Multiple Primary Cancers With Hematologic Malignancies and Germline Predisposition: A Case Series.

The term "multiple primary (MP) cancers" refers to the existence of more than one cancer in the same patient. The combination of MP cancers with hematological malignancies is relatively uncommon. In this study, we present five patients diagnosed with MP cancers concomitant with hematological malignancies. We comprehensively analyzed their clinical characteristics, cytogenetic profiles, and germline and somatic variants. As first primaries, two patients had solid cancer not followed by cytotoxic therapy and three had hematologic cancer, followed by cytotoxic therapy. The second primaries were all hematologic malignancies that did not meet the criteria for therapy-related myeloid neoplasm. Notably, two (40%) out of the five patients harbored pathogenic potential/presumed germline variants in cancer predisposition genes. Therefore, germline variant testing should be considered when MP cancers with hematological malignancies require consideration for related donor stem cell transplantation.

Hema-seq reveals genomic aberrations in a rare simultaneous occurrence of hematological malignancies.

Co-occurrence of multiple myeloma and acute myelogenous leukemia is rare, with both malignancies often tracing back to multipotent hematopoietic stem cells. Cytogenetic techniques are the established baseline for diagnosis and characterization of complex hematological malignancies. In this study, we develop a workflow called Hema-seq to delineate clonal changes across various hematopoietic lineages through the integration of whole-genome sequencing, copy-number variations, cell morphology, and cytogenetic aberrations. In Hema-seq, cells are selected from Wright-stained slides and fluorescent probe-stained slides for sequencing. This technique therefore enables direct linking of whole-genome sequences to cytogenetic profiles. Through this method, we mapped sequential clonal alterations within the hematopoietic lineage, identifying critical shifts leading to myeloma and acute myeloid leukemia (AML) cell formations. By synthesizing data from each cell lineage, we provided insights into the hematopoietic tree's clonal evolution. Overall, this study highlights Hema-seq's capability in deciphering genomic heterogeneity in complex hematological malignancies, which can enable better diagnosis and treatment strategies.

Cryptic KMT2A/MLLT10 fusion detected by next-generation sequencing in a case of pediatric acute megakaryoblastic leukemia.

KMT2A (11q23.3) gene rearrangements are found in acute leukemia and are associated with a poor or intermediate prognosis. MLLT10 is the fourth most common gene fusion partner for KMT2A. A reciprocal translocation t(10;11) is insufficient to produce an in-frame KMT2A/MLLT10 fusion, because the genes involved in the rearrangement have opposite transcriptional orientations. In order to bring KMT2A and MLLT10 into juxtaposition, complex rearrangements are required. Until now, conventional chromosome, fluorescence in situ hybridization (FISH), and reverse transcriptase-polymerase chain reaction (RT-PCR) studies have been used to detect KMT2A/MLLT10 fusions. However, conventional studies have limitations, such as poor and inconsistent resolution, when compared to next-generation sequencing (NGS). In this study, we report a pediatric patient with acute megakaryoblastic leukemia, in whom the cryptic KMT2A/MLLT10 fusion was not detected by KMT2A break-apart probe FISH and chromosome analysis, but detected by NGS. In this patient, NGS showed cryptic insertion of MLLT10 exons 9-24 into intron 9 of KMT2A, resulting in a KMT2A/MLLT10 fusion. Therefore, NGS is a valuable complementary option for the evaluation of structural aberrations, especially those with a cryptic size.

Analysis of clinical and genomic profiles of therapy-related myeloid neoplasm in Korea.

