FISH Still a Very Important Tool to Monitor Breast Cancer: Review of Recent Literature, Alternative Methods, and Proposed Techniques.

OBJECTIVES: Fluorescent in situ hybridization has been the definitive modality in testing for overexpression of the Human Epidermal Growth Factor Receptor 2 (HER2) for decades to guide the appropriate treatment for cancer patients. In more recent years innovation and new techniques have been developed to supplant or even replace FISH as a standard method for biomarker testing. Alternative testing methods such polymerase chain reaction (PCR), next-generation sequencing (NGS), and other in situ hybridization (ISH)-derived techniques such as chromogenic-ISH (CISH) have been shown in multiple publications to have high concordance with FISH in addition to advantages in economics, logistics and practicality to the point where CISH and derived methods appear to have eclipsed FISH as a testing method of choice after immunohistochemistry (IHC). This review assesses the status of FISH compared to other diagnostic techniques such as IHC, CISH, and less common and/or experimental methods. Also addressed are the updates to the guidelines from the American Society of Clinical Oncology (ASCO), College of American Pathologists (CAP), and National Comprehensive Cancer Network (NCCN) regarding FISH and IHC for HER2 testing with the updates reducing the number of equivocal diagnoses in the latest iteration. Though our findings show a constantly changing technological landscape, FISH remains an important primary tool to guide medical treatment and as a solid foundation to build upon for innovation in cancer research.

Fanconi Anemia, AML, and MDS.

OBJECTIVES: The Fanconi anemia (FA) genes are a family of at least 23 known genes that are spread across many chromosomes and participate in interstrand crosslink (ICLs) DNA repair. In this pathway, FA proteins are involved in sensing sites of ICLs, translocating repair enzymes from the cytoplasm to the nucleus, excising the area of damage, and facilitating repair of the fractured DNA. Mutations in these genes lead to Fanconi anemia, a syndrome characterized primarily by pancytopenia but with associated symptoms involving nearly every organ system; the majority of patients present with dermatological symptoms and growth deficits. Additionally, individuals with Fanconi anemia are known to be predisposed individuals to an increased risk of malignancies, particularly acute myeloid dystrophy and myelodysplastic syndrome, but also in the head, neck, esophagus, reproductive organs, brain, skin, liver, and kidneys. In fact, the cytogenetic aberrations seen in those with FA-associated AML differ from those in typical AML. In contrast, the cytogenetic changes seen in FA-associated MDS are similar to those in typical MDS.

ETV6::RUNX1-like Acute Lymphoblastic Leukemia.

OBJECTIVES: ETV6::RUNX1-like acute lymphoblastic leukemia (ALL) is a novel B-cell precursor leukemia subtype with similarities to ETV6::RUNX1 ALL without the presence of the ETV6-RUNX1 fusion gene. In this review, we survey the body of literature surrounding this recently categorized B-ALL type, including biomarkers, frequently associated mutations and prognosis of the disease. Identifying novel subcategories of B-ALL through high-throughput genetic analysis techniques allows for better guidance in management and more accurate prognosis.

A Case of a Patient with Therapy-related Core Binding Factor (CBF) Acute Myeloid Leukemia (CBF-AML).

OBJECTIVES: Identifying therapy-related AML (t-AML) of newly diagnosed acute leukemias is of great interest. Development of t-AML can occur after cytotoxic chemotherapy and/or radiation. We report a case of t-AML with CBFB::MYH11 fusion in a patient with a distant history of treated stage IIIB nodular sclerosing Hodgkin's lymphoma. We present the clinical course of the patient and the methods used to detect and monitor the rearrangement. Core binding factor AML (CBF-AML) after exposure to treatment is considered to be a good prognostic marker. The identification of these favorable AML subtypes such as CBF-AML highlights the importance of identifying genetic alterations, especially with increasing incidences of t-AML due to changes in choice of treatment and prognosis.

The Main Genetic-Molecular Aspects of Penile Cancer.

