Natural history of clonal haematopoiesis seen in real-world haematology settings.

Recursive partitioning of healthy consortia led to the development of the Clonal Hematopoiesis Risk Score (CHRS) for clonal haematopoiesis (CH); however, in the practical setting, most cases of CH are diagnosed after patients present with cytopenias or related symptoms. To address this real-world population, we characterize the clinical trajectories of 94 patients with CH and distinguish CH harbouring canonical DNMT3A/TET2/ASXL1 mutations alone ('sole DTA') versus all other groups ('non-sole DTA'). TET2, rather than DNMT3A, was the most prevalent mutation in the real-world setting. Sole DTA patients did not progress to myeloid neoplasm (MN) in the absence of acquisition of other mutations. Contrastingly, 14 (20.1%) of 67 non-sole DTA patients progressed to MN. CHRS assessment showed a higher frequency of high-risk CH in non-sole DTA (vs. sole DTA) patients and in progressors (vs. non-progressors). RUNX1 mutation conferred the strongest risk for progression to MN (odds ratio [OR] 10.27, 95% CI 2.00-52.69, p = 0.0053). The mean variant allele frequency across all genes was higher in progressors than in non-progressors (36.9% ± 4.62% vs. 24.1% ± 1.67%, p = 0.0064). This analysis in the post-CHRS era underscores the natural history of CH, providing insight into patterns of progression to MN.

Prognostic heterogeneity and clonal dynamics within distinct subgroups of myelodysplastic syndrome and acute myeloid leukemia with TP53 disruptions.

TP53 aberrations constitute the highest risk subset of myelodysplastic neoplasms (MDS) and acute myeloid leukemia (AML). The International Consensus Classification questions the blast threshold between MDS and AML. In this study, we assess the distinction between MDS and AML for 76 patients with TP53 aberrations. We observed no significant differences between MDS and AML regarding TP53 genomics. Median overall survival (OS) was 223 days for the entire group, but prognostic discrimination within subgroups showed the most inferior OS (46 days) for AML with multihit allelic state plus TP53 variant allele frequency (VAF) > 50%. In multivariate analysis, unadjusted Cox models revealed the following variables as independent risk factors for mortality: AML (vs. MDS) (hazard ratio [HR]: 2.50, confidence interval [CI]: 1.4-4.4, p = 0.001), complex karyotype (HR: 3.00, CI: 1.4-6.1, p = 0.003), multihit status (HR: 2.30, CI 1.3-4.2, p = 0.005), and absence of hematopoietic cell transplant (HCT) (HR: 3.90, CI: 1.8-8.9, p = 0.0009). Clonal dynamic modeling showed a significant reduction in TP53 VAF with front-line hypomethylating agents. These findings clarify the impact of specific covariates on outcomes of TP53-aberrant myeloid neoplasms, irrespective of the diagnosis of MDS versus AML, and may influence HCT decisions.

Clinico-genomic profiling and clonal dynamic modeling of TP53-aberrant myelodysplastic syndrome and acute myeloid leukemia.

TP53-aberrant myelodysplastic syndrome and acute myeloid leukemia have dismal outcomes. Here, we define the clinico-genomic landscape of TP53 disruptions in 40 patients and employ clonal dynamic modeling to map the mutational hierarchy against clinical outcomes. Most TP53 mutations (45.2%) localized to the L3 loop or LSH motif of the DNA-binding domain. TP53 disruptions had high co-occurrence with mutations in epigenetic regulators, spliceosome machinery, and cohesin complex and low co-occurrence with mutations in proliferative signaling genes. Ancestral and descendant TP53 mutations constituted measurable residual disease and fueled relapse. High mutant TP53 gene dosage predicted low durability of remission. The median overall survival (OS) was 280 days. Hypomethylating agent-based therapy served as an effective bridge to transplant, leading to improved median OS compared to patients who did not receive a transplant (14.7 vs. 5.1 months). OS was independent of the genomic location of TP53 disruption, which has implications for rational therapeutic design.

Elderly do benefit from induction chemotherapy: High dose mitoxantrone-based ("5 + 1") induction chemotherapy regimen in newly diagnosed acute myeloid leukemia.

