B cell-specific conditional expression of Myd88(p.L252P) leads to the development of diffuse large B cell lymphoma in mice

The adaptor protein MYD88 is critical to relay activation of Toll-like receptor signaling to NF-{kappa}B activation.MYD88 mutations, particularly the p.L265P mutation, have been described in numerous distinct B cell malignancies, including diffuse large B cell lymphoma (DLBCL). 29% of activated B cell (ABC)-type DLBCL, which is characterized by constitutive activation of the NF-{kappa}B pathway, carry the p.L265P mutation. In addition, ABC-DLBCL frequently displays focal copy number gains affecting BCL2. Here, we generated a novel mouse model, in which Cre-mediated recombination, specifically in B cells, leads to the conditional expression of Myd88(p.L252P)(the orthologous position of the human MYD88(p.L265P) mutation) from the endogenous locus. These animals develop a lympho-proliferative disease, and occasional transformation into clonal lymphomas. The clonal disease displays morphological and immunophenotypical characteristics of ABC-DLBCL. Lymphomagenesis can be accelerated by crossing in a further novel allele, which mediates conditional overexpression ofBCL2 Cross-validation experiments in human DLBCL samples revealed that bothMYD88andCD79Bmutations are substantially enriched in ABC-DLBCL, compared to germinal center B cell DLBCL. Furthermore, analyses of human DLBCL genome sequencing data confirmed that BCL2 amplifications frequently co-occur with MYD88 mutations, further validating our approach. Lastly,in silicoexperiments revealed that particularly MYD88-mutant ABC-DLBCL cells display an actionable addiction to BCL2. Altogether, we generated a novel autochthonous mouse model of ABC-DLBCL, which could be used as a preclinical platform for the development and validation of novel therapeutic approaches for the treatment of ABC-DLBCL

Прогностическая значимость sPD-1/sPD-L1 при раке почки в зависимости от фенотипа опухолевых и стромальных клеток

Background. The search for new prognostic markers of renal cell carcinoma is an urgent problem of oncourology. Modern studies demonstrate the need for a comprehensive assessment of the clinical and prognostic significance of many markers.Aim is a comprehensive analysis of the prognostic significance of soluble forms of PD-1 and PD-L1 (sPD-1 and sPD-L1) depending on the phenotype of tumor cells and the microenvironment.Materials and methods. The study included tumor tissue and serum samples from 54 patients with renal cell cancer and from 67 healthy donors. The concentrations of sPD-1 and sPD-L1 were determined in blood serum using ELISA. Tissue expression of PD-L1, PU.1, CD3, and CD20 was assessed by immunohistochemistry. To determine statistically significant differences in independent groups, the Mann–Whitney test and Fisher’s exact test were used. Overall survival was analyzed by constructing survival curves using the Kaplan–Meier method. Differences were considered statistically significant at p <0.05.Results. Increase