EGFR and ERBB2 exon 20 insertion/duplication in advanced non–small cell lung cancer: genomic profiling and clinicopathologic features

BackgroundExon 20 (ex20) in-frame insertions or duplications (ins/dup) in epidermal growth factor receptor (EGFR) and its analog erb-b2 receptor tyrosine kinase 2 (ERBB2) are each detected in 1.5% of non–small cell lung cancer (NSCLC). Unlike EGFR p.L858R or ex19 deletions, ex20 ins/dup is associated with de novo resistance to classic EGFR inhibitors, lack of response to immune checkpoint inhibitors, and poor prognosis. US Food and Drug Administration has approved mobocertinib and amivantamab for targeting tumors with this aberration, but the number of comprehensive studies on ex20 ins/dup NSCLC is limited. We identified 18 cases of NSCLCs with EGFR/ERBB2 ex20 ins/dup and correlated the findings with clinical and morphologic information including programed death-ligand 1 (PD-L1) expression.MethodsA total of 536 NSCLC cases tested at our institution between 2014 and 2023 were reviewed. A custom-designed 214-gene next-generation sequencing panel was used for detecting DNA variants, and the FusionPlex CTL panel (ArcherDx) was used for the detection of fusion transcripts from formalin-fixed, paraffin-embedded tissue. Immunohistochemistry (IHC)for PD-L1 was performed using 22C3 or E1L3N clones.ResultsNine EGFR and nine ERBB2 ex20 ins/dup variants were identified from an equal number of men and women, 14 were non- or light smokers, and 15 had stage IV disease. All 18 cases were adenocarcinomas. Seven of the 11 cases with available primary tumors had acinar predominant pattern, two had lepidic predominant pattern, and the remainder had papillary (one case) and mucinous (one case) patterns. Ex20 ins/dup variants were heterogenous in-frame one to four amino acids spanning A767–V774 in EGFR and Y772–P780 in ERBB2 and were clustered in the loop following the C-helix and α C-helix. Twelve cases (67%) had co-existing TP53 variants. Copy number variation in CDK4 amplification was identified in one case. No fusion or microsatellite instability was identified in any case. PD-L1 was positive in two cases, low positive in four cases, and negative in 11 cases.ConclusionsNSCLCs harboring EGFR/ERBB2 ex20 ins/dup are rare and tend to be acinar predominant, negative for PD-L1, more frequent in non- or light smokers, and mutually exclusive with other driver mutations in NSCLC. The correlation of different EGFR/ERBB2 ex20 ins/dup variants and co-existing mutations with response to targeted therapy and the possibility of developing resistant mutations after mobocertinib treatment warrants further investigation

Organic carbon in surface sediments of Chaunskaya Bay (East Siberian Sea): results of pyrolytic analysis using the Rock-Eval method

Relevance. Dictated by the need to assess functioning of the biogeochemical regime of the Arctic region by studying geochemical properties of organic matter of bottom sediments on the example of the Chaunskaya Bay (East-Siberian Sea). Aim. To study the spatial variability of geochemical parameters of organic matter of bottom sediments of the Chaunskaya Bay using the Rock-Eval method, as well as to identify a possible relationship between the parameter TOC and the pelite fraction. Objects. Samples of bottom sediments of the Chaunskaya Bay (East Siberian Sea). Sampling took place in stages from three horizons (upper 0–2 cm, intermediate 2–5 cm, lower 5–10 cm) during a comprehensive scientific expedition to the R/V "Academician Oparin" in September–October 2020. Methods. Granulometric composition of bottom sediments was determined using the Analysette 22 NanoTec particle analyzer (Fritsch, Germany). The analysis of hydrocarbon compounds of organic matter was performed using pyrolytic analysis on the device (Rock Eval 6 Turbo of Vinci Technologies, France). Results. The results of pyrolytic analysis considered by the authors have shown that such factors as the primary productivity of the waters of the studied water area and the processes of erosion of the coastal zone play a decisive role in the formation of the composition of the TOC in bottom sediments of the Chaunskaya Bay. We also do not exclude the contribution of river runoff to the composition of the TOC in bottom sediments; however, we consider it small due to the insignificant inflow of river sediments into the waters of the studied area. The pyrolytic data obtained by us indicate that both the marine component (primary productivity) and the terrigenous component (erosion of the coastal complex) are present in the composition of the TOC in bottom sediments of the studied area

