Clinical Evaluation of the BIOFIRE SPOTFIRE Respiratory Panel.

The BIOFIRE SPOTFIRE Respiratory (R) Panel is a novel, in vitro diagnostic PCR assay with 15 pathogen targets. The runtime is about 15 min which is the shortest among similar panels in the market. We evaluated the performance of the SPOTFIRE R Panel with 151 specimens, including 133 collected from the upper respiratory tract (URT), 13 from the lower respiratory tract (LRT) and 5 external quality assessment program (EQAP) samples. The respiratory specimens were enrolled throughout the first two post-COVID-19 influenza seasons in Hong Kong (March to December 2023). For URT specimens, full concordance was observed between the SPOTFIRE R Panel and the standard-of-care FilmArray Respiratory 2.1 plus Panel (RP2.1plus) for 109 specimens (109/133, 81.95%). After discrepant analysis, the SPOTFIRE R Panel identified more pathogens than the RP2.1plus in 15 specimens and vice versa in 3 specimens. The per-target negative and positive percentage agreement (NPA and PPA) were 92.86-100% except the PPA of adenovirus (88.24%). For LRT and EQAP samples, all results were fully concordant. To conclude, the performance of the SPOTFIRE R Panel was comparable to the RP2.1plus.

Application of droplet digital PCR in minimal residual disease monitoring of rare fusion transcripts and mutations in haematological malignancies.

Leukaemia of various subtypes are driven by distinct chromosomal rearrangement or genetic abnormalities. The leukaemogenic fusion transcripts or genetic mutations serve as molecular markers for minimal residual disease (MRD) monitoring. The current study evaluated the applicability of several droplet digital PCR assays for the detection of these targets at RNA and DNA levels (atypical BCR::ABL1 e19a2, e23a2ins52, e13a2ins74, rare types of CBFB::MYH11 (G and I), PCM1::JAK2, KMT2A::ELL2, PICALM::MLLT10 fusion transcripts and CEBPA frame-shift and insertion/duplication mutations) with high sensitivity. The analytical performances were assessed by the limit of blanks, limit of detection, limit of quantification and linear regression. Our data demonstrated serial MRD monitoring for patients at molecular level could become "digitalized", which was deemed important to guide clinicians in treatment decision for better patient care.

The Seasonality of Respiratory Viruses in a Hong Kong Hospital, 2014-2023.

We reviewed the multiplex PCR results of 20,127 respiratory specimens tested in a hospital setting from January 2014 to April 2023. The seasonal oscillation patterns of 17 respiratory viruses were studied. Compared with 2014-2019, a prominent drop in PCR positivity (from 64.46-69.21% to 17.29-29.89%, p < 0.001) and virus diversity was observed during the COVID-19 pandemic, with predominance of rhinovirus/enterovirus, sporadic spikes of parainfluenza viruses 3 and 4, respiratory syncytial virus and SARS-CoV-2, and rare detection of influenza viruses, metapneumovirus, adenovirus and coronaviruses. The suppressed viruses appeared to regain activity from the fourth quarter of 2022 when pandemic interventions had been gradually relaxed in Hong Kong. With the co-circulation of SARS-CoV-2 and seasonal respiratory viruses, surveillance of their activity and an in-depth understanding of the clinical outcomes will provide valuable insights for improved public health measures and reducing disease burden.

Effect on Germline Mutation Rate in a High-Risk Chinese Breast Cancer Cohort after Compliance with The National Comprehensive Cancer Network (NCCN) 2023 v.1 Testing Criteria.