of sPD-L1 concentration in serum from patients with renal cell carcinoma compared with healthy donors was demonstrated. The highest concentration of the soluble form of the PD-1 receptor was observed in serum from patients with the non-clear cell renal cell carcinoma. High levels of sPD-L1 in serum and PD-L1 in tumor cells are associated with disease progression (advanced stage, higher malignancy, as well as the presence of regional metastases). It has been shown that the high content of PU.1+ and CD20+ cells in the tumor stroma are significant factors of unfavorable prognosis. No prognostic significance was found for both sPD-L1 and PD-L1 expressed in tumor tissue. However, analysis of a combination of these markers showed that the high concentration of sPD-L1 together with the high tissue expression of PD-L1 is an extremely unfavorable factor.Conclusion. Analysis of sPD-L1 concentration and tissue expression of PD-L1 in combination is a new approach for assessing the prognosis of renal cell carcinoma.Введение. Поиск новых прогностических маркеров почечно-клеточного рака является актуальной проблемой онкоурологии. Современные исследования демонстрируют необходимость комплексной оценки клинической и прогностической значимости многих маркеров.Цель исследования – комплексный анализ прогностической значимости растворимых форм PD-1 и PD-L1 (sPD-1 и sPD-L1) в зависимости от фенотипа опухолевых клеток и их микроокружения.Материалы и методы. В исследование включены образцы опухолей и сывороток крови от 54 пациентов с почечноклеточным раком и от 67 здоровых доноров. Концентрацию sPD-1 и sPD-L1 определяли в сыворотке крови с помощью иммуноферментного анализа. Оценку тканевой экспрессии PD-L1, PU.1, CD3 и CD20 проводили иммуногистохимическим методом. Для определения статистически значимых различий в независимых группах использовали критерий Манна–Уитни и точный критерий Фишера. Анализ общей выживаемости проводили путем построения кривых дожития по методу Каплана–Майера. Статистически достоверными считали различия при р <0,05.Результаты. Показано увеличение содержания sPD-L1 у пациентов с почечно-клеточным раком по сравнению со здоровыми донорами. Наибольшее содержание растворимой формы PD-1 характерно для пациентов с несветлоклеточным вариантом опухоли. Высокое содержание sPD-L1 в сыворотке и PD-L1 в опухолевых клетках ассоциировано с прогрессированием заболевания (высокой стадией и низкой дифференцировкой, а также наличием регионарных метастазов). Показано, что высокое содержание PU.1+- и CD20+-клеток в строме опухоли является значимым фактором неблагоприятного прогноза. Не выявлено прогностической значимости как для растворимой формы PD-L1, так и для тканевой. Однако их комплексный анализ показал, что высокое содержание растворимой формы PD-L1 при высокой тканевой экспрессии данного маркера – крайне неблагоприятный фактор.Заключение. Комплексный анализ прогностической значимости содержания растворимой и тканевой форм PD-L1 является новым подходом к оценке прогноза почечно-клеточного рака