An intensive "5 + 1" regimen, which included bolus high dose cytarabine (HiDAC) at 3 g/m2 once daily over 3 hours on days 1-5 and high dose mitoxantrone (HDM) 80 mg/m2 on day 2, was evaluated in 101 consecutively treated newly diagnosed acute myeloid leukemia (AML) patients at a single center since 2009. The median age was 65 (range 18-90) years. The 4 and 8-week mortality in our cohort was 3/101 (2.9%) and 7/99 (7%), respectively. The overall response (complete remission [CR] + CRi) was 76.2% (77/101). The median overall survival (OS) stratified by age group <60, 60-69 and ≥70 years were 56, 31 and 9 months respectively (log-rank, P = 0.02). 51.7% (45/84) of patients with intermediate/adverse risk category proceeded to allogeneic stem cell transplants. Among these 84 patients, the percentage of patients able to proceed to transplant in age groups <60, 60-69, and ≥ 70 years were 75% (18/24), 60.7% (17/28), and 31.2% (10/32), respectively. In conclusion, HDM-based chemotherapy regimen produces high CR rates, is well tolerated and more patients can undergo curative postremission therapy including stem cell transplant.

Assessing copy number aberrations and copy-neutral loss-of-heterozygosity across the genome as best practice: An evidence-based review from the Cancer Genomics Consortium (CGC) working group for chronic lymphocytic leukemia.

The prognostic role of cytogenetic analysis is well-established in B-cell chronic lymphocytic leukemia (CLL). Approximately 80% of patients have a cytogenetic aberration. Interphase FISH panels have been the gold standard for cytogenetic evaluation, but conventional cytogenetics allows detection of additional abnormalities, including translocations, complex karyotypes and multiple clones. Whole genome copy number assessment, currently performed by chromosomal microarray analysis (CMA), is particularly relevant in CLL for the following reasons: (1) copy number alterations (CNAs) represent key events with biologic and prognostic significance; (2) DNA from fresh samples is generally available; and (3) the tumor burden tends to be relatively high in peripheral blood. CMA also identifies novel copy number variants and copy-neutral loss-of-heterozygosity (CN-LOH), and can refine deletion breakpoints. The Cancer Genomics Consortium (CGC) Working Group for CLL has performed an extensive literature review to describe the evidence-based clinical utility of CMA in CLL. We provide suggestions for the integration of CMA into clinical use and list recurrent copy number alterations, regions of CN-LOH and mutated genes to aid in interpretation.

Rare gene fusion rearrangement SPTNB1-PDGFRB in an atypical myeloproliferative neoplasm.

The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia recognizes a distinct class of myeloid and lymphoid tumors with eosinophilia-related proliferations associated with specific gene rearrangements, one of which involves rearrangements of platelet-derived growth factor receptor B (PDGFRB) gene. We report a case of a rare PDGFRB rearrangement with SPTNB1 (spectrin beta, nonerythrocytic 1) that presented as atypical myeloproliferative neoplasm.

Mammary Analogue Secretory Carcinoma of the Thyroid Mimicking Locally Advanced Papillary Thyroid Carcinoma: A Rare Case Report.

Mammary analogue secretory carcinoma (MASC) harboring ETV6 gene rearrangements was first described in the salivary gland with a relatively favorable prognosis and a possible molecular therapeutic target with pan-Trk inhibitors. Recently, primary MASC of the thyroid gland has been reported. We report a case of a 4.0 cm MASC arising from the left thyroid of a 58-year-old female with extrathyroidal extension. Initially, it was diagnosed by fine needle aspiration as suspicious for papillary thyroid carcinoma (PTC) and subsequently called a poorly differentiated carcinoma on resection. A final diagnosis of primary MASC of the thyroid was confirmed after an expanded immunohistochemical panel and identification of an ETV6 gene rearrangement by fluorescence in situ hybridization. Morphologically, the tumor was composed of solid, microcystic and focally papillary growth with dense fibrotic stroma and necrosis. Overlapping cytological features with PTC were identified, including foci of enlarged cells with irregular nuclear membranes/grooves. However, most of the cells contained prominent nucleoli with intraluminal and intracytoplasmic eosinophilic secretions. Immunohistochemically, the tumor cells were strongly positive for pancytokeratin, cytokeratin 7, PAX8, mammaglobin, and GCDFP-15, with rare staining for GATA3 and S100 and negative for TTF-1 and thyroglobulin. We report a rare case of a primary thyroid MASC, initially misdiagnosed as PTC. Pathologists should be aware of this entity and, given the similarities to PTC, have a high index of suspicion, prompting the addition of immunohistochemical and molecular studies. Furthermore, an accurate diagnosis is important because of the possible prognostic and treatment implications.