BACKGROUND: Therapy-related myeloid neoplasm (T-MN) rarely occurs among cancer survivors, and was characterized by poor prognosis. T-MN has germline predisposition in a considerable proportion. Here, clinical characteristics and germline/somatic variant profiles in T-MN patients were investigated, and the findings were compared with those of previous studies. METHODS: A review of medical records, cytogenetic study, targeted sequencing by next-generation sequencing, and survival analysis were performed on 53 patients with T-MN at a single institution in Korea. RESULTS: The patients were relatively younger compared to T-MN patients in other studies. Our T-MN patients showed a high frequency of complex karyotypes, -5/del(5q), and -7/del(7q), which was similar to the Japanese study group but higher than the Australian study group. The most common primary disease was non-Hodgkin lymphoma, followed by breast cancer. The detailed distributions of primary diseases were different across study groups. Seven patients (13.2%) harbored deleterious presumed/potential germline variants in cancer predisposition genes (CPG) such as BRIP1, CEBPA, DDX41, FANCM, NBN, NF1, and RUNX1. In the somatic variant profile, TP53 was the most frequently mutated gene, which was consistent with the previous studies about T-MN. However, the somatic variant frequency in our study group was lower than in other studies. Adverse factors for overall survival were male sex, older age, history of previous radiotherapy, previous longer cytotoxic therapy, and -5/del(5q). CONCLUSION: The findings of our study corroborate important information about T-MN patients. As well as a considerable predisposition to CPG, the clinical characteristics and somatic variant profile showed distinctive patterns. Germline variant testing should be recommended for T-MN patients. If the T-MN patients harbor pathogenic germline variants, the family members for stem cell donation should be screened for carrier status through germline variant testing to avoid donor-derived myeloid neoplasm. For the prediction of the prognosis in T-MN patients, sex, age, past treatment history, and cytogenetic findings can be considered.

Telomere integrated scoring system of myelodysplastic syndrome.

INTRODUCTION: Recently, multigene target sequencing is widely performed for the purpose of prognostic prediction and application of targeted therapy. Here, we proposed a new scoring system that encompasses gene variations, telomere length, and Revised International Prognostic Scoring System (IPSS-R) together in Asian myelodysplastic syndrome. METHODS: We developed a new scoring model of these variables: age ≥ 65 years + IPSS-R score + ASXL1 mutation + TP53 mutation + Telomere length (<5.37). According to this new scoring system, patients were divided into four groups: very good score cutoff (≤3.0), good (3.0-4.5), poor (4.5-7.0), and very poor (>7.0). RESULTS: The median OS was 170.1, 100.4, 46.0, and 12.0 months for very good, good, poor, and very poor, retrospectively (p < 0.001). Meanwhile, according to the conventional IPSS-R scoring system, the median OS was 141.3, 50.2, 93.0, 36.0, and 16.2 months for very low, low, intermediate, high, and very high, retrospectively (p < 0.001). CONCLUSIONS: The newly developed model incorporating molecular variations and TL yielded more clear separations of the survival curves. By adding the presence of gene mutation and telomere length to the existing IPSS-R, its predictive ability can be further improved in myelodysplastic syndrome.

Novel THPO variant in hereditary thrombocytopenia: A potential candidate variant for predisposition to myeloid neoplasm.

Hereditary thrombocytopenia is a heterogeneous group of congenital disorders with a wide range of symptoms depending on the severity of platelet dysfunction or thrombocytopenia. Because of its clinical phenotypes and the bone marrow morphology associated with this condition, hereditary thrombocytopenia can be misdiagnosed as primary immune thrombocytopenia and myelodysplastic syndrome. Therefore, genetic evidence is necessary for the accurate diagnosis of hereditary thrombocytopenia. Refractory cytopenia of childhood is a subgroup of myelodysplastic syndrome that was added to the World Health Organization classification in 2008. To investigate the germline and somatic variants associated with refractory cytopenia of childhood, we performed targeted multigene sequencing in three patients with refractory cytopenia of childhood. Of the three patients, one progressed from megakaryocytic hypoplasia with thrombocytopenia, and targeted multigene sequencing revealed THPO variants in this patient and his sister. We propose that the monoallelic deletion of THPO is a potential candidate for germline predisposition to myeloid malignancy.

The importance of critically short telomere in myelodysplastic syndrome.

