Archived Cytogenetic Cell Pellets Used to Detect a BCR::ABL1 Driver Mutation Eight Years before Disease Presentation.

Evidence suggests that the earliest genetic events in the evolution of a cancer can predate diagnosis by several years or decades. In chronic myeloid leukemia (CML), the BCR::ABL1 fusion driver mutation can be present for an extended period before clinical disease manifests. The time between the BCR::ABL1 occurrence and symptom onset is referred to as the latency period. Though modeling studies predict this latency period is no more than ten years, it is still unclear how long it can be. We present a case of a patient referred for suspected CML. Both karyotype and FISH analysis identified the t(9;22)(q34;q11.2) translocation resulting in the Philadelphia chromosome formation in 98.5% of cells analyzed. The patient responded to imatinib and achieved a sustained complete hematologic and cytogenetic remission. Clinical history revealed that the same patient presented eight years previously with anemia. Various non-neoplastic conditions were excluded, and a bone marrow biopsy was performed to rule out MDS. Cytogenetic analysis at that time revealed del(20q) as the sole abnormality in all 20 cells analyzed. No treatment was given since the presence of isolated del(20q) is not considered evidence of MDS in the absence of diagnostic morphologic criteria. Retrospective FISH analysis of archived bone marrow pellets from this previous specimen revealed the presence of BCR::ABL1 in 1.8% of cells. A clonal population of cells harboring the BCR::ABL1 fusion was unambiguously detected in this patient's archived bone marrow pellet obtained eight years before the current CML diagnosis. This case demonstrates that Carnoy's fixed nuclear pellets stored in cytogenetic laboratories are suitable for detecting driver mutations years before disease presentation. Such archived material may be useful for the retrospective studies needed to better understand the initiation and subsequent development of hematological malignancies. By identifying individuals who are at increased risk, it may be possible to initiate preventive measures or begin treatment at an earlier stage before disease progression.

A treatment-refractory aggressive MDS-MLD with multiple highly complex chromosome 5 intrachromosomal rearrangements: a case report.

BACKGROUND: A patient with a myelodysplastic neoplasm exhibited a karyotype with multiple complex chromosome 5 rearrangements. This patient appeared to have a catastrophic cytogenetic event that manifested as a treatment-refractory aggressive form of disease, which lead to patient demise within one year. Both the clinical presentation and disease course were unusual based on the medical history and morphologic findings. Such cases of myelodysplastic syndrome with multilineage dysplasia (MDS-MLD) with complex abnormalities are not reported in the literature. CASE PRESENTATION: The patient was a 62-year-old female who presented with pancytopenia and dyspnea. The morphologic appearance of the peripheral blood smear and bone marrow biopsy, along with flow cytometric findings, favored the diagnosis of MDS-MLD unclassifiable. Myelodysplastic syndrome (MDS) with multilineage dysplasia (MDS-MLD), is an MDS characterized by one or more cytopenias and dysplastic changes in two or more of the myeloid lineages (i.e., erythroid, granulocytic, and megakaryocytic). The bone marrow, in particular, showed prominent dysplasia, including the presence of atypical megakaryocytes with small hypolobated morphology reminiscent of those typically seen in MDS with isolated 5q deletion. Cytogenetic analysis, including interphase and metaphase FISH, karyotype and SNP chromosomal microarray were performed, as well as DNA sequencing studies. Cytogenetic analysis showed a very complex karyotype featuring multiple 5q intrachromosomal rearrangements including a pericentric inversion with multiple interspersed deletions and monosomy 7. FISH studies showed a partial deletion of the PDGFRß gene, and SNP chromosomal microarray and targeted panel-based sequencing identified biallelic loss of function of the TP53 gene. Based on the pathologic findings, the patient was treated for MDS but did not respond to either lenalidomide or azacitidine. CONCLUSION: The genetic changes described, in particular, the complex intrachromosomal rearrangements of chromosome 5, suggest the occurrence of a sudden catastrophic event that led to an aggressive course in the patient's disease. Conventional karyotyping, metaphase and interphase FISH, SNP chromosomal microarray and NGS helped to identify the complex genetic changes seen in this case. This highlights the importance of utilizing a multimodality approach to fully characterize complex chromosomal events that may significantly impact disease progression, treatment and survival.

Spatial analysis of drug absorption, distribution, metabolism, and toxicology using mass spectrometry imaging.