OBJECTIVES: Penile cancer, while relatively rare compared to other male malignancies, has seen an increased global incidence, with 36,068 new cases reported in 2020. This condition primarily affects regions with low human development indexes, notably India, China and Brazil. The mainstay of treatment is often partial or total penectomy, which has a profound impact on patients' emotional and social lives. Due to limited options for early diagnosis, non-surgical treatments, restricted healthcare funding and the negative consequences of mutilating surgeries, penile cancer is often considered a neglected disease. Penile cancer exhibits various histological types, but penile squamous cell carcinoma (SCC) is the most prevalent, accounting for 95% of cases worldwide. Multiple risk factors are associated with this condition, largely tied to lifestyle behaviors, such as promiscuous sexual behavior, zoophilia, poor hygiene, phototherapy, smoking and obesity. Human papillomavirus (HPV) infection is a significant etiological factor, particularly in squamous cell carcinomas. The prevalence of HPV in penile neoplasia varies widely, and its association with mortality remains uncertain.

The Key Role of the RPS14 Gene in Neoplasms and Solid Tumors.

OBJECTIVES: The ribosomal protein S14 (RPS14) gene located at 5q33 codes for a protein involved in ribosomal biogenesis. The RPS14 gene has a length of 5.9 kb of DNA comprising 5 exons and 4 introns. It is possible that RPS14 is involved in the formation of pre-RNA 18s, an intermediate RNA that serves for the formation of the 40S small subunit of the ribosome. RPS14 haploinsufficiency (HI) produces alterations in intermediate RNA levels (pre-RNA 30S/18SE/18S), which are found in del(5q) MDS. In addition, RPS14 haploinsufficiency results in the formation of the MDM2 (double minute mouse E3 ubiquitin ligase)-RP (ribosomal protein) complex that prevents the MDM2-p53 interaction, generating an accumulation of p53 levels. This accumulation produces cell cycle arrest, impaired DNA repair, senescence, and apoptosis. RPS14 haploinsufficiency has been seen in MDS. Altered expression levels of RPS14 have also been reported in glioma, colorectal cancer, hepatocellular carcinoma, breast cancer, renal cell carcinoma, and primary myelofibrosis.

An Isochromosome 9q: A Rare Event in Pediatric B-ALL.

OBJECTIVES: B-cell acute lymphoblastic leukemia (B-ALL) is one of the most common leukemias affecting the pediatric population. It represents ~25% of cancer diagnoses among children. Specific genetic changes predict the prognosis in B-ALL with recurrent genetic changes. Here we present a case report of a 20-year-old male with B-ALL. The patient presented with acute onset worsening upper extremity pain with pallor, weight loss, dizziness, fatigue, and abnormal complete blood count (CBC). Conventional cytogenetics showed a karyotype of 46,XY,add(9)(q13),i(9)(q10)[19]. DNA FISH analysis performed on the bone marrow showed hemizygous deletion of the 9p21(CDKN2A) in 15.5% of the nuclei examined. The presence of an isochromosome 9q [i(9)(q10) is a rare event in pediatric B-ALL. An isochromosome 9q occurs in 0.6% of the patients studied in the literature. The significance of this abnormality in pediatric B-ALL is not clear. Profiling cases like this to understand the molecular mechanisms of rare chromosomal abnormalities and rare mutations in children with B-ALL could help us to better treat them.

A B-ALL Pediatric Patient with a Cryptic IGH Rearrangement Within the Context of a Complex Karyotype.