BACKGROUND: The National Comprehensive Cancer Network (NCCN) testing criteria for the high-penetrance breast cancer susceptibility genes, specifically BRCA1, BRCA2, CDH1, PALB2, PTEN, and TP53, have been recently modified in 2023 to 2023 v.1. The following criteria have been changed: (1) from a person diagnosed with breast cancer at ≤45 to ≤50; (2) from aged 45-50 of personal breast diagnosis to any age of diagnosis with multiple breast cancers; and (3) from aged ≥51 of personal breast diagnosis to any age of diagnosis with family history listed in NCCN 2022 v.2. METHODS: High-risk breast cancer patients (n = 3797) were recruited from the Hong Kong Hereditary Breast Cancer Family Registry between 2007 and 2022. Patients were grouped according to NCCN testing criteria 2023 v.1 and 2022 v.2. A 30-gene panel for hereditary breast cancer was performed. The mutation rates on high-penetrance breast cancer susceptibility genes were compared. RESULTS: About 91.2% of the patients met the 2022 v.2 criteria, while 97.5% of the patients met the 2023 v.1 criteria. An extra 6.4% of the patients were included after the revision of the criteria, and 2.5% of the patients did not meet both testing criteria. The germline BRCA1/2 mutation rates for patients meeting the 2022 v.2 and 2023 v.1 criteria were 10.1% and 9.6%, respectively. The germline mutation rates of all 6 high-penetrance genes in these two groups were 12.2% and 11.6%, respectively. Among the additional 242 patients who were included using the new selection criteria, the mutation rates were 2.1% and 2.5% for BRCA1/2 and all 6 high-penetrance genes, respectively. Patients who did not meet both testing criteria were those with multiple personal cancers, a strong family history of cancers not listed in the NCCN, unclear pathology information, or the patient's voluntary intention to be tested. The mutation rates of BRCA1/2 and the 6 high-penetrance genes in these patients were 5.3% and 6.4%, respectively. CONCLUSION: This study provided a real-world application of the revision of NCCN guidelines and its effect on the germline mutation rate in the Chinese population. Applying the updated criteria for further genetic investigation would increase the positive detection rate, and potentially more patients would benefit. The balance between the resource and outcome requires careful consideration.

HFE promotes mitotic cell division through recruitment of cytokinetic abscission machinery in hepatocellular carcinoma.

HFE (Hemochromatosis) is a conventional iron level regulator and its loss of function due to gene mutations increases the risk of cancers including hepatocellular carcinoma (HCC). Likewise, studies focusing on HFE overexpression in cancers are all limited to linking up these events as a consequence of iron level deregulation. No study has explored any iron unrelated role of HFE in cancers. Here, we first reported HFE as an oncogene in HCC and its undescribed function on promoting abscission in cytokinesis during mitotic cell division, independent of its iron-regulating ability. Clinical analyses revealed HFE upregulation in tumors linking to large tumor size and poor prognosis. Functionally and mechanistically, HFE promoted cytokinetic abscission via facilitating ESCRT abscission machinery recruitment to the abscission site through signaling a novel HFE/ALK3/Smads/LIF/Hippo/YAP/YY1/KIF13A axis. Pharmacological blockage of HFE signaling axis impeded tumor phenotypes in vitro and in vivo. Our data on HFE-driven HCC unveiled a new mechanism utilized by cancer cells to propel rapid cell division. This study also laid the groundwork for tumor intolerable therapeutics development given the high cytokinetic dependency of cancer cells and their vulnerability to cytokinetic blockage.

How does re-classification of variants of unknown significance (VUS) impact the management of patients at risk for hereditary breast cancer?

BACKGROUND: The popularity of multigene testing increases the probability of identifying variants of uncertain significance (VUS). While accurate variant interpretation enables clinicians to be better informed of the genetic risk of their patients, currently, there is a lack of consensus management guidelines for clinicians on VUS. METHODS: Among the BRCA1 and BRCA2 mutations screening in 3,544 subjects, 236 unique variants (BRCA1: 86; BRCA2: 150) identified in 459 patients were being reviewed. These variants consist of 231 VUS and 5 likely benign variants at the initial classification. RESULTS: The variants in 31.8% (146/459) patients were reclassified during the review, which involved 26 unique variants (11.0%). Also, 31 probands (6.8%) and their family members were offered high-risk surveillance and related management after these variants were reclassified to pathogenic or likely pathogenic. At the same time, 69 probands (15%) had their VUS downgraded to cancer risk equivalent to the general population level. CONCLUSION: A review of archival variants from BRCA1 and BRCA2 genetic testing changed the management for 31.8% of the families due to increased or reduced risk. We encourage regular updates of variant databases, reference to normal population and collaboration between research laboratories on functional studies to define the clinical significances of VUS better.

Germline mutations in Chinese ovarian cancer with or without breast cancer.