Mass spectrometry imaging (MSI) is emerging as a powerful analytical tool for detection, quantification, and simultaneous spatial molecular imaging of endogenous and exogenous molecules via in situ mass spectrometry analysis of thin tissue sections without the requirement of chemical labeling. The MSI generates chemically specific and spatially resolved ion distribution information for administered drugs and metabolites, which allows numerous applications for studies involving various stages of drug absorption, distribution, metabolism, excretion, and toxicity (ADMET). MSI-based pharmacokinetic imaging analysis provides a histological context and cellular environment regarding dynamic drug distribution and metabolism processes, and facilitates the understanding of the spatial pharmacokinetics and pharmacodynamic properties of drugs. Herein, we discuss the MSI's current technological developments that offer qualitative, quantitative, and spatial location information of small molecule drugs, antibody, and oligonucleotides macromolecule drugs, and their metabolites in preclinical and clinical tissue specimens. We highlight the macro and micro drug-distribution in the whole-body, brain, lung, liver, kidney, stomach, intestine tissue sections, organoids, and the latest applications of MSI in pharmaceutical ADMET studies.

Double Robertsonian translocations in an infertile patient with macrocytic anemia: a case report.

BACKGROUND: Constitutional heterologous double Robertsonian translocations (DRT) between chromosomes 13/14 and chromosomes 14/15 with 44 chromosomes are extremely rare. In this case report, we present the karyotype analysis of metaphases prepared from bone marrow, peripheral blood and cultured skin tissue cells. These showed only 44 chromosomes with DRT involving chromosomes 13, 14 and 15. To our knowledge this is the first reported case with DRT involving chromosomes 14 and 15. CASE PRESENTATION: The patient is an 81-year-old infertile male with a history of persistent macrocytic anemia (MA). The patient presented with fatigue, paleness of the skin, shortness of breath, lightheadedness and occasional dizziness. Work-up for common causes of macrocytic anemias in this case were excluded: folate/vitamin B12 deficiency, hypothyroidism, liver diseases, hemolysis, bleeding, alcoholism, exposure, HIV infection, chemotherapy or blood loss, drug-toxicity effect, or myelodysplasia. This individual with DRT had only six nucleolus organizer regions (NORs), instead of the usual ten, of which 50% of the 6 NORs were inactive (n = 3). CONCLUSION: In this case, macrocytic anemia (MA) appeared to be due to reduction in active NORs in DRT. We postulate that the marked reduction in active NORs leads to reduction in active nucleoli formation, which may be limiting ribosomal RNA synthesis, contributing to MA. It is probable that reduction in NOR activity affected normal DNA synthesis and cellular functions.

Comparison of Purified ß-glucuronidases in Patient Urine Samples Indicates a Lack of Correlation Between Enzyme Activity and Drugs of Abuse Metabolite Hydrolysis Efficiencies Leading to Potential False Negatives.

Pain management laboratories analyze biological fluids (urine, saliva or blood) from patients treated for chronic pain to ensure compliance and to detect undisclosed drug use. The quantitation of multi-panel drugs in urine and tissues utilizes ß-glucuronidase to cleave the glucuronic acid and liberate the parent drug for mass spectrometry analysis. This work focuses on the comparison of three different, purified and commercially available ß-glucuronidases across 83 patient urine samples. One enzyme is genetically modified, expressed in bacteria and the other two enzymes are purified from abalone. The results indicate that the source of ß-glucuronidase plays an important role in substrate specificity which in turn dictates hydrolysis efficiency. Contaminants in the enzyme solutions also interfere with analyte detection. Altogether, these factors impact precision and accuracy of data interpretation, leading up to 13% positive/negative disagreement.

c-MYC Amplification in Acute Myelogenous Leukemia Evolving from Double Minutes (dmins) to Homogeneously Staining Region (hsr).

A bone marrow biopsy of a 68-year-old woman revealed 59% blasts and immature monocytes, consistent with acute myeloid leukemia (AML) with monocytic features. Occasional hypolobated megakaryocytes and decreased iron stores were also present. A peripheral blood sample showed 7% blasts in addition to monocytosis, macrocytic anemia and thrombocytopenia. Molecular testing was negative for FLT3-ITD, NPM1 and CEBPA. Fluorescence in situ hybridization (FISH) probes were negative for t(8;21), t(15;17), inversion 16 and 11q23 rearrangements. The karyotype was 46,XX,del(20)(q11.2q13.1),~50dmin[3]/47,idem,+4[13]/47,idem,+22[2]/46,XX[2]. FISH confirmed that the double minutes were c-MYC positive with cryptic deletion of the c-MYC FISH signal on one of the chromosome 8s. Two months post-diagnosis, 57% of our patient's cells were still positive for c-MYC amplification; however, by four months only 8% of cells were positive for c-MYC amplification. After seven months, the patient's karyotype had the 20q deletion, X chromosome loss and a ring chromosome that consisted of a homogeneously staining region (hsr) containing c-MYC amplification. This case demonstrates that gene amplification in the form of double minutes can transform into a more stable hsr.