Clinical efficacy of tumor organoid-guided cancer therapy for locally advanced unresectable or metastatic breast cancer.

Patient-derived organoids (PDOs) may facilitate treatment selection. This retrospective cohort study evaluated the feasibility and clinical benefit of using PDOs to guide personalized treatment in metastatic breast cancer (MBC). Patients diagnosed with MBC were recruited between January 2019 and August 2022. PDOs were established and the efficacy of customized drug panels was determined by measuring cell mortality after drug exposure. Patients receiving organoid-guided treatment (OGT) were matched 1:2 by nearest neighbor propensity scores with patients receiving treatment of physician's choice (TPC). The primary outcome was progression-free survival. Secondary outcomes included objective response rate and disease control rate. Targeted gene sequencing and pathway enrichment analysis were performed. Forty-six PDOs (46 of 51, 90.2%) were generated from 45 MBC patients. PDO drug screening showed an accuracy of 78.4% (95% CI 64.9%-91.9%) in predicting clinical responses. Thirty-six OGT patients were matched to 69 TPC patients. OGT was associated with prolonged median progression-free survival (11.0 months vs. 5.0 months; hazard ratio 0.53 [95% CI 0.33-0.85]; p = .01) and improved disease control (88.9% vs. 63.8%; odd ratio 4.26 [1.44-18.62]) compared with TPC. The objective response rate of both groups was similar. Pathway enrichment analysis in hormone receptor-positive, human epidermal growth factor receptor 2-negative patients demonstrated differentially modulated pathways implicated in DNA repair and transcriptional regulation in those with reduced response to capecitabine/gemcitabine, and pathways associated with cell cycle regulation in those with reduced response to palbociclib. Our study shows that PDO-based functional precision medicine is a feasible and effective strategy for MBC treatment optimization and customization.

Organoid forming potential as complementary parameter for accurate evaluation of breast cancer neoadjuvant therapeutic efficacy.

BACKGROUND: 13-15% of breast cancer/BC patients diagnosed as pathological complete response/pCR after neoadjuvant systemic therapy/NST suffer from recurrence. This study aims to estimate the rationality of organoid forming potential/OFP for more accurate evaluation of NST efficacy. METHODS: OFPs of post-NST residual disease/RD were checked and compared with clinical approaches to estimate the recurrence risk. The phenotypes of organoids were classified via HE staining and ER, PR, HER2, Ki67 and CD133 immuno-labeling. The active growing organoids were subjected to drug sensitivity tests. RESULTS: Of 62 post-NST BC specimens, 24 were classified as OFP-I with long-term active organoid growth, 19 as OFP-II with stable organoid growth within 3 weeks, and 19 as OFP-III without organoid formation. Residual tumors were overall correlated with OFP grades (P < 0.001), while 3 of the 18 patients (16.67%) pathologically diagnosed as tumor-free (ypT0N0M0) showed tumor derived-organoid formation. The disease-free survival/DFS of OFP-I cases was worse than other two groups (Log-rank P < 0.05). Organoids of OFP-I/-II groups well maintained the biological features of their parental tumors and were resistant to the drugs used in NST. CONCLUSIONS: The OFP would be a complementary parameter to improve the evaluation accuracy of NST efficacy of breast cancers.

Validation of fetal DNA fraction estimation and its application in noninvasive prenatal testing for aneuploidy detection in multiple pregnancies.

OBJECTIVE: To analyze the fetal fraction, fetal sex, and chromosomal aneuploidy in multiple pregnancies using noninvasive prenatal testing (NIPT). METHOD: A total of 362 pregnant women including 203 singleton pregnancies, 69 twins, and 90 higher-order multiple pregnancies were recruited. Fetal fractions estimated by size ratio-based and Y chromosome-based approaches in singleton pregnancies with male fetus were used as source data to establish the model. The model was then applied to multiple pregnancies for fetal fraction estimation. By comparing the fetal fractions estimated by size ratio to those estimated by Y chromosome or autosomal chromosomes, fetal sex and chromosomal aneuploidy can be analyzed. RESULTS: The size ratio-based approach has been well established in estimating fetal fractions for twin and higher-order multiple pregnancies. Fetal fraction had a positive correlation with gestational age in twin and triplet pregnancies. Fetal sex was determined with accuracies of 98.6% (95% CI, 92.19%-99.96%) in twins and 97.6% (95% CI, 91.76%-99.71%) in triplet pregnancies. Four trisomy 21, one trisomy 18, and one trisomy 13 cases were detected by NIPT. Two trisomy 21 singleton pregnancies and one trisomy 21 twin pregnancy were confirmed by karyotyping. CONCLUSION: Fetal sex and chromosomal aneuploidy in multiple pregnancies can be determined using NIPT.