A few critically short telomeres trigger genomic instability regardless of average telomere length (TL). Recently, the telomere shortest length assay (TeSLA) was developed to detect critically short telomeres and measure absolute telomeres. Using TeSLA with the internally labeled biotin probe, we measured the TL of bone marrow (BM) aspirates from 52 patients with myelodysplastic syndrome (MDS). A percentage of shortest telomeres (< 1.0 kb (ShTL1.0)) were calculated. ShTL1.0 was correlated to IPSS-R risk (spearman's rho = 0.35 and p = 0.0196), and ShTL1.0 and BM blast (2.61% in < 5% blast, 4.15% in 5-10% blast, and 6.80% in 10-20% blast, respectively, p = 0.0332). Interestingly, MDS patients with a shortest TL ≥ 0.787 kb at the time of diagnosis showed better overall survival (OS) and progression-free survival (PFS) than patients with a shortest TL < 0.787 kb in the multivariate analyses (HR = 0.13 and 0.30, p = 0.011 and 0.048 for OS and PFS, respectively). Our results clearly show the presence and abundance of critically short telomeres in MDS patients. These pathologic telomeres are associated with IPSS-R which is a validated prognostic scoring system in MDS. Furthermore, they are independent prognostic factors for OS in MDS patients. Future prospective studies are needed to validate our results.

Increased Apoptotic Activity in Low-Risk Myelodysplastic Syndrome.

Myelodysplastic syndrome (MDS) is a heterogeneous hematopoietic disorder associated with cellular proliferative and apoptotic activity. We retrospectively investigated these activities in bone marrow samples from 76 MDS patients using immunohistochemical staining for Ki-67 and cleaved caspase-3. We divided cleaved caspase-3 into two groups based on median value and compared the differences according to MDS risk scoring systems. We compared MDS patient indices with idiopathic cytopenia of undetermined significance (ICUS) and healthy control (HC) indices using our previously published data. Cleaved caspase-3 immunohistochemistry was highest in MDS patients, followed by ICUS patients and HCs. Similarly, the mean Ki-67 grade was also highest in MDS patients, followed by ICUS patients and HCs. Higher cleaved caspase-3 grade was significantly associated with lower IPSS-R score (p = 0.020), whereas Ki-67 was not associated with MDS. Interestingly, TET2 mutation was associated with decreased cleaved caspase-3 levels (p = 0.03). However, there was no significant association between proliferative/apoptotic activity and survival. Our results suggest that apoptotic activity gradually increases from healthy controls and ICUS patients to MDS patients. Furthermore, higher apoptotic activity was associated with better MDS patient prognostic scores. Further studies are needed to reveal the differences in apoptotic activity between lower- and higher-risk MDS.

Heterogeneous genetic landscape of congenital neutropenia in Korean patients revealed by whole exome sequencing: genetic, phenotypic and histologic correlations.

Congenital neutropenia (CN) is a hematological disease heterogeneous in its genetic, phenotypic and histologic aspects. We aimed to identify the genetic etiology of Korean CN patients in the context of bone marrow (BM) histology and clinical phenotype. Whole-exome sequencing (WES) or targeted sequencing was performed on the BM or peripheral blood specimens of 16 patients diagnosed with CN based on BM exam from 2009 to 2018. Absolute count of myeloperoxidase (MPO)-positive cells was calculated using ImageJ software. Semi-quantitation of MPO-positive cells in BM sections was performed by MPO grading (grades 0-3). Comprehensive retrospective review on real-world data of 345 pediatric patients with neutropenia including 16 patients in this study during the same period was performed. Seven disease-causing variants were identified in ELANE, G6PC3 and CXCR4 in 7 patients. A novel homozygous G6PC3 variant (K72fs) of which the mechanism was copy-neutral loss of heterozygosity was detected in two brothers. A low myeloid-to-erythroid ratio (0.5-1.5) was consistently observed in patients with ELANE mutations, while MPO-positive cells (40%-50%) with MPO grade 1 or 2 were detected in myelokathexis caused by G6PC3 and CXCR4 mutations. Meanwhile, disease-causing variants were detected in ELANE, TAZ and SLC37A4 in 5 patients by retrospective review of medical records. Our results suggest that following the immunological study and BM exam, WES or an expanded next generation sequencing panel that covers genes related to immunodeficiency and other inherited bone marrow failures as well as CN is recommended for neutropenia patient diagnosis.