OBJECTIVES: B-cell acute lymphoblastic leukemia (B-ALL) can afflict both adult and pediatric patients and is characterized by a build-up of B lymphoblasts. Here we present a case of a 25-year-old male patient with a history of B-ALL. Ninety percent of the bone marrow revealed pancytopenia with sheets of B lymphoblasts consistent with the diagnosis of B-ALL for acute pre-B lymphoblastic leukemia. The immunophenotype also presented predominant immature precursor B lymphoid cells positive for CD19, CD10, CD34, CD58, CD38, CD9, and TdT. Chromosome analysis of the bone marrow showed a complex karyotype described as 45~47,XY,i(8)(q10),der(10)add(10)(p11.1)add(10)(q23),-20,+1~2mar[cp3]/46,XY[36]. While IGH rearrangements were cryptic cytogenetically, DNA FISH analysis showed evidence of the IGH (14q32.2) gene rearrangement in 96.5% of the nuclei examined. These results were described as nuc ish(IGHx2)(5'IGH sep 3'IGHx1)[187/200],(5'IGH,3'IGH)x1~4(5'IGH con 3'IGHx0~2) [6/200]. The remaining probes were normal. Further studies using the MYC/IGH DC, DF probe from Abbott showed a gain of IGH signal in 7.5% of the nuclei examined: nuc ish(MYCx2,IGHx3)[15/200]. Metaphase FISH also showed that what appeared to be an isochromosome 8q was a derivative chromosome 8 defined as add(8)(p11.2) that contained a green IGH signal. In light of these results the karyotype was characterized as 45~47,XY,add(8)(p11.2),der(10)add(10)(p11.1)add(10)(q23),-20,+1~2mar[cp3].ish add(8) (p11.2) IgH+. IgH abnormalities are rare in B-ALL and are usually associated with a poor prognosis. However, at the present time our patient presented no evidence of persistent or residual disease and a cytogenetic response to the present therapy.

A Highly Complex Hyperdiploid Karyotype in a Patient with MDS: A Case Report and Review of the Literature.

OBJECTIVES: We present a case study of a 73-year-old female with a history of pancytopenia. The bone marrow core biopsy was suggestive of a myelodysplastic syndrome, unspecified (MDS-U). Chromosomal analysis of the bone marrow revealed an abnormal karyotype including gain of chromosomes 1, 4, 6, 8, 9, 19, and 20 in addition to loss of chromosomes 11, 13, 15, 16, 17, and 22. Also, additional material of unknown origin was found on 3q, 5p, 9p, 11p, 13p, 14p, and 15p; there were two copies of 19p, a deletion of 8q, and numerous unidentified rings and markers were present. This was characterized as: 75~77,XXX,+1,der(1;6)(p10;p10),add(3)(q27),+4,add(5)(p15.1),+6,+8,del(8)(q24.1),+add(9)(p24),-11,add (11) (p13),-13,add(13)(p10),add(14)(p11.2),-15,add(15)(p11.2), -16,-17,+19,add(19)(p13.3)x2,+20,-22, +0~4r,+4~10mar[cp11]/46,XX[8]. The cytogenetic analysis correlates with the concurrent FISH study which was positive for additional signals of EVI1(3q26.2), TAS2R1 (5p15.31), EGR1 (5q31.2), RELN (7q22), TES (7q31) RUNX1T1 (8q21.3), ABL1 (9q34), KMT2A (11q23), PML (15q24.1), CBFB (16q22), RARA (17q21), PTPRT (20q12), MYBL2 (20q13.12), RUNX1 (21q22.12) and BCR (22q11.2). Hyperdiploid karyotypes within the context of complex structural abnormalities are rare events usually associated with a poor prognosis in MDS.

JAK2 in Ph-like B-Acute Lymphoblastic Leukemia.

OBJECTIVES: The Janus Kinase 2 gene (JAK2) provides instructions for generating a protein that promotes the division and growth, or what is referred to as the proliferation, of cells. This generated protein relays signals in cells in order to promote cell growth, as well as help manage the count of white blood cells, red blood cells, and platelets that are generated within the bone marrow. Mutations and rearrangements of JAK2 are found in 3.5% of B-acute lymphoblastic leukemia (B-ALL) cases and in 18.9% of Down syndrome B-ALL patients, and are associated with a Ph-like ALL and a poor prognosis. However, there have been great challenges in understanding their role in this pathogenesis. In this review, we will discuss the most recent literature and trends associated with JAK2 mutations in patients with B-ALL.

A CRLF2 Rearrangement in a Pediatric Patient with B-ALL Detected by FISH Within the Context of a Complex Abnormal Karyotype.