BACKGROUND: Ovarian and breast cancers are known to have significant genetic components. Considering the differences in the mutation spectrum across ethnicity, it is important to identify hereditary breast and ovarian cancer (HBOC) genes mutation in Chinese for clinical management. METHODS: Two cohorts of 451 patients with ovarian cancer only (OV) and 93 patients with both breast and ovarian (BROV) cancers were initially screened for BRCA1, BRCA2, TP53, and PTEN. 109 OV and 43 BROV patients with extensive clinical risk and were being tested negative, were then further characterized by 30-gene panel analysis. RESULTS: Pathogenic BRCA1/2 variants were identified in 45 OV patients and 33 BROV patients, giving a prevalence of 10% and 35.5%, respectively. After the extended screening, mutations in other HBOC genes were identified in an additional 12.8% (14/109) of the OV cohort and 14% (6/43) in the BROV cohort. The most commonly mutated genes in the OV cohort were MSH2 (4.6%) while in the BROV cohort were MSH2 (4.7%) and PALB2 (4.7%). With this extended multigene testing strategy, pathogenic mutations were detected in 12.8% of OV patients (BRCAs: 10%; additional genes: 12.8%) and 40.9% (BRCAs: 35.5%; additional genes: 14%) of BROV patients. CONCLUSION: Extended characterization of the contributions of HBOC genes to OV and BROV patients has significant impacts on further management in patients and their families, expanding the screening net for more asymptomatic individuals.

Germline PALB2 Mutation in High-Risk Chinese Breast and/or Ovarian Cancer Patients.

The prevalence of the PALB2 mutation in breast cancer varies across different ethnic groups; hence, it is of intense interest to evaluate the cancer risk and clinical association of the PALB2 mutation in Chinese breast and/or ovarian cancer patients. We performed sequencing with a 6-gene test panel (BRCA1, BRCA2, TP53, PTEN, PALB2, and CDH1) to identify the prevalence of the PALB2 germline mutation among 2631 patients with breast and/or ovarian cancer. In this cohort, 39 mutations were identified with 24 types of mutation variants, where the majority of the mutations were frame-shift mutations and resulted in early termination. We also identified seven novel PALB2 mutations. Most of the PALB2 mutation carriers had breast cancer (36, 92.3%) and were more likely to have family history of breast cancer (19, 48.7%). The majority of the breast tumors were invasive ductal carcinoma (NOS type) (34, 81.0%) and hormonal positive (ER: 32, 84.2%; PR: 23, 60.5%). Pathogenic mutations of PALB2 were found in 39 probands with a mutation frequency of 1.6% and 1% in breast cancer and ovarian cancer patients, respectively. PALB2 mutation carriers were more likely have hormonal positive tumors and were likely to have familial aggregation of breast cancer.

A Case Report of Germline Compound Heterozygous Mutations in the BRCA1 Gene of an Ovarian and Breast Cancer Patient.

The germline carrier of the BRCA1 pathogenic mutation has been well proven to confer an increased risk of breast and ovarian cancer. Despite BRCA1 biallelic pathogenic mutations being extremely rare, they have been reported to be embryonically lethal or to cause Fanconi anemia (FA). Here we describe a patient who was a 48-year-old female identified with biallelic pathogenic mutations of the BRCA1 gene, with no or very subtle FA-features. She was diagnosed with ovarian cancer and breast cancer at the ages of 43 and 44 and had a strong family history of breast and gynecological cancers.

Mutation screening of germline TP53 mutations in high-risk Chinese breast cancer patients.

BACKGROUND: Germline TP53 mutations are associated with Li-Fraumeni syndrome, a severe and rare hereditary cancer syndrome. Despite the rarity of germline TP53 mutations, the clinical implication for mutation carriers and their families is significant. The risk management of TP53 germline mutation carriers is more stringent than BRCA carriers, and radiotherapy should be avoided when possible. METHODS: TP53 gene mutation screening was performed in 2538 Chinese breast cancer patients who tested negative for BRCA mutations. RESULTS: Twenty TP53 mutations were identified with high next-generation sequencing concerning for germline mutations in Chinese breast cancer families. The majorities of the TP53 carriers had early-onset, hormone receptor-positive breast cancer, and had strong family history of cancer. Among all, 11 patients carried a germline mutation and 6 of which were likely de novo germline mutations. In addition, 1 case was suspected to be induced by chemotherapy or radiation, as this patient had no significant family history of cancer and aberrant clonal expansion can commonly include TP53 mutations. Furthermore, we have identified one mosaic LFS case. Two novel mutations (c.524_547dup and c.529_546del) were identified in patients with early-onset. CONCLUSIONS: In view of the high lifetime risk of malignancy, identification of patients with germline TP53 mutations are important for clinicians to aid in accurate risk assessment and offer surveillance for patients and their families.

Somatic mutation profiling in BRCA-negative breast and ovarian cancer patients by multigene panel sequencing.