Clinical and Genomic Profiles of Korean Patients with MECOM Rearrangement and the t(3;21)(q26.2;q22.1) Translocation.

The translocation (3;21)(q26.2;q22.1) is a unique cytogenetic aberration that characterizes acute myeloid leukemia with myelodysplasia-related changes (AML-MRC) in patients with AML and myelodysplastic syndrome (MDS) or a therapy-related myeloid neoplasm. Using multigene target sequencing and FISH, we investigated the clinical and genomic profiles of patients with t(3;21) over the past 10 years. The frequency of t(3;21) among myeloid malignancies was very low (0.2%). Half of the patients had a history of cancer treatment and the remaining patients had de novo MDS. Twenty-one somatic variants were detected in patients with t(3;21), including in CBL, GATA2, and SF3B1. Recurrent variants in RUNX1 (c.1184A>C, p.Glu395Ala) at the same site were detected in two patients. None of the patients with t(3;21) harbored germline predisposition mutations for myeloid neoplasms. MECOM rearrangement was detected at a higher rate using FISH than using G-banding, suggesting that FISH is preferable for monitoring. Although survival of patients with t(3;21) is reportedly poor, the survival of patients with t(3;21) in this study was not poor when compared with that of other AML patients in Korea.

Application of Interphase Fluorescent in Situ Hybridization: a Screening Tool for the Diagnosis of Microdeletion Syndrome.

BACKGROUND: Most laboratories adopt the results of metaphase fluorescent in situ hybridization (FISH) for the diagnosis of microdeletion syndromes. To investigate the discrepancy between the results of interphase and metaphase, we compared the quantitative results of FISH for 5 kinds of microdeletion syndrome and gender determination disorders (SDD). METHODS: A total of 282 (135 for DiGeorge syndrome, 20 for Kalmann syndrome, 7 for Miller-Dieker syndrome, 38 for Prader Willi/Angelman syndrome, 62 for Williams syndrome, and 20 for SDD (SRY FISH)) were enrolled. For SRY FISH, we artificially mixed fresh blood of male and female with various ratios and then compared the results of metaphase and interphase SRY FISH. Using a bio-cell chip, we performed interphase FISH in 168 patients with microdeletion syndromes and compared the results with manual interphase. RESULTS: The concordance rate between the results of metaphase and interphase was 100% in microdeletion syndrome. In the disorders of gender development, SRY FISH showed 100% concordance between interphase and metaphase when we counted 50 metaphase cells and 100 interphase cells. Comparison with mixtures of male and female blood at various ratios also showed 100% concordance. The results of bio-cell chip showed 100% concordance between previous interphase FISH results. CONCLUSIONS: Considering the complete concordance between interphase and metaphase in microdeletion syndrome, the application of interphase FISH without performing metaphase FISH can be a screening test for microdeletion syndrome. Confirmation by metaphase FISH can be performed only in cases with abnormal results by interphase FISH.

Genetic profile of primary plasma cell leukemia in Korea: comparison with plasma cell myeloma.

Plasma cell leukemia (PCL) is clinically and genetically distinct from multiple myeloma (MM), despite controversies regarding the disease definition. To determine the distinct features of PCL, the genetic property of primary PCL (pPCL) was compared with that of secondary PCL (sPCL) and MM. In patients with pPCL, Eighty-nine non-synonymous mutations were observed in 68 genes. The most frequently mutated genes were TP53, TSC2, and TYK2. In comparison with genetic abnormalities of sPCL and MM, 45 genes were present only in pPCL while 28 genes were only in sPCL and 22 genes only in MM. Among the common genes between pPCL and MM, a higher prevalence of TP53 was observed in pPCL, compared to MM (p < 0.05), while similar, compared to sPCL (p = 0.64). In summary, pPCL patients showed a higher level of genetic heterogeneity and distinctive genetic signature in their mutational profile compared to patients with MM and sPCL.