Prenatal detection and molecular cytogenetic characterization of Xp deletion and Xq duplication: a case report and literature review.

BACKGROUND: Copy number variation (CNV) of X chromosome can lead to a variety of neonatal abnormalities, especially for male fetuses. In recent years, due to the high sensitivity and high specificity of NIPS, its application has gradually expanded from chromosome aneuploidy to CNV. Few prenatal cases involving the detection of Xq duplication and deletion by NIPS have been reported, but it is of great significance for genetic counseling. CASE PRESENTATION: A 36-year-old woman was referred for prenatal diagnosis and genetic counseling at 17 weeks of gestation because of abnormal result of noninvasive prenatal screening (NIPS). Multiple congenital malformations, hydrocephalus, and enlarged gallbladder were observed by prenatal ultrasound. Amniocentesis revealed the karyotype of the fetus as 46, XN, add(X) (p22.2) and the result of chromosomal microarray analysis was arr[hg19] Xq27.1q28(138,506,454-154896094) × 2 and arr[hg19] Xp22.33p22.32(168,551-5,616,964) × 1. CNV-seq showed that the mother shares a 16.42 Mb duplication in the Xq27.1-q28 region and a 2.97 Mb deletion in the Xp22.33-p22.32 region. After genetic counseling, the couple chose to terminate the pregnancy. CONCLUSION: The combination of NIPS and CMA would be of values in detection of subchromosomal duplications and/or deletions at fetal stage. The detection of X chromosome aberration in a male fetus should give suspicion of the possibility of maternal inheritance.

Feasibility study of semiconductor sequencing for noninvasive prenatal detection of fetal aneuploidy.

BACKGROUND: Noninvasive prenatal detection of common fetal aneuploidies with cell-free DNA from maternal plasma has been achieved with high-throughput next-generation sequencing platforms. Turnaround times for previously tested platforms are still unsatisfactory for clinical applications, however, because of the time spent on sequencing. The development of semiconductor sequencing technology has provided a way to shorten overall run times. We studied the feasibility of using semiconductor sequencing technology for the noninvasive detection of fetal aneuploidy. METHODS: Maternal plasma DNA from 13 pregnant women, corresponding to 4 euploid, 6 trisomy 21 (T21), 2 trisomy 18 (T18), and 1 trisomy 13 (T13) pregnancies, were sequenced on the Ion Torrent Personal Genome Machine sequencer platform with 318 chips. The data were analyzed with the T statistic method after correcting for GC bias, and the T value was calculated as an indicator of fetal aneuploidy. RESULTS: We obtained a mean of 3 524 401 high-quality reads per sample, with an efficiency rate of 77.9%. All of the T21, T13, and T18 fetuses could be clearly distinguished from euploid fetuses, and the time spent on library preparation and sequencing was 24 h. CONCLUSIONS: Semiconductor sequencing represents a suitable technology for the noninvasive prenatal detection of fetal aneuploidy. With this platform, sequencing times can be substantially reduced; however, a further larger-scale study is needed to determine the imprecision of noninvasive fetal aneuploidy detection with this system.

Noninvasive prenatal testing for ß-thalassemia by targeted nanopore sequencing combined with relative haplotype dosage (RHDO): a feasibility study.

