A molecular approach to triple-negative breast cancer: targeting the Notch signaling pathway.

INTRODUCTION: Triple-negative breast cancer is an aggressive subtype of breast cancer characterized by the absence of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 expression. This phenotype renders triple-negative breast cancer cells refractory to conventional therapies, resulting in poor clinical outcomes and an urgent need for novel therapeutic approaches. Recent studies have implicated dysregulation of the Notch receptor signaling pathway in the development and progression of triple-negative breast cancer. OBJECTIVE: This study aimed to conduct a comprehensive literature review to identify potential therapeutic targets of the Notch pathway. Our analysis focused on the upstream and downstream components of this pathway to identify potential therapeutic targets. RESULTS: Modulating the Notch signaling pathway may represent a promising therapeutic strategy to treat triple-negative breast cancer. Several potential therapeutic targets within this pathway are in the early stages of development, including upstream (such as Notch ligands) and downstream (including specific molecules involved in triple-negative breast cancer growth). These targets represent potential avenues for therapeutic intervention in triple-negative breast cancer. COMMENTS: Additional research specifically addressing issues related to toxicity and improving drug delivery methods is critical for the successful translation of these potential therapeutic targets into effective treatments for patients with triple-negative breast cancer.

A molecular approach to triple-negative breast cancer: targeting the Notch signaling pathway

ABSTRACT Introduction Triple-negative breast cancer is an aggressive subtype of breast cancer characterized by the absence of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 expression. This phenotype renders triple-negative breast cancer cells refractory to conventional therapies, resulting in poor clinical outcomes and an urgent need for novel therapeutic approaches. Recent studies have implicated dysregulation of the Notch receptor signaling pathway in the development and progression of triple-negative breast cancer. Objective This study aimed to conduct a comprehensive literature review to identify potential therapeutic targets of the Notch pathway. Our analysis focused on the upstream and downstream components of this pathway to identify potential therapeutic targets. Results Modulating the Notch signaling pathway may represent a promising therapeutic strategy to treat triple-negative breast cancer. Several potential therapeutic targets within this pathway are in the early stages of development, including upstream (such as Notch ligands) and downstream (including specific molecules involved in triple-negative breast cancer growth). These targets represent potential avenues for therapeutic intervention in triple-negative breast cancer. Comments Additional research specifically addressing issues related to toxicity and improving drug delivery methods is critical for the successful translation of these potential therapeutic targets into effective treatments for patients with triple-negative breast cancer.

Real-world genomic profiling of acute myeloid leukemia and the impact of European LeukemiaNet risk stratification 2022 update.

BACKGROUND: Acute myeloid leukemia (AML) is a myeloid neoplasm associated with a high morbidity and mortality. The diagnosis, risk stratification and therapy selection in AML have changed substantially in the last decade with the progressive incorporation of clinically relevant molecular markers. METHODS: In this work, our aim was to describe a real-world genomic profiling experience in AML and to demonstrate the impact of the European Leukemia Net 2022 update on risk stratification in AML. RESULTS AND DISCUSSION: One hundred and forty-one patients were evaluated with an amplicon-based multi-gene next-generation sequencing (NGS) panel. The most commonly mutated genes were FLT3, DNMT3A, RUNX1, IDH2, NPM1, ASXL1, SRSF2, NRAS, TP53 and TET2. Detection of FLT3 ITD with NGS had a sensitivity of 96.3% when compared to capillary electrophoresis. According to ELN 2017, 26.6%, 20.1%, and 53.3% of patients were classified as having a good, moderate, or unfavorable risk. When ELN 2022 was used, 15.6%, 27.8%, and 56.6% of patients were classified as favorable, moderate, or unfavorable risk, respectively. When ELN 2022 was compared to ELN 2017, thirteen patients (14.4%) exhibited a different risk classification, with a significant decrease in the number of favorable risk patients, what has immediate clinical impact. CONCLUSIONS: In conclusion, we have described a real-world genomic profiling experience in AML and the impact of the 2022 ELN update on risk stratification.

Molecular profile of patients with myelofibrosis: a 10-year experience.