Prognostic value of integrated cytogenetic, somatic variation, and copy number variation analyses in Korean patients with newly diagnosed multiple myeloma.

BACKGROUND: To investigate the prognostic value of gene variants and copy number variations (CNVs) in patients with newly diagnosed multiple myeloma (NDMM), an integrative genomic analysis was performed. METHODS: Sixty-seven patients with NDMM exhibiting more than 60% plasma cells in the bone marrow aspirate were enrolled in the study. Whole-exome sequencing was conducted on bone marrow nucleated cells. Mutation and CNV analyses were performed using the CNVkit and Nexus Copy Number software. In addition, karyotype and fluorescent in situ hybridization were utilized for the integrated analysis. RESULTS: Eighty-three driver gene mutations were detected in 63 patients with NDMM. The median number of mutations per patient was 2.0 (95% confidence interval [CI] = 2.0-3.0, range = 0-8). MAML2 and BHLHE41 mutations were associated with decreased survival. CNVs were detected in 56 patients (72.7%; 56/67). The median number of CNVs per patient was 6.0 (95% CI = 5.7-7.0; range = 0-16). Among the CNVs, 1q gain, 6p gain, 6q loss, 8p loss, and 13q loss were associated with decreased survival. Additionally, 1q gain and 6p gain were independent adverse prognostic factors. Increased numbers of CNVs and driver gene mutations were associated with poor clinical outcomes. Cluster analysis revealed that patients with the highest number of driver mutations along with 1q gain, 6p gain, and 13q loss exhibited the poorest prognosis. CONCLUSIONS: In addition to the known prognostic factors, the integrated analysis of genetic variations and CNVs could contribute to prognostic stratification of patients with NDMM.

Ultradeep Sequencing Analysis of Paroxysmal Nocturnal Hemoglobinuria Clones Detected by Flow Cytometry: PIG Mutation in Small PNH Clones.

OBJECTIVES: We aimed to determine whether small paroxysmal nocturnal hemoglobinuria (PNH) clones detected by flow cytometry (FCM) harbor PIG gene mutations with quantitative correlation. METHODS: We analyzed 89 specimens from 63 patients whose PNH clone size was ≥0.1% by FCM. We performed ultradeep sequencing for the PIGA, PIGM, PIGT, and PIGX genes in these specimens. RESULTS: A strong positive correlation between PNH clone size by FCM and variant allele frequency (VAF) of PIG gene mutation was identified (RBCs: r = 0.77, P < .001; granulocytes: r = 0.68, P < .001). Granulocyte clone size of 2.5% or greater and RBCs 0.4% or greater by FCM always harbored PIG gene mutations. Meanwhile, in patients with clone sizes of less than 2.5% in granulocytes or less than 0.4% in RBCs, PIG gene mutations were present in only 15.9% and 12.2% of cases, respectively. In addition, there was not a statistically significant positive correlation between FCM clone size and VAF or the presence or absence of a PIG mutation. CONCLUSIONS: Our results showed that in small PNH clones PIG gene mutations were present in only a small portion without significant correlation to VAF or the presence or absence of a PIG mutation.

OPENchip: an on-chip in situ molecular profiling platform for gene expression analysis and oncogenic mutation detection in single circulating tumour cells.