Targeted therapeutic agents such as poly (ADP-ribose) polymerases (PARP) inhibitors have emerged in treating cancers associated with germline BRCA mutations. Recently studies demonstrated the effectiveness of PARP inhibitors in treating patients with somatic BRCA mutations. Somatic mutations in 122 Chinese breast or ovarian cancer patients without BRCA, PTEN and TP53 mutations were screened using multigene sequencing panel. The five most frequent pathogenic or likely pathogenic mutated genes identified in breast cancer patients were PIK3CA (28.6%), TP53 (16.9%), MAP3K1 (14.3%), GATA3 (14.3%) and PTEN (5.2%). The five most frequently mutated genes identified in ovarian patients were TP53 (52.9%), KRAS (23.5%) and PIK3CA (11.8%), BRCA1 (5.9%) and RB1 (5.9%). Somatic PIK3CA and TP53 mutations were common events in both germline BRCA-negative breast and ovarian cancer patients. In contrast, somatic screening of BRCA mutations in BRCA-negative breast cancer patients has limited value. The results highlight the benefit of somatic testing to guide future research directions on other targeted therapies for breast and ovarian malignancies.

An economical Nanopore sequencing assay for human papillomavirus (HPV) genotyping.

BACKGROUND: Human papillomavirus (HPV) testing has been employed by several European countries to augment cytology-based cervical screening programs. A number of research groups have demonstrated potential utility of next-generation sequencing (NGS) for HPV genotyping, with comparable performance and broader detection spectrum than current gold standards. Nevertheless, most of these NGS platforms may not be the best choice for medium sample throughput and laboratories with less resources and space. In light of this, we developed a Nanopore sequencing assay for HPV genotyping and compared its performance with cobas HPV Test and Roche Linear Array HPV Genotyping Test (LA). METHODS: Two hundred and one cervicovaginal swabs were routinely tested for Papanicolaou smear, cobas HPV Test and LA. Residual DNA was used for Nanopore protocol after routine testing. Briefly, HPV L1 region was amplified using PGMY and MGP primers, and PCR-positive specimens were sequenced on MinION flow cells (R9.4.1). Data generated in first 2 h were aligned with reference sequences from Papillomavirus Episteme database for genotyping. RESULTS: Nanopore detected 96 HPV-positive (47.76%) and 95 HPV-negative (47.26%) specimens, with 10 lacking ß-globin band and not further analyzed (4.98%). Substantial agreement was achieved with cobas HPV Test and LA (κ: 0.83-0.93). In particular, Nanopore appeared to be more sensitive than cobas HPV Test for HPV 52 (n = 7). For LA, Nanopore revealed higher concordance for high-risk (κ: 0.93) than non-high risk types (κ: 0.83), and with similar high-risk positivity in each cytology grading. Nanopore also provided better resolution for HPV 52 in 3 specimens co-infected with HPV 33 or 58, and for HPV 87 which was identified as HPV 84 by LA. Interestingly, Nanopore identified 5 additional HPV types, with an unexpected high incidence of HPV 90 (n = 12) which was reported in North America and Belgium but not in Hong Kong. CONCLUSIONS: We developed a Nanopore workflow for HPV genotyping which was economical (about USD 50.77 per patient specimen for 24-plex runs), and with comparable or better performance than 2 reference methods in the market. Future prospective study with larger sample size is warranted to further evaluate test performance and streamline the protocol.

Sorafenib and omacetaxine mepesuccinate as a safe and effective treatment for acute myeloid leukemia carrying internal tandem duplication of Fms-like tyrosine kinase 3.

BACKGROUND: Omacetaxine mepesuccinate (OME) has antileukemic effects against acute myeloid leukemia (AML) carrying an internal tandem duplication of Fms-like tyrosine kinase 3 (FLT3-ITD). A phase 2 clinical trial was conducted to evaluate a combination treatment of sorafenib and omacetaxine mepesuccinate (SOME). METHODS: Relapsed or refractory (R/R) or newly diagnosed patients were treated with sorafenib (200-400 mg twice daily) and OME (2 mg daily) for 7 (first course) or 5 days (second course onward) every 21 days until disease progression or allogeneic hematopoietic stem cell transplantation (HSCT). The primary endpoint was composite complete remission, which was defined as complete remission (CR) plus complete remission with incomplete hematologic recovery (CRi). Secondary endpoints were leukemia-free survival (LFS) and overall survival (OS). RESULTS: Thirty-nine R/R patients and 5 newly diagnosed patients were recruited. Among the R/R patients, 28 achieved CR or CRi. Two patients showed partial remission, and 9 patients did not respond. Among the 5 newly diagnosed patients, 4 achieved CR, and 1 achieved CRi. The median LFS and OS were 5.6 and 10.9 months, respectively. Prior Fms-like tyrosine kinase 3 (FLT3) inhibitor exposure (P = .007), 2 or more inductions (P = .001), and coexisting IDH2 (P = .008) and RUNX1 mutations (P = .003) were associated with lower CR/CRi rates. HSCT consolidation and deep molecular responses (defined as an FLT3-ITD variant allelic frequency [VAF] ≤ 0.1% or a nucleophosmin 1 [NPM1] mutant VAF ≤ 0.01%) were associated with better OS and LFS. Prior FLT3 inhibitor exposure and 2 or more inductions were associated with inferior LFS. CONCLUSIONS: SOME was safe and effective for R/R and newly diagnosed FLT3-ITD AML.

