser scan microscopy of eGFP tagged GIRK1 subunits, expressed with and without GIRK4 in oocytes of Xenopus laevis. GIRK ion currents were activated via coexpressed m2Rs and measured by the Two Electrode Voltage Clamp technique. Magnitude of the total GIRK current, as well as the fraction of current inducible by the agonist, were measured. Ion selectivity was gauged by assessment of the PNa+/PK+ ratio, calculated by the GIRK current reversal potential in extracellular media at different Na+ and K+ concentrations. None of the tested mutations was able to form functional GIRK1 homooligomeric ion channels. One of the mutations, G145A, which locates directly to the ion selectivity signature, exerted an increased PNa+/PK+ ratio. Generally, the missense mutations studied can be categorized into three groups: (i) normal/reduced expression accompanied by reduced/absent function (S132Y, F136L, E139K, G145A, R149Q, R149P, G178D, S185Y, Q186R), (ii) normal/increased expression as well as increased function (E140M, A142T, M184I) and (iii) miniscule expression but increased function relative to expression levels (I151N, G158S). We conclude, that gain of function mutations, identical or similar to categories (ii) and (iii), may potentially be involved in genesis and progression of malignancies in tissues that exert a high rate of occurrence of somatic mutations of KCNJ3.</p

DataSheet_3_Consequences of somatic mutations of GIRK1 detected in primary malign tumors on expression and function of G-protein activated, inwardly rectifying, K+ channels.pdf

A search in the GDC Data Portal revealed 304 documented somatic mutations of the KCNJ3 gene in primary tumors (out of 10.202 cases). Most affected tumor types were carcinomas from uterus, skin and lung, while breast cancer exerted the lowest number of somatic mutations. We focused our research on 15 missense mutations within the region between TM1 and TM2, comprising the pore helix and ion selectivity signature. Expression was measured by confocal laser scan microscopy of eGFP tagged GIRK1 subunits, expressed with and without GIRK4 in oocytes of Xenopus laevis. GIRK ion currents were activated via coexpressed m2Rs and measured by the Two Electrode Voltage Clamp technique. Magnitude of the total GIRK current, as well as the fraction of current inducible by the agonist, were measured. Ion selectivity was gauged by assessment of the PNa+/PK+ ratio, calculated by the GIRK current reversal potential in extracellular media at different Na+ and K+ concentrations. None of the tested mutations was able to form functional GIRK1 homooligomeric ion channels. One of the mutations, G145A, which locates directly to the ion selectivity signature, exerted an increased PNa+/PK+ ratio. Generally, the missense mutations studied can be categorized into three groups: (i) normal/reduced expression accompanied by reduced/absent function (S132Y, F136L, E139K, G145A, R149Q, R149P, G178D, S185Y, Q186R), (ii) normal/increased expression as well as increased function (E140M, A142T, M184I) and (iii) miniscule expression but increased function relative to expression levels (I151N, G158S). We conclude, that gain of function mutations, identical or similar to categories (ii) and (iii), may potentially be involved in genesis and progression of malignancies in tissues that exert a high rate of occurrence of somatic mutations of KCNJ3.</p

Changes in Colorectal Carcinoma Genomes under Anti-EGFR Therapy Identified by Whole-Genome Plasma DNA Sequencing

<div><p>Monoclonal antibodies targeting the Epidermal Growth Factor Receptor (EGFR), such as cetuximab and panitumumab, have evolved to important therapeutic options in metastatic colorectal cancer (CRC). However, almost all patients with clinical response to anti-EGFR therapies show disease progression within a few months and little is known about mechanism and timing of resistance evolution. Here we analyzed plasma DNA from ten patients treated with anti-EGFR therapy by whole genome sequencing (plasma-Seq) and ultra-sensitive deep sequencing of genes associated with resistance to anti-EGFR treatment such as <i>KRAS</i>, <i>BRAF</i>, <i>PIK3CA</i>, and <i>EGFR</i>. Surprisingly, we observed that the development of resistance to anti-EGFR therapies was associated with acquired gains of <i>KRAS</i> in four patients (40%), which occurred either as novel focal amplifications (<i>n</i> = 3) or as high level polysomy of 12p (<i>n</i> = 1). In addition, we observed focal amplifications of other genes recently shown to be involved in acquired resistance to anti-EGFR therapies, such as <i>MET</i> (<i>n</i> = 2) and <i>ERBB2</i> (<i>n</i> = 1). Overrepresentation of the <i>EGFR</i> gene was associated with a good initial anti-EGFR efficacy. Overall, we identified predictive biomarkers associated with anti-EGFR efficacy in seven patients (70%), which correlated well with treatment response. In contrast, ultra-sensitive deep sequencing of <i>KRAS</i>, <i>BRAF</i>, <i>PIK3CA</i>, and <i>EGFR</i> did not reveal the occurrence of novel, acquired mutations. Thus, plasma-Seq enables the identification of novel mutant clones and may therefore facilitate early adjustments of therapies that may delay or prevent disease progression.</p></div

High-level focal amplification of <i>ERBB2</i> in patient #7 treated with cetuximab.

<p>(a) Copy number profile of the plasma sample (P7_1) and the locations of the <i>EGFR</i>, <i>MET</i>, <i>KRAS</i>, and <i>ERBB2</i> genes. (b) The time line illustrates the duration of cetuximab treatment and the date (red bar) of our blood collection. Below the time line are the ERBB2 immunohistochemistry and silver in situ hybridization (SISH) images of the biopsy material from the primary tumor (month 0) showing an immunoreactive score 3+ and high amplification, respectively. Her2/neu signals are black in the SISH technique. Furthermore, a plot of chromosome 17 from P7_1 obtained at month 14 is illustrated (note that the scale of the Y-axis reaches till 5 to illustrate the high-level gain; color designation as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004271#pgen-1004271-g001" target="_blank">Figure 1</a>).</p