Клиническое значение молекулы повреждения почек KIM-1 в плазме крови больных почечно-клеточным раком

Background. The most important task in the field of renal-cell cancer (RCC) treatment results improvement is the search and validation of the markers for its early diagnostics still absent in the clinical practice. It was established that even before the onset and/or detection of RCC the level of kidney injury molecule-1 (KIM-1) in blood plasma did increase.Objective of the study — comparative evaluation of KIM-1 levels in blood plasma of practically healthy persons, RCC cancer, benign kidney tumor patients, patients with non-oncological renal pathologies, and analysis of its role in RCC diagnostics and prognosis.Materials and methods. 125 RCC (age 33—81 years), 14 — benign kidney neoplasms (29—84 years) patients, 90patients with chronic nephritis (28—82 years) and 68 practically healthy persons (18—71 years) were included in the study. Plasma KIM-1 content was measured using Human Serum TIM-1/KIM-1/HAVCR Quantikine® ELISA kit (R&D Systems Biotechne®, USA).Results. KIM-1 level in blood plasma of RCC and chronic nephritis patients was significantly higher than in control (medians 305, 282 and 37.8pg/ml respectively, p <0.0001). The rate of KIM-1 elevation over cut-offvalue 90pg/ml corresponding to the upper 95 % confidence interval of control in RCC patients comprised 79.2 %, in patients with nephritis — 83 %, in those with benign renal tumors — 50 %. Specificity in relation to healthy control was 96 %. KIM-1 level highly significantly increased with RCC progression, and already at stage I was 4.3-fold higher by median than in control (p <0.0001). Sensitivity of stage I—IIRCC detection at cut-off 90pg/ml comprised 75 %; stage III—IV — 94 %. The highest plasma KIM-1 levels were detected in papillary cancer patients (median 644pg/ml), that was more than 2-fold higher than in clear-cell and 32-fold higher than in chromophobic RCC. Plasma KIM-1 median level was 7-fold higher in patients with G3 4RCC than in those with G12 (p <0.0001). At the cut-off KIM-1 value of 163pg/ml, corresponding to the median at stage I, significant differences in 3.5-years overall survival both in the total group: 49 % at high, 95 % at low marker level (p <0.01), and at stage I RCC: 62 % and 100 % respectively (p <0.05) — were revealed.Conclusion. Plasma KIM-1 may become the first highly sensitive marker for the early detection of RCC, but it does not allow differentiating between oncologic and non-oncologic renal pathologies. Increased basal plasma KIM-1 is an unfavorable prognostic factor irrespective of the stage of tumor progression.Введение. Важнейшая задача в области улучшения результатов лечения почечно-клеточного рака (ПКР) — поиск и валидация маркеров для его ранней диагностики, которых в клинической практике пока нет. Установлено, что еще до возникновения и/или выявления ПКР повышается уровень молекулы повреждения почек 1 (kidney injury molecule 1, KIM-1) в плазме крови.Цель исследования — сравнительная оценка уровней KIM-1 в плазме крови здоровых доноров, больных раком, пациентов с доброкачественными новообразованиями и неопухолевыми патологиями почек и ее роли в диагностике и прогнозе рака почки.Материалы и методы. Обследованы 125 больных ПКР (возраст 33—81 год), 14 пациентов с доброкачественными новообразованиями почки (29—84года), 90пациентов с хроническими нефритами (28—82года) и 68практически здоровых доноров (18—71 год). Содержание KIM-1 в плазме крови определено с помощью наборов Human Serum TIM-1/KIM-1/HAVCR Quantikine® ELISA kit (R&D Systems Biotechne®, США).Результаты. Содержание KIM-1 в плазме крови больных ПКР и хроническими нефритами статистически значимо выше, чем в контроле (медиана 305, 282 и 37,8 пг/мл соответственно; p <0,0001). Частота превышения порогового уровня 90 пг/мл, соот-ветствующего верхнему 95 % доверительному интервалу контроля, у больных ПКР составила 79,2 %, у больных нефритами — 83 %, у пациентов с доброкачественными опухолями — 50 %. Специфичность относительно здоровых доноров составила 96 %. Уровень KIM-1 высокозначимо повышался с увеличением распространенности рака почки и уже при I стадии в 4,3 раза по медиане превышал показатели группы контроля (р <0,0001). Частота выявления I—II стадии рака почки при пороговом уровне 90 пг//м — 75 %; III—IVстадии — 94 %. Наиболее высокие уровни KIM-1 обнаружены у больных папиллярным раком (медиана 644 пг/мл), что более чем вдвое выше, чем при светлоклеточном, и в 32 раза выше, чем при хромофобном раке. Уровень KIM-1 в 7 раз выше по медиане при степени злокачественности G3_4, чем при Gi2 (p <0,0001). При пороговом уровне KIM-1163 пг/мл, соответствующем медиане при I стадии, выявлены значимые различия в показателях 3,5-летней выживаемости как в общей группе (при высоком уровне маркера — 49 %, при низком — 95 %; p <0,01), так и при Iстадии ПКР (62 и 100 % соответственно; p <0,05).Заключение. KIM-1 плазмы крови может стать первым высокочувствительным маркером для раннего выявления ПКР, но не позволяет провести дифференциальную диагностику опухолевых и неопухолевых поражений почек. Повышенный уровень KIM-1 в плазме является фактором неблагоприятного прогноза независимо от степени распространенности опухолевого процесса