OBJECTIVE: To analyze the karyotype test and myeloid panel with next-generation sequencing findings in patients with myelofibrosis, and to compare transplant characteristics in patients referred for bone marrow transplantation. METHODS: Retrospective, single-center study with patients diagnosed with myelofibrosis treated at Hospital Israelita Albert Einstein between 2010 and 2020. RESULTS: A total of 104 patients with myelofibrosis were examined. Patients who had not been submitted to tests in our service were excluded. The final sample comprised 69 patients. Of these 69, 56 were submitted to karyotyping and 22 to myeloid panel with next-generation sequencing. Karyotype was normal in 60% of the patients and altered in 40%. The prevalence of high-risk molecular mutations was higher in patients referred for bone marrow transplantation (100% versus 50%). The median follow-up of transplant patients was 2.4 years and the overall survival at 2 years was 80% (95%CI: 62-100%). CONCLUSION: The molecular analysis enables estimating the patient's risk and thus instituting more aggressive treatment such as bone marrow transplant for patients at higher risk, being a relevant tool to guide therapy. Given the significance of molecular analysis for therapeutic decision-making in myelofibrosis, collection and disclosure of data on the prevalence of cytogenetic changes and findings of next-generation sequencing in affected patients is important.

Molecular profile of patients with myelofibrosis: a 10-year experience

ABSTRACT Objective To analyze the karyotype test and myeloid panel with next-generation sequencing findings in patients with myelofibrosis, and to compare transplant characteristics in patients referred for bone marrow transplantation. Methods Retrospective, single-center study with patients diagnosed with myelofibrosis treated at Hospital Israelita Albert Einstein between 2010 and 2020. Results A total of 104 patients with myelofibrosis were examined. Patients who had not been submitted to tests in our service were excluded. The final sample comprised 69 patients. Of these 69, 56 were submitted to karyotyping and 22 to myeloid panel with next-generation sequencing. Karyotype was normal in 60% of the patients and altered in 40%. The prevalence of high-risk molecular mutations was higher in patients referred for bone marrow transplantation (100% versus 50%). The median follow-up of transplant patients was 2.4 years and the overall survival at 2 years was 80% (95%CI: 62-100%). Conclusion The molecular analysis enables estimating the patient's risk and thus instituting more aggressive treatment such as bone marrow transplant for patients at higher risk, being a relevant tool to guide therapy. Given the significance of molecular analysis for therapeutic decision-making in myelofibrosis, collection and disclosure of data on the prevalence of cytogenetic changes and findings of next-generation sequencing in affected patients is important.

Standards for the classification of pathogenicity of somatic variants in cancer (oncogenicity): Joint recommendations of Clinical Genome Resource (ClinGen), Cancer Genomics Consortium (CGC), and Variant Interpretation for Cancer Consortium (VICC).

PURPOSE: Several professional societies have published guidelines for the clinical interpretation of somatic variants, which specifically address diagnostic, prognostic, and therapeutic implications. Although these guidelines for the clinical interpretation of variants include data types that may be used to determine the oncogenicity of a variant (eg, population frequency, functional, and in silico data or somatic frequency), they do not provide a direct, systematic, and comprehensive set of standards and rules to classify the oncogenicity of a somatic variant. This insufficient guidance leads to inconsistent classification of rare somatic variants in cancer, generates variability in their clinical interpretation, and, importantly, affects patient care. Therefore, it is essential to address this unmet need. METHODS: Clinical Genome Resource (ClinGen) Somatic Cancer Clinical Domain Working Group and ClinGen Germline/Somatic Variant Subcommittee, the Cancer Genomics Consortium, and the Variant Interpretation for Cancer Consortium used a consensus approach to develop a standard operating procedure (SOP) for the classification of oncogenicity of somatic variants. RESULTS: This comprehensive SOP has been developed to improve consistency in somatic variant classification and has been validated on 94 somatic variants in 10 common cancer-related genes. CONCLUSION: The comprehensive SOP is now available for classification of oncogenicity of somatic variants.