Improved detection rate of cytogenetic abnormalities in chronic lymphocytic leukemia and other mature B-cell neoplasms with use of CpG-oligonucleotide DSP30 and interleukin 2 stimulation.

Detection of cytogenetic abnormalities requires successful culture of the clonal population to obtain metaphase chromosomes for study, and as such, has been hampered by low mitotic indices of mature B cells in culture. Our study presents data on the improved abnormality detection rate with the use of a CpG-oligonucleotide/interleukin 2 (OL/IL-2) culture protocol for mature B-cell neoplasms, including chronic lymphocytic leukemia (CLL) and non-CLL specimens. The increased detection rate of abnormalities, compared with unstimulated culture and traditional pokeweed mitogen culture, was statistically significant for both CLL and non-CLL neoplasms. For CLL specimens, our data also showed that for cytogenetically visible aberrations, OL/IL-2 was as, if not more, sensitive than detection with interphase fluorescence in situ hybridization (iFISH). Use of OL/IL-2 allowed a number of abnormalities to be detected, which were not covered by specific iFISH panels, especially balanced translocations. Therefore, OL/IL-2 stimulation improves diagnostic sensitivity and increases discovery rate of novel prognostic findings.

Systemic mastocytosis with associated clonal hematological non-mast cell lineage disease: clinical significance and comparison of chomosomal abnormalities in SM and AHNMD components.

Some patients with systemic mastocytosis have concurrent hematological neoplasms, designated in the World Health Organization (WHO) classification as systemic mastocytosis with associated clonal hematological non-mast cell lineage disease (SM-AHNMD). In this study, we analyzed 29 patients with SM-AHNMD and compared them to 40 patients with pure SM. The AHNMDs were classified as chronic myelomonocytic leukemia (CMML) (n = 10), myelodysplastic syndrome (MDS) (n = 7), myeloproliferative neoplasms (n = 4), B-cell lymphoma/leukemia/plasma cell neoplasms (n = 7), and acute myeloid leukemia (n = 1). Patients with SM-AHNMD were older, more frequently had constitutional symptoms and hematological abnormalities, less often had skin lesions, and had an inferior overall survival compared with pure SM patients (48 months vs. not-reached, P < 0.001). Karyotypic abnormalities were detected in 9/28 (32%) patients with SM-AHNMD but not in pure SM patients (P < 0.001). Combined imaging/ fluorescence-in-situ hybridization performed in four SM-AHNMD cases revealed shared abnormal signals in mast cells and myeloid cells in two patients with SM-CMML and one patient with SM-MDS, but not in the mast cells of a case SM-associated with chronic lymphocytic leukemia with ATM-deletion. Quantitative mutation analysis showed higher levels of mutant KIT D816V in SM-CMML and SM-MDS than in pure SM (P < 0.001). Our data indicate that the SM-AHNMD category in the WHO classification is heterogeneous, including clonally related and unrelated forms of AHNMD. The presentation, treatment, and outcome of patients with SM-AHNMD is often dictated by the type of AHNMD.

Chronic basophilic leukemia: a rare form of chronic myeloproliferative neoplasm.

Chronic basophilic leukemia is a rare and poorly characterized entity. Only a limited number of cases have been described. Herein, we report a patient who presented with fatigue, weight loss, leukocytosis, persistent prominent basophilia, and mild eosinophilia. The bone marrow showed features characteristic of a myeloproliferative neoplasm with a marked increase in maturing basophils. The basophils exhibited nuclear hypersegmentation, abnormal granulation, and abnormally low CD38 expression. Conventional karyotyping revealed a t(5;12)(q31;p13). ETV6 but not PDGFRB rearrangement was detected by fluorescence in situ hybridization.

Interphase FISH in plasma cell dyscrasia: increase in abnormality detection with plasma cell enrichment.