Прогностическая роль экспрессии маркера PBRM1 при светлоклеточном раке почки

Background. Clear-cell renal-cell carcinoma (CCRCC) is the most common histological type of cancer of this localization. Changes in 16 genes were identified as significant in carcinogenesis of CCRCC. After VHL suppressor gene, PBRM1 gene is the second by frequency of genetic abnormalities in CCRCC and it is mutated in 40—50 % cases of CCRCC.The study objectiveis to analyze the effect of abnormalities in PBRM1 protein expression on survival of patients with CCRCC.Materials and methods. The study included 137patients with newly diagnosed and histologically confirmed CCRCC. For all study participant, detailed medical history and questionnaire data were acquired. Prior to treatment, blood samples and tumor tissue removed during surgery were obtainedfrom all patients. All patients are annually followed up for current information on their life status, disease dynamics, treatment. Minimalfollow-up time is 22 months, maximal is 128 months, mean is 61.8 months, median is 48 months. Immunohistochemical (IHC) testing of PBRM1 expression was performed using standard technique with polyclonal rabbit antibodies PB1[N1N2] N-term (GeneTex 100781) with 1:50 dilution, DAB staining. Normally, protein product of the wild type PBRM1 gene is functioning and can be detected in the nucleus. Absence of nuclear expression of PBRM1 points to genetic or epigenetic abnormalities.Results.Renal cancer-specific survival is significantly lower in patients without expression of the PRBM1 protein in tumor cells. The longest 5- (84 %) and 10-year (84 %) survival was observed in patients with diffuse nuclear expression of the PBRM1 protein. Difference in survival of these patients compared to patients without PRBM1 protein expression is statistically significant (p = 0.004). We have performed an analysis of the association between survival of patients with CCRCC andfocal nuclear PBRM1 expression. In these patients, survival is lower than in patients with diffuse expression but higher than in patients without nuclear expression of PBRM1 (p = 0.02). Cytoplasmic expression of PBRM1 doesn’t affect survival.Conclusion.The obtained results point to prognostic value of PBRM1 gene activity which is abnormal in almost half of all CCRCC cases. IHC testing is an appropriate, reliable and affordable method for determination of PBRM1 protein expression and therefore can be used in practice. Favorable course and prognosis in patients with stage I—II CCRCC and preserved nuclear expression of the PBRM1 protein should be noted: 5-year survival for these patients is 100 %. This observation is crucial for making decisions on treatment of these patients.Введение. Светлоклеточный почечно-клеточный рак (скПКР) является наиболее частым гистологическим типом рака этой локализации. Выделяют 16 генов, нарушения которых играют значительную роль в канцерогенезе скПКР. Вторым по частоте генетических нарушений в скПКР после гена-супрессора VHL является ген PBRM1, который мутирует в 40—50 % случаев скПКР.Цель исследования — анализ влияния нарушений экспрессии белка PBRM1 на выживаемость пациентов со скПКР.Материалы и методы. В исследование были включены 137пациентов с впервые выявленным и гистологически верифицированным диагнозом скПКР. Для каждого участника исследования были собраны детальная медицинская информация и данные анкетирования. От всех больных до начала лечения были получены образцы крови и удаленной во время хирургической операции опухолевой ткани. Все пациенты ежегодно прослеживаются в целях получения актуальной информации об их жизненном статусе, динамике заболевания, лечении. Минимальное время прослеживания — 22мес, максимальное — 128мес, среднее — 61,8мес, медиана — 48мес. Иммуногистохимическое (ИГХ) исследование экспрессии PBRM1 было выполнено по стандартной методике c поликлональными кроличьими антителами PB1[N1N2] N-term (GeneTex 100781) в разведении 1:50, проявление проводилось с использованием DAB. Белковый продукт гена PBRM1 дикого типа в норме функционирует и выявляется в ядре. Отсутствие ядерной экспрессии PBRM1 указывает на генетические или эпигенетические нарушения.Результаты. Специфическая для рака почки выживаемость статистически достоверно ниже у больных, в опухолевых клетках которых нет экспрессии белка PRBM1. Наилучшая 5- (84 %) и 10-летняя (84 %) выживаемость отмечена у больных с диф­фузной ядерной экспрессией белка PBRM1. Различия в выживаемости этих больных и тех, у которых нет экспрессии белка PBRM1, статистически высоко достоверны (р = 0,004). Нами впервые проведен анализ выживаемости больных скПКР с фо­кальной ядерной экспрессией PBRM1. У этих пациентов выживаемость ниже, чем у больных с диффузной экспрессией, но выше, чем у больных с отсутствием ядерной экспрессии PBRM1 (р = 0,02). Цитоплазматическая экспрессия PBRM1 на выживаемость не влияет.Заключение. Таким образом, полученные нами результаты указывают на прогностическую значимость активности гена PBRM1, нарушение функции которого встречается почти в половине случаев скПКР. ИГХ-исследование является адекватным, надежным и доступным методом для определения экспрессии белка PBRM1 и, соответственно, может применяться на практике. Особен­но следует отметить благоприятное течение и прогноз болезни у пациентов с I—II стадиями скПКР, у которых сохранена ядер­ная экспрессия белка PBRM1: 5-летняя выживаемость у них составляет 100 %. Это наблюдение крайне важно для принятия решения по тактике лечения таких больных