Noninvasive prenatal testing (NIPT) for single gene disorders remains challenging. One approach that allows for accurate detection of the slight increase of the maternally inherited allele is the relative haplotype dosage (RHDO) analysis, which requires the construction of parental haplotypes. Recently, the nanopore sequencing technologies have become available and may be an ideal tool for direct construction of haplotypes. Here, we explored the feasibility of combining nanopore sequencing with the RHDO analysis in NIPT of ß-thalassemia. Thirteen families at risk for ß-thalassemia were recruited. Targeted region of parental genomic DNA was amplified by long-range PCR of 10 kb and 20 kb amplicons. Parental haplotypes were constructed using nanopore sequencing and next generation sequencing data. Fetal inheritance of parental haplotypes was classified by the RHDO analysis using data from maternal plasma DNA sequencing. Haplotype phasing was achieved in 12 families using data from 10 kb library. While data from the 20 kb library gave a better performance that haplotype phasing was achieved in all 13 families. Fetal status was correctly classified in 12 out of 13 families. Thus, targeted nanopore sequencing combined with the RHDO analysis is feasible to NIPT for ß-thalassemia.

Identification of GPC3 mutation and upregulation in a multidrug resistant osteosarcoma and its spheroids as therapeutic target.

BACKGROUND: Drug resistance and the lack of molecular therapeutic target are the main challenges in the management of osteosarcomas (OSs). Identification of novel genetic alteration(s) related with OS recurrence and chemotherapeutic resistance would be of scientific and clinical significance. METHODS: To identify potential genetic alterations related with OS recurrence and chemotherapeutic resistance, the biopsies of a 20-year-old male osteosarcoma patient were collected at primary site (p-OS) and from its metastatic tumor (m-OS) formed after 5 months of adjuvant chemotherapy. Both OS specimens were subjected to cancer-targeted next generation sequencing (NGS) and their cell suspensions were cultured under three-dimensional condition to establish spheroid therapeutic model. Transcript-oriented Sanger sequencing for GPC3, the detected mutated gene, was performed on RNA samples of p-OS and m-OS tissues and spheroids. The effects of anti-GPC3 antibody and its combination with cisplatin on m-OS spheroids were elucidated. RESULTS: NGS revealed 4 mutations (GPC3, SOX10, MDM4 and MAPK8) and 6 amplifications (MDM2, CDK4, CCND3, RUNX2, GLI1 and FRS2) in p-OS, and 3 mutations (GPC3, SOX10 and EGF) and 10 amplifications (CDK4, CCND3, MDM2, RUNX2, GLI1, FRS2, CARD11, RAC1, SLC16A7 and PMS2) in m-OS. Among those alterations, the mutation abundance of GPC3 was the highest (56.49%) in p-OS and showed 1.54 times increase in m-OS. GPC3 transcript-oriented Sanger sequencing confirmed the mutation at 1046 in Exon 4, and immunohistochemical staining showed increased GPC3 production in m-OS tissues and its spheroids. EdU cell proliferation and Calcein/PI cell viability assays revealed that of the anti-OS first line drugs (doxorubicin, cisplatin, methotrexate, ifosfamide and carboplatin), 10 µM carboplatin exerted the best inhibitory effects on the p-OS but not the m-OS spheroids. 2 µg/mL anti-GPC3 antibody effectively committed m-OS spheroids to death by itself (76.43%) or in combination with cisplatin (92.93%). CONCLUSION: This study demonstrates increased abundance and up-regulated expression of mutant GPC3 in metastatic osteosarcoma and its spheroids with multidrug resistance. As GPC3-targeting therapy has been used to treat hepatocellular carcinomas and it is also effective to OS PDSs, GPC3 would be a novel prognostic parameter and therapeutic target of osteosarcomas.

Noninvasive prenatal diagnosis of ß-thalassemia by relative haplotype dosage without analyzing proband.