Использование низкобелковых обогащенных крахмаломучных продуктов в диетотерапии больных фенилкетонурией детей в возрасте старше 1 года

Background. The nutrition of children with phenylketonuria includes specialized starch-based products, the range of which is constantly expanding. Our aim was to study the safety of the composition of starchy flakes enriched with a complex of fat-soluble vitamins, natural fruit and berry additives used in the food of children with phenylketonuria. Methods. The study included children under the age of 14 years who were compliant with the previously conducted hypophenylalanine diet, without acute infectious, severe somatic or neurological diseases. The investigated products (starch-rye, wheat, and wheat fruit flakes with a complex of provitamin A and vitamin E) were prescribed instead of previously used low-protein confectionery products in the amount of 20–25 g/day for children under 6 years, 30–40 g — for children aged 6 years and over. The products were given with the recommendation to use alternately, with a duration of at least 10 days, totally for 30 days of the study. The safety of the products was assessed by phenylalanine concentration in the blood (determined by the fluorimetric method). In addition, we assessed the organoleptic qualities of the products and the dynamics of physical development of children. Results. The study included 15 children, mean age 4.4 ± 1.9 years. The initial concentration of phenylalanine in the blood varied from 1.6 to 3.9 mg%, the median — 2.2 mg% (2.0; 2.8). In 30 days after inclusion of starchy flakes in the diet, the content of phenylalanine in the blood did not change and was 2.5 mg% (2.2; 2.7); p = 0.859. The organoleptic properties of the products were rated «excellent» by all patients and their parents (in children under 6 years, only according to the parents’ assessment). The indicators of physical development did not change. There was no adverse events (allergic reactions, dyspepsia, refusal to take food). Conclusion. Introduction of new functional products — low-protein starchy flakes enriched with a vitamin complex and natural fruit and berry additives — in the diet of children with phenylketonuria allows to maintain the level of phenylalanine in the blood at the level of reference values.Обоснование. В питании детей с фенилкетонурией широко используют специализированные продукты на основе крахмалов, ассортимент которых постоянно расширяется.Цель исследования — изучить безопасность состава хлопьев крахмаломучных, обогащенных комплексом жирорастворимых витаминов, натуральными плодовыми и ягодными добавками, используемых в пище детей с фенилкетонурией.Методы. В исследование включали детей в возрасте до 14 лет, комплаентных к ранее проводимой гипофенилаланиновой диете, без острых инфекционных, тяжелых соматических или неврологических заболеваний. Исследуемые продукты — крахмалоржаные, пшеничные и пшеничные плодово-ягодные хлопья с комплексом провитамина А и витамина Е — назначали взамен применявшихся ранее низкобелковых кондитерских изделий в количестве 20–25 г/сут детям младше 6 лет, по 30–40 г — детям, достигших возраста или старше 6 лет. Продукты выдавали с рекомендацией использовать поочередно, продолжительностью не менее 10 сут, всего на 30 сут исследования. Безопасность продуктов оценивали по концентрации фенилаланина в крови (определяли флюориметрическим методом). Дополнительно оценивали органолептические качества продуктов и динамику физического развития детей.Результаты. В исследование включили 15 детей, средний возраст 4,4±1,9 года. Исходная концентрация фенилаланина в крови варьировала от 1,6 до 3,9 мг%, медиана — 2,2 мг% (2,0; 2,8). Через 30 сут после включения в рацион крахмаломучных хлопьев содержание фенилаланина в крови не изменилось и составило 2,5 мг% (2,2; 2,7); р=0,859. Органолептические свойства продуктов были оценены на «отлично» всеми пациентами и их родителями (у детей в возрасте до 6 лет — только согласно оценке родителей). Показатели физического развития не изменились. Нежелательные явления (аллергические реакции, диспепсии, отказ от приема продуктов) не зафиксированы.Заключение. Введение в рацион детей с фенилкетонурией новых функциональных продуктов — хлопьев крахмаломучных низкобелковых, обогащенных витаминным комплексом и натуральными плодовыми и ягодными добавками, позволяет сохранять уровень фенилаланина в крови на уровне референсных значений.ИСТОЧНИК ФИНАНСИРОВАНИЯ Работа выполнена при поддержке гранта Федерального государственного научного учреждения «Всероссийский научно-исследовательский институт крахмалопродуктов» Федерального агентства научных организаций (Московская область). Для целей исследования использовались продукты, безвозмездно предоставленные производителем (опытное производство ФГНУ «ВНИИК» ФАНО).КОНФЛИКТ ИНТЕРЕСОВ Т.Э. Боровик, Н.Н. Семёнова, О.Л. Лукоянова, Н.Г. Звонкова, Т.В. Бушуева, Т.Н. Степанова, В.А. Скворцова — проведение научно-исследовательских работ при поддержке компаний Heinz, Semper, Хипрока Нутришион Ист Лимитед. И.М. Гусева, Е.А. Рославцева, А.К. Геворкян, С.Т. Быкова, Т.Г. Калинина, С.Г. Калиненкова подтвердили отсутствие конфликта интересов.ВЫРАЖЕНИЕ ПРИЗНАТЕЛЬНОСТИ Выражаем благодарность к.м.н. С.Г. Калиненковой (Московский областной научно-исследовательский клинический институт им. М.Ф. Владимирского) за участие в выполнении лабораторной части данного исследования. 