Laryngeal paraganglioma: clinical case presentation

We present rare clinical case of laryngeal paraganglioma. Modern possibilities of the disease diagnostics, determination of the prevalence of tumor process, methods, and assessment of the effectiveness of treatment are shown in this paper

Clinical case of oropharyngeal metastasis of kidney cancer

Oropharyngeal metastases are very rare and occur in approximately 1.0–1.5 % of all malignant tumors of the oral cavity and oropharynx. Information on oropharyngeal localization of metastases of kidney cancer is almost absent in the literature. A description of a rare clinical observation of metastasis of clear cell renal cell carcinoma into the oropharynx is presented

Key VEGF signaling system components and matrix metalloproteinases in the diagnosis and prognosis of overall survival of patients with renal cell cancer

Background: Evaluation and search for new molecular markers of renal cell cancer, first of all, associated with angiogenic and invasive activity, continue to be highly relevant. In our previous publications, we have assessed the potential diagnostic value of matrix metalloproteinases (MMP) 2, 7, 8, and 9, their tissue inhibitor type 1 (TIMP1) and components of the VEGF signaling system in renal cell cancer. Aim: To assess the role of serum VEGF, VEGFR1, VEGFR2, MMP2, 7, 8, 9, and TIMP1 levels in renal cell patients as diagnostic and prognostic markers of overall survival. Materials and methods: 99 renal cell cancer patients (94 primary and 5 at progression) were recruited into the study. The control group included 97 healthy control blood donors. Ninety three (93) primary patients with renal cell cancer were followed for 1 to 45 (median, 26) months for assessment of their overall survival. Serum concentrations of the study proteins were measured by direct immunoenzyme analysis (Quantikine® ELISA kits, R&D Systems, США). Results: Serum VEGF, VEGFR1, VEGFR2, MMP7, MMP8, and TIMP1 levels in renal cell cancer patients are significantly higher than those in the control group. The diagnostic characteristics of the markers are considerably different, the most reliable marker with 84% sensitivity at 87.5% specificity being MMP7. VEGFR1, MMP7, MMP8, and TIMP1 were positively associated with disease stage and TNM indices. MMP7 and TIMP1 levels also increased with a higher tumor grade. MMP7 was found to be a significant unfavorable prognostic factor for overall survival: the 3-years survival in those with low (< 6.3 ng/ml) marker level amounted to 93%, whereas with high, 51% (p < 0.001). MMP7 prognostic value remained significant also in stage I renal cancer: after 3-years' follow-up, all patients with low MMP7 were alive, while survival of those with high marker levels was 72% (p=0.02). Increased serum MMP8 level (> 51 ng/ml) also had an unfavorable prognostic value in the whole renal cell cancer patient group, with 3-years' survival being 78 and 58% for low and high levels, respectively (p < 0.01). The components of VEGF signaling system, MMP2, MMP9, and TIMP1 had no significant prognostic values. Conclusion: MMP7 should be viewed as the most promising diagnostic and prognostic renal cell cancer marker. VEGF and its soluble receptors could be useful for monitoring of patients receiving anti-angiogenic treatments and prediction of their sensitivity to these agents

Matrix metalloproteinases 2, 7, 8, 9 and their type 1 tissue inhibitor in serum of renal cancer patients: clinical and pathologic correlations