Genomic characterization and prognostication applied to a Brazilian cohort of patients with myelofibrosis.

Genomic characterization of patients with myeloproliferative neoplasms (MPN) may lead to better diagnostic classification, prognostic assessment, and treatment decisions. These goals are particularly important in myelofibrosis (MF). We performed target Next Generation Sequencing for a panel of 255 genes and Chromosome Microarray Analysis (CMA) in 27 patients with MF. Patients were classified according to genomic findings and we compared the performance of a personalized prognostication system with IPSS, MIPSS70 and MIPSS70 + v2. Twenty-six patients presented mutations: 11.1% had single driver mutations in either JAK2, CALR or MPL; 85.2% had mutations in non-restricted genes (median: 2 per patient). CMA was abnormal in 91.7% of the 24 cases with available data. Copy-Number-Neutral Loss-of-Heterozygosity was the most common finding (66.7%). Del13q was the most frequent copy number variation, and we could define a 2.4 Mb minimally affected region encompassing RB1, SUCLA2 and CLLS2 loci. The largest genomic subgroup consisted of patients with mutations in genes involved with chromatin organization and splicing control (40.7%) and the personalized system showed better concordance and accuracy than the other prognostic systems. Comprehensive genomic characterization reveals the striking genetic complexity of MF and, when combined with clinical data, led, in our cohort, to better prognostication performance.

Autophagy inhibition potentiates ruxolitinib-induced apoptosis in JAK2V617F cells.

JAK2V617F can mimic growth factor signaling, leading to PI3K/AKT/mTOR activation and inhibition of autophagy. We hypothesized that selective inhibition of JAK1/2 by ruxolitinib could induce autophagy and limit drug efficacy in myeloproliferative neoplasms (MPN). Therefore, we investigated the effects of ruxolitinib treatment on autophagy-related genes and cellular processes, to determine the potential benefit of autophagy inhibitors plus ruxolitinib in JAK2V617F cells, and to verify the frequency and clinical impact of autophagy-related gene mutations in patients with MPNs. In SET2 JAK2V617F cells, ruxolitinib treatment induced autophagy and modulated 26 out of 79 autophagy-related genes. Ruxolitinib treatment reduced the expressions of important autophagy regulators, including mTOR/p70S6K/4EBP1 and the STAT/BCL2 axis, in a dose- and time-dependent manner. Pharmacological inhibition of autophagy was able to significantly suppress ruxolitinib-induced autophagy and increased ruxolitinib-induced apoptosis. Mutations in autophagy-related genes were found in 15.5% of MPN patients and were associated with increased age and a trend towards worse survival. In conclusion, ruxolitinib induces autophagy in JAK2V617F cells, potentially by modulation of mTOR-, STAT- and BCL2-mediated signaling. This may lead to inhibition of apoptosis. Our results suggest that the combination of ruxolitinib with pharmacological inhibitors of autophagy, such as chloroquine, may be a promising strategy to treat patients with JAK2V617F-mutated MPNs.

Prognostic impact of RAS-pathway mutations in patients with myelofibrosis.

RAS-pathway mutations are recurrent events in myeloid malignancies. However, there is limited data on the significance of RAS-pathway mutations in patients with myelofibrosis (MF). We analyzed next-generation sequencing data of 16 genes, including RAS-pathway genes, from 723 patients with primary and secondary MF across three international centers and evaluated their significance. N/KRAS variants were present in 6% of patients and were typically sub-clonal (median VAF = 20%) relative to other genes variants. RAS variants were associated with advanced MF features including leukocytosis (p = 0.02), high somatic mutation burden (p < 0.01) and the presence of established "molecular high-risk" (MHR) mutations. MF patients with N/KRAS mutations had shorter 3-year overall survival (OS) (34% vs 58%, p < 0.001) and higher incidence of acute myeloid leukemia at 3 years (18% vs 11%, p = 0.03). In a multivariate Cox model, RAS mutations were associated with decreased OS (HR 1.93, p < 0.001). We created a novel score to predict OS incorporating RAS mutations, and it predicted OS across training and validation cohorts. Patients with intermediate risk/high-risk DIPSS with RAS mutations who received ruxolitinib had a nonsignificant longer 2-year OS relative to those who did not receive ruxolitinib. These data demonstrate the importance of identifying RAS mutations in MF patients.