Historically, cytogenetic studies of plasma cell neoplasms have been hampered by the fact that terminally differentiated plasma cells do not proliferate well in vitro. Although the use of interphase FISH (iFISH) has greatly improved the ability to detect cytogenetic abnormalities, cases with low numbers of neoplastic cells often do not demonstrate abnormalities. Using a four-assay, nine-probe iFISH panel, we compared the abnormality detection rate for overnight unstimulated bone marrow cultures (ONC) to that for plasma-cell enriched fractions obtained with use of CD138-coated immunomagnetic beads (PCE). In the ONC, an abnormality was detected in 11 of 29 cases (38%); in the PCE, an abnormality was detected in 30 of 33 cases (91%). For 28 cases in which iFISH results from ONC were compared directly with PCE samples, the overall abnormality rate was 36% for ONC and 89% for PCE (P < 0.01). The conventional GTG-banded chromosome analysis revealed only 2 of 34 cases with an abnormal karyotype (6%); both cases were hyperdiploid. We conclude that the plasma cell enrichment step for iFISH should be incorporated into the routine cytogenetic work-up for all patients with plasma cell neoplasms.

Cytogenetic abnormalities in a series of 1,029 patients with primary myelodysplastic syndromes: a report from the US with a focus on some undefined single chromosomal abnormalities.

BACKGROUND: Conventional karyotype has an established role in myelodysplastic syndrome (MDS) and is included in the International Prognostic Scoring System (IPSS) for patient risk stratification and treatment selection. Although some chromosomal abnormalities have been well characterized, the significance of several miscellaneous, infrequent, single chromosomal abnormalities remains to be defined. In addition, the emerging therapeutic agents may change the natural course of disease in patients with MDS and the cytogenetic impact on risk stratification. METHODS: Clinicopathologic data were retrieved on 1029 patients who had a diagnosis of primary MDS and had available cytogenetic data (karyotype) on file. RESULTS: Cytogenetic abnormalities were identified in 458 patients (45%) and occurred most frequently in patients who had refractory anemia with excess blasts (62%). Overall, the 3 cytogenetic risk groups defined by the IPSS -- good, intermediate, and poor -- effectively stratified the patients' overall survival (OS) (64 months, 31 months, and 12 months, respectively; P < .001). With the exception of gain of chromosome 8, single cytogenetic abnormalities within the intermediate group were extremely infrequent in the series but demonstrated variable OS ranging from 10 months for patients who had isochromosome (17q) to 69 months for patients who had deletion of 12p [del(12p)], suggesting different prognostic significance. In the poor cytogenetic risk group, patients with isolated del(7q) and derivative (1;7)(q10;p10) had a significantly better median OS than patients who had either loss of chromosome 7 or a complex karyotype (P < .05). CONCLUSIONS: The current data generated from a large cohort of patients with primary MDS indicated that some specific cytogenetic abnormalities carry different risk than their IPSS cytogenetic risk-group assignment, especially in the new treatment era. Because of the extreme low frequency, additional combined studies are needed to better categorize some rare single cytogenetic abnormalities within the intermediate cytogenetic risk group.

Mosaic tetraploidy and transient GFI1 mutation in a patient with severe chronic neutropenia.

This report presents the case of a 15-year-old male with severe chronic neutropenia, leukopenia, and persistent tetraploid mosaicism in the bone marrow and peripheral blood. His father had mild neutropenia and bone marrow tetraploidy. Flow cytometric analysis of DNA content peripheral blood showed tetraploidy in 20% of granulocytes and 15% of monocytes. Sequence analysis of the ELA2 gene was normal, but the GFI1 gene exhibited transient appearance of single base changes the coding region and promoter. We speculate that an underlying genetic defect, inherited in an autosomal dominant pattern, leads to both disordered mitosis and neutropenia in this kindred.

Validation of DNA probes for preimplantation genetic diagnosis (PGD) by fluorescence in situ hybridization (FISH) R1.

BACKGROUND: Preimplantation genetic diagnosis (PGD) by fluorescence in situ hybridization (FISH) is being employed increasingly by medical centers and private companies. Validation of any clinical assay, particularly one with novel applications such as PGD by FISH, is of critical importance in the clinical setting. This importance is recognized by both the College of American Pathologists (CAP) and the American College of Medical Genetics (ACMG), who recommend validation of FISH assays in the clinical setting. Validation of FISH assays for PGD is especially significant, as only one or two cells (blastomeres) will be available for testing of a given embryo. METHODS: We have developed validation protocols for a variety of FISH assays, including sex identification, structural chromosomal aneusomy, and aneuploidy screening with the Vysis, Inc., PGT probe panel. RESULTS: Our validation results show good individual performance of commercially available probes, and decreasing overall efficiency as the number of probes included in an assay increases.