An In Vitro Reporter Cell System for Analysis of Functional Ly49 Receptor Binding of Cognate Ligands in Cis and Tran

Activation of natural killer (NK) cells requires the integration of stimulatory and inhibitory signals mediated in part by members of the allelic Ly49 receptor family. Inhibitory Ly49 receptors bind cognate MHC class I ligands in trans (ITIM activation) and in cis (no ITIM activation). We have developed an in vitro reporter cell system for analysis of the functional interaction between the activating Ly49H receptor (C57BL/6 strain, B6) and its murine cytomegalovirus (MCMV)-encoded ligand, m157. Since m157 also binds inhibitory Ly49 receptors, including Ly49I from 129 mice, we exploited our reporter cell system to determine: 1) whether Ly49HB6 and/or Ly49I129 bind the GPI-linked m157 ligand in cis, and 2) whether the induction of beta-galactosidase (b-gal) reporter activity of Ly49H-expressing HD12 cells could be inhibited or attenuated by the co-expression of Ly49I129 (HD12-I129 cells) binding to the same m157 ligand. We found that Ly49HB6, but not Ly49I129, binds m157 in cis, as measured by flow cytometry and by activation of Ly49H reporter (HD12) cells. When Ly49I129 is co-expressed in cis with Ly49H and m157, partial m157 staining is restored, possibly reflecting trogocytosis of m157 by Ly49I129. When Ly49H is co-expressed with Ly49I129 and m157, the mean fluorescence intensity of m157 is slightly reduced and correlates with a minor reduction in HD12 activation (in trans). We also show that co-expression of Ly49HB6 and Ly49I129 on HD12-I129 cells results in lower b-gal induction following co-incubation with m157-expressing stimulator cells compared with HD12 cells (expressing only Ly49H). These findings represent a novel demonstration of cis ligand binding for an activating Ly49 receptor, and also demonstrate the utility of this reporter cell system for analysis of other relevant inhibitory and activating Ly49 receptor interactions (e.g. Ly49G2 and H-2Dk)

The Glycophosphatidylinositol Anchor of the MCMV Evasin, m157, Facilitates Optimal Cell Surface Expression and Ly49 Receptor Recognition.