BACKGROUND: ß-thalassemia is one of the most common monogenic diseases in the world. Southeast China is a highly infected area affected by four ß-thalassemia mutation types (HBB:c.-78A>G, HBB:c.52A>T, HBB:c.126_129delCTTT, and HBB:c.316-197C>T). Relative haplotype dosage (RHDO), a haplotype-based approach, has shown promise as an application for noninvasive prenatal diagnosis (NIPD); however, additional family members (such as the proband) are required for haplotype construction. The abovementioned circumstances make RHDO-based NIPD cost prohibitive; additionally, the genetic information of the proband is not always available. Thus, it is necessary to find a practical method to solve these problems. METHODS: Targeted sequencing was applied to sequence parental genomic DNA and cell-free fetal DNA (cffDNA). Parental haplotypes were constructed with the SHAPEIT software based on the 1000 Genomes Project (1000G) Phase 3 v5 Southern Han Chinese (CHS) haplotype dataset. Single-nucleotide polymorphisms (SNPs) in the target region were called and classified, and the fetal mutation inheritance status was deduced using the RHDO method. RESULTS: Construction of the parental haplotypes and detection of the inherited parental mutations were successfully achieved in five families, despite a suspected recombination event. The status of the affected fetuses is consistent with the results of traditional reverse dot blot (RDB) diagnosis. CONCLUSION: This research introduced SHAPEIT into the classical RHDO workflow and proved that it is applicable to construct parental haplotypes without information from other family members.

A reliable, high-resolution and high-throughput genotyping method for HLA-DRB1.

Human leukocyte antigen (HLA) DNA typing is an essential part to identify a donor who may be a good match for the patients who need haematopoietic stem cells from bone marrow. Thus, fetching quickly high-resolution genotype is very important at present. However, current HLA DNA typing methods especially for HLA-DRB1 generally yielded ambiguous typing results because of high degree of heterozygosity on exome region and primer pairs design limitations, which generally amplified only exon2 of HLA-DRB1 and the position of its primer pairs is on exome region. To solve this problem, we developed a reliable, high-resolution and high-throughput (RHH) sequence based typing approach for HLA-DRB1 through massively parallel paired-end sequencing. Several primer pairs were designed to amplify three exon regions, which are part of exon1, the whole region of exon2 and exon3 of HLA-DRB1, to genotyping for HLA-DRB1 by synthesis. 94 samples were included to analyze and the highly successful genotyping ratio (98.94%) and no ambiguous genotyping result demonstrated the accurate performance of our method for HLA-DRB1 genotyping.

Effects of Maternal and Fetal Characteristics on Cell-Free Fetal DNA Fraction in Maternal Plasma.

OBJECTIVE: To study factors that influence the concentration of cell-free fetal DNA (fetal fraction) using a large clinical data set of pregnancies with male fetus. METHOD: A retrospective analysis of 23 067 pregnancies that received noninvasive prenatal testing from January 2012 to October 2013, including 22 650 normal singleton pregnancies (control group) and 417 pregnancies with aneuploidy, twin pregnancy, or various maternal conditions including preexisting hypertension, preexisting diabetes, hyperthyroidism, and carrier of the surface antigen of the hepatitis B virus (HBsAg; study group). Multiples of the median (MoM) analysis was performed in the control group to derive gestation and body mass index (BMI)-corrected fetal fraction. The effects of study group conditions on fetal fraction were examined by calculating the ratio of MoM (RMoM) values. RESULTS: Fetal fraction showed a positive correlation with gestational age (r(2) = .10, P < .001) and increased rapidly after the 21 weeks of gestation (r(2) = .26, P < .001). Negative association with maternal BMI was found with fetal fraction (r(2) = .04, P < .001). In study group, fetal fraction was higher among pregnant women with a trisomy 21 fetus (RMoM = 1.24, P < .001) and lower among trisomy 18 (RMoM = 0.84, P < .001). A 1.6-fold incensement of fetal fraction was observed in twin fetuses comparing to singleton pregnancy (RMoM = 1.62, P < .001). Women with preexisting hypertension had significantly lower fetal fraction (RMoM = 0.85, P = .02). Preexisting diabetes, hyperthyroidism, or carrier of HBsAg did not affect fetal fraction. CONCLUSION: The fetal fraction was affected by fetal aneuploidy, maternal BMI, and the number of gestation. Maternal preexisting of hypertension appeared to reduce fetal fraction.