ZAP-70 expression is associated with increased CD4 central memory T cells in chronic lymphocytic leukemia: cross-sectional study.

BACKGROUND: Although chronic lymphocytic leukemia is basically a B cell disease, its pathophysiology and evolution are thought to be significantly influenced by T cells, as these are probably the most important interaction partner of neoplastic B cells, participating in their expansion, differentiation and survival. Chronic lymphocytic leukemia B cells may also drive functional and phenotypic changes of non-malignant T cells. There are few data about the association between memory T cells and prognosis, especially related to ZAP-70, a common reliable surrogate of the gold standard chronic lymphocytic leukemia prognostic markers. OBJECTIVE: The aim of this study was to investigate whether the expression of ZAP-70 in chronic lymphocytic leukemia patients is associated with abnormal patterns of the distribution of naïve and memory T cells related to crosstalk between these cells. METHODS: In this cross-sectional, controlled study, patients with chronic lymphocytic leukemia were compared with healthy blood donors regarding the expression of ZAP-70 and the distribution of naïve and memory T cell subsets in peripheral blood as measured by flow cytometry. RESULTS: ZAP-70 positive patients presented an increased frequency and absolute number of central memory CD4+ T cells, but not CD8+ T cells, compared to ZAP-70 negative patients and age-matched apparently healthy donors. CONCLUSIONS: Because central memory CD4+ T cells are located in lymph nodes and express CD40L, we consider that malignant ZAP-70-positive B cells may receive beneficial signals from central memory CD4+ T cells as they accumulate, which could contribute to more aggressive disease.

ZAP-70 expression is associated with increased CD4 central memory T cells in chronic lymphocytic leukemia: cross-sectional study

ABSTRACT Background: Although chronic lymphocytic leukemia is basically a B cell disease, its pathophysiology and evolution are thought to be significantly influenced by T cells, as these are probably the most important interaction partner of neoplastic B cells, participating in their expansion, differentiation and survival. Chronic lymphocytic leukemia B cells may also drive functional and phenotypic changes of non-malignant T cells. There are few data about the association between memory T cells and prognosis, especially related to ZAP-70, a common reliable surrogate of the gold standard chronic lymphocytic leukemia prognostic markers. Objective: The aim of this study was to investigate whether the expression of ZAP-70 in chronic lymphocytic leukemia patients is associated with abnormal patterns of the distribution of naïve and memory T cells related to crosstalk between these cells. Methods: In this cross-sectional, controlled study, patients with chronic lymphocytic leukemia were compared with healthy blood donors regarding the expression of ZAP-70 and the distribution of naïve and memory T cell subsets in peripheral blood as measured by flow cytometry. Results: ZAP-70 positive patients presented an increased frequency and absolute number of central memory CD4+ T cells, but not CD8+ T cells, compared to ZAP-70 negative patients and age-matched apparently healthy donors. Conclusions: Because central memory CD4+ T cells are located in lymph nodes and express CD40L, we consider that malignant ZAP-70-positive B cells may receive beneficial signals from central memory CD4+ T cells as they accumulate, which could contribute to more aggressive disease.

Overall survival of Brazilian acute myeloid leukemia patients according to the European LeukemiaNet prognostic scoring system: a cross-sectional study.

Prognostic stratification in acute myeloid leukemia (AML) relies, mostly, on cytogenetics and molecular features of leukemic blasts. The LeukemiaNet prognostic scoring system has been proposed as a standardized way of evaluating prognosis in AML. We have analysed outcomes in 65 AML cases (median age of 54 years, range 18-82) treated at five hematology centers in Brazil stritified according to the European Leukemia Net (ELN) recommendations for cytogenetic and molecular analysis. We classified patients as favorable (N = 13), intermediate-1 (N = 25), intermediate-2 (N = 15), or adverse risk (N = 9). Bone marrow transplantation (BMT) was performed in 13 patients (21%). Median follow-up was 12 months. The median overall survival (OS) for all patients was 12.4 months. Median OS was 19.8, 12.4, 10.1, and 10.4 months (p = 0.24) for patients in the favorable, intermediate-1, intermediate-2, and adverse groups, respectively. Among patients receiving BMT, median OS was 26.8 months. The ELN is a valuable tool for prognostic stratification of AML patients treated in Brazil. Nevertheless, its usefulness is limited when compared to data from developed countries.