OBJECTIVES: B-cell acute lymphoblastic leukemia (B-ALL) is one of the prevalent pediatric leukemias, accounting for 26% of cancers diagnosed in children 0-14 years of age. We present a case report of an 11-year-old girl with B-ALL. The patient was in complete remission nine months after diagnosis but passed away a month later from chemotherapy-induced hepatic failure, renal failure, and febrile neutropenia. Conventional cytogenetics showed a karyotype of 46,XX,del(5)(q31q35),add(6)(q23),del(7)(q32q36),add(11)(q23),ider(21)(q10)add(21) (q22),inc[20]. DNA FISH analysis performed on the bone marrow showed variant rearrangement of CRLF2, as well as loss of ETV6 signals and gain of RUNX1 signals. The presence of CRLF2 rearrangements within the context of a complex karyotype is often associated with CRLF2 overexpression and poor prognosis. The heterogeneity of B-ALL and the variability in the outcomes of patients that lack characteristic genetic abnormalities highlight the importance of profiling unusual genetic cases such as this one and continuing research to understand the molecular mechanisms of rarer mutations.

Amplification of RUNX1 in a Patient With AML.

OBJECTIVES: Acute myeloid leukemia (AML) is a heterogeneous disease, characterized by clonal expansion of undifferentiated myeloid precursors, leading to alterations in hematopoiesis and bone marrow failure. Characteristic chromosomal abnormalities in AML are translocations t(8;21), inv(16), t(15;17), t(9;22), as well as mutations of genes that regulate proliferation and survival (FLT 3, PTPN 11, ETV 6/PDGFB), or genes responsible for differentiation and apoptosis (RUNX-1/RUNX1T1, PML/RARA, KMT2A, CEBPA and CBFB). Amplification of RUNX1 is a rare event in AML. Herein we described a 60-year-old patient that was admitted to the hospital due to a clinical picture of symptoms of acute anemia, thrombocytopenia, leukocytosis, and profuse nasal bleeding, hepatomegaly, splenomegaly, and gallstones. The blood cell count indicated the presence of 72% blasts. The bone marrow also showed 97% of blasts of myeloid lineage. The flow cytometry study also showed findings compatible with AML (MPOneg/+, CD34+, CD19neg /+d, CD117+, CD38neg /+, HLA-DR ++, CD13neg /+, CD33neg, CD15neg, D56neg, CD123+, CD7neg, CD11bneg, CD64neg, CD41aneg, which represented 68% of the pathological cellularity). Chromosome analysis showed additional copies of an isochromosome 21q. FISH studies revealed five copies of RUNX1. Amplification of RUNX1 is a rare event in AML with only a few cases reported in the literature (mainly therapy related AML) and it is usually associated with poor prognosis.

CRLF2 Gene in B-cell Acute Lymphoblastic Leukemia.

OBJECTIVES: B-cell acute lymphoblastic leukemia (B-ALL) is a subset of ALL that comprises 75% of ALL cases. There are a variety of chromosome aneuploidy or chromosomal rearrangements implicated in B-ALL. Deregulation of CRLF2 expression is seen in 5-15% of B-ALL patients and occurs primarily via a reciprocal translocation with immunoglobulin heavy chain (IGH), rearrangements of CRLF2, deletion within the PAR1 region of the X and Y chromosomes, and CRLF2 mutations as well as mutations of the CRLF2-involved pathways and are seen in Ph-like B-ALL. They are associated with a poor prognosis. Blinatumomab is an available immunotherapy, and there are currently a few ongoing clinical trials to treat CRLF2 B-ALL. This review focuses on the role of CRLF2 in B-ALL and summarizes the literature regarding its molecular pathways, clinical significance, incidence rates across demographics, therapies, and areas of further research.

A Patient with Myxoid/Round Cell Liposarcoma (MRCL) Involving the Well-known Translocation t(12;22): A Case Report with the Cytogenomic Landscape of this Rearrangement.