Potential utility of metagenomic sequencing for improving etiologic diagnosis of infective endocarditis.

Aim: To explore potential utility of metagenomic sequencing for improving etiologic diagnosis of infective endocarditis (IE) caused by fastidious bacteria. Materials & methods: Plasma and heart valves of two patients, who were diagnosed with IE caused by Bartonella quintana and Propionibacterium species, were sequenced by using Illumina MiSeq and Nanopore MinION. Results: For patient 1, B. quintana was detected in the plasma pool collected 4 days before valvular replacement surgery. For patient 2, Propionibacterium sp. oral taxon 193 was detected in the plasma sample collected on hospital day 1. Nearly complete bacterial genomes (>98%) were retrieved from resected heart valves of both patients, enabling detection of antibiotic resistance-associated features. Real-time sequencing of heart valves identified both pathogens within the first 16 min of sequencing runs. Conclusion: Metagenomic sequencing may be a helpful supplement to IE diagnostic workflow, especially when conventional tests fail to yield a diagnosis.

Rapid detection of chromosomal translocation and precise breakpoint characterization in acute myeloid leukemia by nanopore long-read sequencing.

Detection of chromosomal translocation is a key component in diagnosis and management of acute myeloid leukemia (AML). Targeted RNA next-generation sequencing (NGS) is emerging as a powerful and clinically practical tool, but it depends on expression of RNA transcript from the underlying DNA translocation. Here, we show the clinical utility of nanopore long-read sequencing in rapidly detecting DNA translocation with exact breakpoints. In a newly diagnosed patient with AML, conventional karyotyping showed translocation t(10;12)(q22;p13) but RNA NGS detected NUP98-NSD1 fusion transcripts from a known cryptic translocation t(5;11)(q35;p15). Rapid PCR-free nanopore whole-genome sequencing yielded a 26,194 bp sequencing read and revealed the t(10;12) breakpoint to be DUSP13 and GRIN2B in head-to-head configuration. This translocation was then classified as a passenger structural variant. The sequencing also yielded a 20,709 bp sequencing read and revealed the t(5;11) breakpoint of the driver NUP98-NSD1 fusion. The identified DNA breakpoints also served as markers for molecular monitoring, in addition to fusion transcript expression by digital PCR and sequence mutations by NGS. We illustrate that third-generation nanopore sequencing is a simple and low-cost workflow for DNA translocation detection.

Next-generation sequencing and molecular cytogenetic characterization of ETV6-LYN fusion due to chromosomes 1, 8 and 12 rearrangement in acute myeloid leukemia.

In a newly diagnosed patient with acute myeloid leukemia (AML) and complex cytogenetics and negative for gene mutations associated with myeloid neoplasms, RNA sequencing by next-generation sequencing (NGS) through a large cancer-related gene panel showed ETV6-LYN leukemic fusion transcript. Breakpoint analysis of the NGS reads showed fusion of exon 5 of the ETV6 gene to exon 8 of the LYN gene. Metaphase fluorescence in situ hybridization (FISH) inferred a four-break rearrangement of three chromosomes, namely 1, 8 and 12. First, there was a balanced translocation t(1;12)(p13;p13.2) in which the ETV6 was split between der(1) and der(12). Second, an inverted insertion of 8q12.1~q24.21 into 1p13 occurred, thus bringing ETV6 and LYN into juxtaposition in the correct 5' to 3' orientation to produce an in-frame chimeric fusion gene on der(1). Notwithstanding two previous reports of ETV6-LYN fusion in myeloproliferative neoplasms (MPN), we report the first case of this fusion in AML and hence broaden its disease association. We also illustrate the clinical utility of NGS based detection of gene fusion in the setting of complex karyotype or cryptic aberration, since this method does not require a priori knowledge of the translocation partner and exact breakpoints to guide the application of appropriate primers or probes.