Ferritin light chain gene mutations in two Brazilian families with hereditary hyperferritinemia-cataract syndrome / Mutações no gene da cadeia leve da ferritina em duas famílias brasileiras com síndrome hereditária hiperferritinemia-catarata

ABSTRACT Hereditary hyperferritinemia-cataract syndrome is an autosomal dominant genetic disorder associated with mutations in the 5'UTR region of the ferritin light chain gene. These mutations cause the ferritin levels to increase even in the absence of iron overload. Patients also develop bilateral cataract early due to accumulation of ferritin in the lens, and many are misdiagnosed as having hemochromatosis and thus not properly treated. The first cases were described in 1995 and several mutations have already been identified. However, this syndrome is still a poorly understood. We report two cases of unrelated Brazilian families with clinical suspicion of the syndrome, which were treated in our department. For the definitive diagnosis, the affected patients, their parents and siblings were submitted to Sanger sequencing of the 5'UTR region for detection of the ferritin light gene mutation. Single nucleotide polymorphism-like mutations were found in the affected patients, previously described. The test assisted in making the accurate diagnosis of the disease, and its description is important so that the test can be incorporated into clinical practice.

RESUMO A síndrome hereditária hiperferritinemia-catarata é uma doença genética autossômica dominante associada a mutações na região 5'UTR do gene da cadeia leve da ferritina. Estas mutações elevam os níveis de ferritina, mesmo na ausência de sobrecarga de ferro. Os pacientes também desenvolvem catarata bilateral precocemente, devido ao acúmulo de ferritina no cristalino, e muitos são erroneamente diagnosticados como portadores de hemocromatose, sendo tratados de maneira inadequada. Os primeiros casos foram descritos em 1995, e diversas mutações já foram identificadas. Entretanto, essa síndrome ainda é pouco conhecida. Relatamos dois casos de famílias brasileiras, não relacionadas, com suspeita clínica da síndrome, que foram atendidas em nosso serviço. Para o diagnóstico definitivo, os pacientes afetados, seus pais e irmãos foram submetidos à pesquisa de mutação do gene ferritina, por sequenciamento de Sanger da região 5'UTR. Foram encontradas mutações do tipo polimorfismo de nucleotídeo único nos pacientes afetados, já descritas anteriormente. O teste auxiliou no diagnóstico preciso da doença e é importante ser divulgado, para ser incorporado na prática clínica.

Successful treatment of post-transplant relapsed acute myeloid leukemia with FLT3 internal tandem duplication using the combination of induction chemotherapy, donor lymphocyte infusion, sorafenib and azacitidine. Report of three cases / Tratamento bem-sucedido de leucemia mieloide aguda recorrente após transplante com duplicação interna em tandem FLT3 usando combinação de indução por quimioterapia, infusão de linfócitos de doador, soferanib e azacitidina. Relato de três casos

ABSTRACT Acute myeloid leukemia is a hematopoietic stem cell neoplastic disease associated with high morbidity and mortality. The presence of FLT3 internal tandem duplication mutations leads to high rates of relapse and decreased overall survival. Patients with FLT3 internal tandem duplication are normally treated with hematopoietic stem cell transplantation in first complete remission. Nevertheless, the incidence of post-transplant relapse is considerable in this group of patients, and the management of this clinical condition is challenging. The report describes the outcomes of patients with FLT3 internal tandem duplication positive acute myeloid leukemia who relapsed after allogeneic hematopoietic stem cell transplantation and were treated with the combination of re-induction chemotherapy, donor lymphocyte infusion, sorafenib and azacitidine. Three cases are described and all patients achieved prolonged complete remission with the combined therapy. The combination of induction chemotherapy followed by donor lymphocyte infusion, and the maintenance with azacitidine and sorafenib can be effective approaches in the treatment of post-hematopoietic stem cell transplant and relapsed FLT3 internal tandem duplication positive acute myeloid leukemia patients. This strategy should be further explored in the context of clinical trials.