OBJECTIVES: Myxoid/Round Cell Liposarcoma (MRCL) is characterized as a soft tissue sarcoma that is associated with unusual patterns of metastasis to extrapulmonary sites, such as bones and other soft tissue sites. Here, we present a case of a 48-year-old male patient, diagnosed with MRCL. The patient presented with a grade 1 myxoid liposarcoma in his left leg. DNA FISH analysis showed variant rearrangements of the EWSR1 (22q12) gene and loss of the 5' DDIT3 (CHOP 12q13) gene. The variant rearrangement showed one or two fusions with multiple separated (rearranged) signals. The EWSR1-DDIT3 rearrangement has been reported in MRCL. The variant rearrangements of the EWSR1 (22q12) gene findings correlate with concurrent conventional cytogenetic findings and were described as nuc ish(EWSR1x2) (5'EWSR1 sep 3'EWSR1x1)[128/100],(5'EWSR1,3'EWSR1)x1~3(5'EWSR1 con 3'EWSR1x1~2)[57/100]. The variant rearrangements of the DDIT3 (CHOP 12q13) gene findings were described as nuc ish(5'DDIT3x1,3'DDIT3x2)(5'DDIT3 con 3'DDIT3x1)[195/200]. Molecular cytogenetic studies also showed a rearrangement of EWSR1 (22q12) in 64% of nuclei and variant rearrangement in 31.5% of nuclei. A loss of DDIT3's (12q13) 5' signal was found in 97.5% of interphase nuclei. Molecular pathology results indicated the patient was positive for EWSR1 (exon 7) and DDIT3 (exon 2) fusion. The patient underwent radiation therapy pre-resection of the myxoid liposarcoma. The most common form of MRCL is associated with t(12;16)(q13;p11), leading to FUS-CHOP and EWS-CHOP fusion proteins acting as aberrant transcription factors. The key element here is that this EWSR1-DDIT3 rearrangement led to a translocation t(12;22)(q13;q12) which is a rare cytogenetic event that led to the development of MRCL in this patient.

Molecular Cytogenetic Characterization of a Structural Abnormal Chromosome 16 in a Patient with Acute Myeloid Leukemia Leading to Inversion Chromosome 16 with Concomitant 3'CBFB Deletion.

OBJECTIVES: Acute myeloid leukemia (AML) presents as a heterogeneous blood cancer characterized by the proliferation of immature myeloid cells. We present the case of an 18-year-old female with AML whose symptoms include marked leukocytosis, anemia, as well as thrombocytopenia with spontaneous cerebellar and intracerebral bleeds. The bone marrow biopsy is hypercellular and is expunged by sheets of blast cells with dispersed chromatin, prominent nucleoli, highly irregular nuclei, and moderate cytoplasm. Chromosome analysis reveals an abnormal karyotype with a derivative trisomy 8 and a derivative chromosome 16. The karyotype is described as 47,XX,+der(8)add(8)(q24.3),der(16) inv(16)(p13.1q22)del(16)(q22)[21]/46,XX[1]. DNA FISH analysis reveals abnormalities for RUNX1T1 (8q21.3) and CBFB (16q22) genes. These findings align with that of conventional cytogenetics. The National Comprehensive Cancer Network guidelines for AML state that CBFB gene rearrangements indicate that the patient falls under the favorable risk category. However, AML with core binding factor molecular aberrations is a heterogeneous group and thus the interaction with further cytogenetic abnormalities may result in further pathogenesis. Clinical correlation was suggested.

EGFR Amplification in a Patient with Glioblastoma: A Case Report and Review of the Literature.

OBJECTIVES: Glioblastoma Multiforme (GBM) is the most malignant and frequently occurring primary brain tumor out of the different types of primary astrocytomas. It presents with an extremely poor prognosis, with a median survival of 14 to 15 months from the diagnosis. Herein, we present an 83-year-old female patient with a right frontal brain mass. A craniotomy for the frontal brain mass was performed, which revealed a tumor with high-grade glioma, necrosis, atypia, and vascular proliferation. The patient was subsequently diagnosed with Glioblastoma Multiforme Grade IV (GBM). Molecular cytogenetic studies showed an amplification of the EGFR gene in 100% nuclei scored. Amplification of EGFR appears in around 40-50% of individuals with Glioblastoma Multiforme Grade IV, leading to high levels of EGFR protein levels that contribute to tumorigenesis. Chromosomal deletions involving 1p36 and 19q13 are characteristic molecular features of solid tumors such as oligodendrocytes and mixed oligoastrocytomas, but in this case there was no evidence of a co-deletion of 1p36/19q13 in this case of glioblastoma.