Background: The cause of late diagnosis of renal cancer lies in its durable, almost asymptomatic course. Due to the use of antiangiogenic therapies much progress has been made in its treatment in recent years. Yet, many questions concerning the diagnosis, prognosis and prediction of the efficiency of targeted therapy remain unsolved. Therefore, exploration of new renal cancer molecular markers, especially those related to its angiogenic and invasive activities, are still on the agenda. Such markers include the family of matrix metalloproteinases (MMPs) that degrade the majority of extracellular matrix components and are involved at all stages of tumor progression. Aim: Comparative evaluation of MMP2, 7, 8, 9 and type 1 tissue inhibitor (TIMP-1) levels in serum of healthy individuals and patients with renal cancer or benign renal tumors, analysis of their associations with the main clinical and pathologic characteristics of the disease. Materials and methods: We examined 99  renal cancer patients (of those 94 with primary tumor and 5 at progression) and 10 patients with benign renal tumors. The control group included 97 healthy individuals. Levels of the proteins studied were measured using respective direct ELISA kits (Quantikine®, R&D Systems, USA). Results: MMP-7, MMP-8 and TIMP-1 levels in the sera of renal cancer patients were significantly higher than in the control group and in benign renal tumor patients. MMP-2 and MMP-9 levels did not differ significantly between the study and control groups. At MMP-7 cut-off level of 3.0  ng/mL, its diagnostic sensitivity for primary renal cancer was 84%, specificity in relation to “healthy” control – 87.5%, in relation to the pathologic control (healthy donors+benign renal tumor patients) – 73%. The best sensitivity: specificity ratio for TIMP-1 was 67 and 65% at the cut-off level of 315 ng/ml. No cut-off value with acceptable sensitivity: specificity ratio was found for MMP-8. Serum levels of all these 3 markers were positively associated with disease stage and TNM indices; MMP-7 and TIMP-1 levels also increased with lower differentiation grade. In 5 patients evaluated at disease progression the levels of all the markers studied were markedly higher than in the primary patients, and exceeded the estimated cut-off values. Conclusion: MMP-7 should be regarded as the most promising serological renal cancer marker; its serum levels exceed the cut-off value even in 84% stage I patients. TIMP-1 has acceptable sensitivity (70% and above) only from stage II renal cancer onwards, while MMP-8 levels are increased only at stage III–IV of the disease

Prognostic role of PBRM1 marker expression in clear-cell renal-cell carcinoma

Background. Clear-cell renal-cell carcinoma (CCRCC) is the most common histological type of cancer of this localization. Changes in 16 genes were identified as significant in carcinogenesis of CCRCC. After VHL suppressor gene, PBRM1 gene is the second by frequency of genetic abnormalities in CCRCC and it is mutated in 40—50 % cases of CCRCC.The study objectiveis to analyze the effect of abnormalities in PBRM1 protein expression on survival of patients with CCRCC.Materials and methods. The study included 137patients with newly diagnosed and histologically confirmed CCRCC. For all study participant, detailed medical history and questionnaire data were acquired. Prior to treatment, blood samples and tumor tissue removed during surgery were obtainedfrom all patients. All patients are annually followed up for current information on their life status, disease dynamics, treatment. Minimalfollow-up time is 22 months, maximal is 128 months, mean is 61.8 months, median is 48 months. Immunohistochemical (IHC) testing of PBRM1 expression was performed using standard technique with polyclonal rabbit antibodies PB1[N1N2] N-term (GeneTex 100781) with 1:50 dilution, DAB staining. Normally, protein product of the wild type PBRM1 gene is functioning and can be detected in the nucleus. Absence of nuclear expression of PBRM1 points to genetic or epigenetic abnormalities.Results.Renal cancer-specific survival is significantly lower in patients without expression of the PRBM1 protein in tumor cells. The longest 5- (84 %) and 10-year (84 %) survival was observed in patients with diffuse nuclear expression of the PBRM1 protein. Difference in survival of these patients compared to patients without PRBM1 protein expression is statistically significant (p = 0.004). We have performed an analysis of the association between survival of patients with CCRCC andfocal nuclear PBRM1 expression. In these patients, survival is lower than in patients with diffuse expression but higher than in patients without nuclear expression of PBRM1 (p = 0.02). Cytoplasmic expression of PBRM1 doesn’t affect survival.Conclusion.The obtained results point to prognostic value of PBRM1 gene activity which is abnormal in almost half of all CCRCC cases. IHC testing is an appropriate, reliable and affordable method for determination of PBRM1 protein expression and therefore can be used in practice. Favorable course and prognosis in patients with stage I—II CCRCC and preserved nuclear expression of the PBRM1 protein should be noted: 5-year survival for these patients is 100 %. This observation is crucial for making decisions on treatment of these patients