RESUMO A leucemia mieloide aguda é uma doença neoplásica de células-tronco hematopoiéticas com alta morbimortalidade. A presença de mutações de duplicação em tandem de FLT3 leva a altas taxas de recorrência e a menor sobrevida global. Os pacientes com duplicação em tandem de FLT3 são normalmente tratados com transplante de células-tronco hematopoiéticas na primeira remissão completa. No entanto, a incidência de recidiva pós-transplante é considerável neste grupo de pacientes, e a conduta, nestes casos, é um desafio. O relato descreve os resultados do tratamento de pacientes com leucemia mieloide aguda positiva e duplicação em tandem de FLT3 que recidivaram depois do transplante alogênico de células-tronco hematopoiéticas e que foram tratados com combinação de quimioterapia de reindução, infusão de linfócitos de doador, sorafenib e azacitidina. São descritos três casos, e todos os pacientes apresentaram remissão completa prolongada com a terapia combinada. A combinação de quimioterapia de indução, seguida de infusão de linfócitos do doador, e a manutenção com azacitidina e sorafenib podem ser abordagens eficazes no tratamento da recorrência pós-transplante em pacientes com leucemia mieloide aguda e duplicação em tandem de FLT3. Essa estratégia deve ser mais explorada no contexto de ensaios clínicos.

Successful treatment of post-transplant relapsed acute myeloid leukemia with FLT3 internal tandem duplication using the combination of induction chemotherapy, donor lymphocyte infusion, sorafenib and azacitidine. Report of three cases.

Acute myeloid leukemia is a hematopoietic stem cell neoplastic disease associated with high morbidity and mortality. The presence of FLT3 internal tandem duplication mutations leads to high rates of relapse and decreased overall survival. Patients with FLT3 internal tandem duplication are normally treated with hematopoietic stem cell transplantation in first complete remission. Nevertheless, the incidence of post-transplant relapse is considerable in this group of patients, and the management of this clinical condition is challenging. The report describes the outcomes of patients with FLT3 internal tandem duplication positive acute myeloid leukemia who relapsed after allogeneic hematopoietic stem cell transplantation and were treated with the combination of re-induction chemotherapy, donor lymphocyte infusion, sorafenib and azacitidine. Three cases are described and all patients achieved prolonged complete remission with the combined therapy. The combination of induction chemotherapy followed by donor lymphocyte infusion, and the maintenance with azacitidine and sorafenib can be effective approaches in the treatment of post-hematopoietic stem cell transplant and relapsed FLT3 internal tandem duplication positive acute myeloid leukemia patients. This strategy should be further explored in the context of clinical trials.

Ferritin light chain gene mutations in two Brazilian families with hereditary hyperferritinemia-cataract syndrome.

Hereditary hyperferritinemia-cataract syndrome is an autosomal dominant genetic disorder associated with mutations in the 5'UTR region of the ferritin light chain gene. These mutations cause the ferritin levels to increase even in the absence of iron overload. Patients also develop bilateral cataract early due to accumulation of ferritin in the lens, and many are misdiagnosed as having hemochromatosis and thus not properly treated. The first cases were described in 1995 and several mutations have already been identified. However, this syndrome is still a poorly understood. We report two cases of unrelated Brazilian families with clinical suspicion of the syndrome, which were treated in our department. For the definitive diagnosis, the affected patients, their parents and siblings were submitted to Sanger sequencing of the 5'UTR region for detection of the ferritin light gene mutation. Single nucleotide polymorphism-like mutations were found in the affected patients, previously described. The test assisted in making the accurate diagnosis of the disease, and its description is important so that the test can be incorporated into clinical practice.