The murine cytomegalovirus-encoded protein m157 is a cognate ligand for both inhibitory and activating receptors expressed by natural killer cells. Additionally, m157 is expressed on the surface of infected cells by a glycophosphatidylinositol (GPI) anchor. Although endogenous GPI-anchored proteins are known to be ligands for the NK cell receptor, NKG2D, the contribution of the GPI anchor for viral m157 ligand function is unknown. To determine whether the GPI anchor for m157 is dispensable for m157 function, we generated m157 variants expressed as transmembrane fusion proteins and tested cells expressing transmembrane m157 for the capacity to activate cognate Ly49 receptors. We found that the GPI anchor is required for high-level cell surface expression of m157, and that the transmembrane m157 ligand retains the capacity to activate reporter cells and NK cells expressing Ly49H, as well as Ly49I(129) reporter cells, but with reduced potency. Importantly, target cells expressing the transmembrane form of m157 were killed less efficiently and failed to mediate Ly49H receptor downregulation on fresh NK cells compared to targets expressing GPI-anchored m157. Taken together, these results show that the GPI anchor for m157 facilitates robust cell surface expression, and that NK cells are sensitive to the altered cell surface expression of this potent viral evasin

Transmembrane m157 stimulates both activating and inhibitory Ly49 reporter cells.

<p>C1498 cells transfected with wt-m157 or m157₃₀₆CD4 were incubated with (<b>A</b>) Ly49H^B6-expressing HD12 cells, (<b>B</b>) Ly49HI¹²⁹-expressing cells, or (<b>C</b>) Ly49HI^B6-expressing cells overnight in the presence or absence of anti-m157 mAb (6D5). Results are expressed as the frequency of maximum β-gal production (determined by stimulating cells with PMA and ionomycin). NS, not significant (<i>P</i>>0.05); *** <i>P</i><0.001 (two-tailed unpaired Student’s t-test). Results are representative of 3 experiments (error bars depict SEM).</p

Expression of m157 as a transmembrane protein results in lower surface expression than GPI-anchored m157.

<p>C1498 cells were transfected with GPI-anchored m157 (wt-m157) or transmembrane-anchored m157 (m157₃₀₆CD4). (<b>A</b>) Schematic of the m157₃₀₆CD4 construct. (<b>B</b>) Surface expression of m157 as determined by flow cytometry using the anti-m157 mAb 6D5 in C1498 cells, representative of 7 experiments. Shaded grey histogram shows untransduced C1498 cells, solid line illustrates wt-m157, and dotted line represents m157₃₀₆CD4. (<b>C</b>) Mean fluorescent intensity of surface wt-m157 and m157₃₀₆CD4; data are accumulated from 7 experiments. (<b>D</b>) C1498 transfectants were treated with PI-PLC and then surface expression of m157 was determined by flow cytometry as in <b>B</b>. Data are representative of 3 experiments. (<b>E</b>) Mean fluorescent intensity of surface wt-m157 or m157₃₀₆CD4 before and after PI-PLC treatment; data are accumulated from 3 experiments. NS, not significant (<i>P</i>>0.05); * <i>P</i><0.05; ** <i>P</i><0.005; *** <i>P</i><0.001 (two-tailed unpaired Student’s t-test). Error bars <b>C</b>, <b>E</b>, depict standard error of the mean, SEM.</p

Transmembrane m157 expression impairs NK cell Ly49H receptor downregulation and cytotoxicity, but not IFN-γ production.

<p>C1498 transfectants were incubated with B6 RAG1^−/− splenocytes. (A) IFN-γ production of NK1.1⁺CD3⁻Ly49H⁺ gated cells was determined by flow cytometry. (B) Representative plots showing downregulation of Ly49H, and mean fluorescent intensity of Ly49H on NK1.1⁺CD3⁻IFN-γ⁺ cells (bar graph). Numbers in upper right quadrant are equal to the frequency of IFN-γ⁺ cells of Ly49H⁺ NK cells. NS, not significant (<i>P</i>>0.05); * <i>P</i><0.05; ** <i>P</i><0.005;*** <i>P</i><0.001 (two-tailed unpaired Student’s t-test). (C) NK cell cytotoxicity against m157 transfectants was determined by chromium release assay. B6 RAG1^−/− splenocytes were incubated with Cr⁵¹-labeled targets at indicated ratios. *** <i>P</i><0.001 (two-way ANOVA with Bonferroni post-tests). Data are representative of at least two independent experiments (error bars A, B, depict SEM).</p