BAMClipper: removing primers from alignments to minimize false-negative mutations in amplicon next-generation sequencing.

Amplicon-based next-generation sequencing (NGS) has been widely adopted for genetic variation detection in human and other organisms. Conventional data analysis paradigm includes primer trimming before read mapping. Here we introduce BAMClipper that removes primer sequences after mapping original sequencing reads by soft-clipping SAM/BAM alignments. Mutation detection accuracy was affected by the choice of primer handling approach based on real NGS datasets of 7 human peripheral blood or breast cancer tissue samples with known BRCA1/BRCA2 mutations and >130000 simulated NGS datasets with unique mutations. BAMClipper approach detected a BRCA1 deletion (c.1620_1636del) that was otherwise missed due to edge effect. Simulation showed high false-negative rate when primers were perfectly trimmed as in conventional practice. Among the other 6 samples, variant allele frequencies of 5 BRCA1/BRCA2 mutations (indel or single-nucleotide variants) were diluted by apparently wild-type primer sequences from an overlapping amplicon (17 to 82% under-estimation). BAMClipper was robust in both situations and all 7 mutations were detected. When compared with Cutadapt, BAMClipper was faster and maintained equally high primer removal effectiveness. BAMClipper is implemented in Perl and is available under an open source MIT license at https://github.com/tommyau/bamclipper.

INDELseek: detection of complex insertions and deletions from next-generation sequencing data.

BACKGROUND: Complex insertions and deletions (indels) from next-generation sequencing (NGS) data were prone to escape detection by currently available variant callers as shown by large-scale human genomics studies. Somatic and germline complex indels in key disease driver genes could be missed in NGS-based genomics studies. RESULTS: INDELseek is an open-source complex indel caller designed for NGS data of random fragments and PCR amplicons. The key differentiating factor of INDELseek is that each NGS read alignment was examined as a whole instead of "pileup" of each reference position across multiple alignments. In benchmarking against the reference material NA12878 genome (n = 160 derived from high-confidence variant calls), GATK, SAMtools and INDELseek showed complex indel detection sensitivities of 0%, 0% and 100%, respectively. INDELseek also detected all known germline (BRCA1 and BRCA2) and somatic (CALR and JAK2) complex indels in human clinical samples (n = 8). Further experiments validated all 10 detected KIT complex indels in a discovery cohort of clinical samples. In silico semi-simulation showed sensitivities of 93.7-96.2% based on 8671 unique complex indels in >5000 genes from dbSNP and COSMIC. We also demonstrated the importance of complex indel detection in accurately annotating BRCA1, BRCA2 and TP53 mutations with gained or rescued protein-truncating effects. CONCLUSIONS: INDELseek is an accurate and versatile tool for complex indel detection in NGS data. It complements other variant callers in NGS-based genomics studies targeting a wide spectrum of genetic variations.

Homoharringtonine (omacetaxine mepesuccinate) as an adjunct for FLT3-ITD acute myeloid leukemia.

An in vitro drug-screening platform on patient samples was developed and validated to design personalized treatment for relapsed/refractory acute myeloid leukemia (AML). Unbiased clustering and correlation showed that homoharringtonine (HHT), also known as omacetaxine mepesuccinate, exhibited preferential antileukemia effect against AML carrying internal tandem duplication of fms-like tyrosine kinase 3 (FLT3-ITD). It worked synergistically with FLT3 inhibitors to suppress leukemia growth in vitro and in xenograft mouse models. Mechanistically, the effect was mediated by protein synthesis inhibition and reduction of short-lived proteins, including total and phosphorylated forms of FLT3 and its downstream signaling proteins. A phase 2 clinical trial of sorafenib and HHT combination treatment in FLT3-ITD AML patients resulted in complete remission (true or with insufficient hematological recovery) in 20 of 24 patients (83.3%), reduction of ITD allelic burden, and median leukemia-free and overall survivals of 12 and 33 weeks. The regimen has successfully bridged five patients to allogeneic hematopoietic stem cell transplantation and was well tolerated in patients unfit for conventional chemotherapy, including elderly and heavily pretreated patients. This study validated the principle and clinical relevance of in vitro drug testing and identified an improved treatment for FLT3-ITD AML. The results provided the foundation for phase 2/3 clinical trials to ascertain the clinical efficacy of FLT3 inhibitors and HHT in combination.