Liquid biopsy holds promise towards practical implementation of personalized theranostics of cancer. In particular, circulating tumour cells (CTCs) can provide clinically actionable information that can be directly linked to prognosis or therapy decisions. In this study, gene expression patterns and genetic mutations in single CTCs are simultaneously analysed by strategically combining microfluidic technology and in situ molecular profiling technique. Towards this, the development and demonstration of the OPENchip (On-chip Post-processing ENabling chip) platform for single CTC analysis by epithelial CTC enrichment and subsequent in situ molecular profiling is reported. For in situ molecular profiling, padlock probes that identify specific desired targets to examine biomarkers of clinical relevance in cancer diagnostics were designed and used to create libraries of rolling circle amplification products. We characterize the OPENchip in terms of its capture efficiency and capture purity, and validate the probe design using different cell lines. By integrating the obtained results, molecular analyses of CTCs from metastatic breast cancer (HER2 (ERBB2) gene expression and PIK3CA mutations) and metastatic pancreatic cancer (KRAS gene mutations) patients were demonstrated without any off-chip processes. The results substantiate the potential implementation of early molecular detection of cancer through sequencing-free liquid biopsy.

Mutation of ten-eleven translocation-2 is associated with increased risk of autoimmune disease in patients with myelodysplastic syndrome.

BACKGROUND/AIMS: Myelodysplastic syndrome (MDS) is caused by genetic and epigenetic alteration of hematopoietic precursors and immune dysregulation. Approximately 20% of patients with MDS develop an autoimmune disease (AID). Here, we investigated whether particular genetic mutations are associated with AID in patients with MDS. METHODS: Eighty-eight genetic mutations associated with myeloid malignancy were sequenced in 73 MDS patients. The association between these mutations and AID was then analyzed. RESULTS: The median age of the 73 MDS patients was 70 years (interquartile range, 56 to 75), and 49 (67.1%) were male. AID was observed in 16 of 73 patients (21.9%). Mutations were detected in 57 (78.1%) patients. The percentage (68.8% vs. 80.7%, p = 0.32) and the mean number of mutations (1.8 ± 1.6 vs. 2.2 ± 1.8, p = 0.34) in MDS patients with or without AID were similar. However, the ten-eleven translocation- 2 (TET2) mutation rate was significantly higher in patients with AID than in those without (31.3% vs. 5.3%, respectively; p = 0.001). All TET2 mutations were variants of strong clinical significance. CONCLUSION: Mutation of TET2 in patients with MDS may be associated with increased risk of developing AID.

The Implication of Cytogenetic Alterations in Pancreatic Ductal Adenocarcinoma and Intraductal Papillary Mucinous Neoplasm Identified by Fluorescence In Situ Hybridization and Their Potential Diagnostic Utility.

Background/Aims: We investigated chromosomal aberrations in patients with pancreatic ductal adenocarcinoma (PDAC) and intraductal papillary mucinous neoplasm (IPMN) by fluorescence in situ hybridization (FISH) to identify cytogenetic changes and molecular markers that may be useful for preoperative diagnosis. Methods: Tissue samples from 48 PDAC and 17 IPMN patients were investigated by FISH analysis using probes targeting chromosomes 7q, 17p, 18q, 20q, and 21q and the pericentromeric region of chromosome 18 (CEP18). Results: The PDAC samples harbored 17p deletion (95.8%), 18q deletion (83.3%), CEP18 deletion (81.2%), 20q gain (81.2%), 21q deletion (77.1%), and 7q gain (70.8%). The IPMN samples had 17p deletion (94.1%), CEP18 deletion (94.1%), 21q deletion (70.6%), 18q deletion (58.8%), 20q gain (58.8%), and 7q gain (58.8%). A significant difference in CEP18 gain was identified between the PDAC and IPMN groups (p=0.029). Detection of 17p or 18q deletion had the highest diagnostic accuracy (80.0%) for PDAC. Conclusions: Chromosomal alterations were frequently identified in both PDAC and IPMN with similar patterns. CEP18 gain and 17p and 18q deletions might be involved in the later stages of PDAC tumorigenesis. Chromosome 17p and 18q deletions might be excellent diagnostic markers.