NMYC Amplification in Neuroblastoma: The Molecular Landscape.

OBJECTIVES: Neuroblastoma remains one of the most clinically diverse cancers common in pediatric patients. An important prognostic indicator for neuroblastoma involves the NMYC gene, which is the differentiating factor between high-risk and low-risk disease; the five-year survival rates for patients with and without NMYC mutations are 40% and 95%, respectively. This review assesses our current understanding of the molecular role and function of NMYC in risk stratification and disease progression and highlights key areas of research to improve existing and identify novel targets for neuroblastoma treatments.

Amplification of CCND1 in Urothelial Carcinoma.

OBJECTIVES: Urothelial carcinoma (UC) is the most prevalent form of bladder cancer and a significant cause of mortality in the world each year. As molecular genetic techniques improve, researchers and medical professionals are turning toward finding potential biomarkers to diagnose and characterize UC, guide treatment decisions, and use as therapeutic targets. Located on chromosome 11q13.2, the CCND1 gene encodes Cyclin D1, a CDK-regulating protein that plays a critical role in cell cycle progression. Amplification of CCND1 is seen in about 10% of all bladder cancer patients and has been a target of research due to its potential as a prognostic biomarker and a therapeutic target. However, existing literature on CCND1 amplification and Cyclin D1 expression report conflicting information about their clinical significance and association with disease staging, pointing to the need for more research to determine mechanistic pathways. Additionally, while there are currently no approved therapies or drugs that directly target CCND1 or Cyclin D1 in UC, several clinical trials with drugs targeting CDK4/CDK6 in the Cyclin D1 pathway are already underway. This paper aims to provide an update on the amplification of CCND1 in urothelial carcinoma, including an overview of recent research on elucidated pathways, clinical significance, relevant therapies under development, and directions for future research.

Monosomy 21, a Sole Abnormality in an Elderly Man with Non-CLL-Type Monoclonal B-cell Lymphocytosis.

OBJECTIVES: Monoclonal B-cell lymphocytosis (MBL) is a light-chain restricted proliferation of mature B cells fewer than 5000 cells/µL without additional clinical or hematologic abnormalities. Sibling studies of individuals genetically susceptible to chronic lymphocytic leukemia (CLL) first identified monoclonal B cells in otherwise healthy persons, and studies show a 3% to 14% prevalence for MBL in persons over 40 years of age. Non-CLL-type MBL accounts for less than 20% of all MBL cases, and its progression is incompletely characterized. Here we present the case of an 85-year-old man with CD5-, CD19+, CD20 bright, and lambda-restricted lymphoid cells whose immunophenotypic findings are suggestive for a precursor lesion to marginal zone lymphoma (MZL). Karyotyping showed monosomy 21 without additional cytogenetic changes in three of the 35 cells examined. Monosomy 21 as a sole abnormality in CLL has been detected in just 11 cases between 1984 and 2003. As a sole abnormality in splenic and nodal marginal zone lymphoma, only three instances of monosomy 21 have been recorded on the Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer. The significance of monosomy 21 as a marker for oncogenesis remains unclear.

c-MYC Amplification in AML.

OBJECTIVES: Acute myeloid leukemia (AML) is a clonal disorder of myeloid lineage precursors. Identification of cytogenetic aberrations is essential for classification and risk stratification of AML, with many demonstrating unique associations with various clinicopathologic features. One such abnormality is MYC amplification, a rare occurrence identified in less than 1% of AML patients. MYC is most commonly amplified in the form of double minutes, but may also occur via ring and marker chromosomes or homogeneously staining regions. Amplification of MYC often involves various chromosomal aberrations, including trisomies 4 and 6 and aneusomy of the sex chromosomes. In many cases, the presence of MYC amplicons is also associated with other negative prognostic factors, including complex karyotype and advanced age. Although MYC has been extensively investigated as a therapeutic target in various cancers, there are few studies examining the clinical significance of MYC amplification in AML. In this review, we explore recurrent cytogenetic abnormalities and demographic characteristics associated with amplification of MYC in patients with AML and discuss their diagnostic and therapeutic implications.