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1710 Part XI: Malignant Lymphoid Diseases Chapter 105: Plasma Cell Neoplasms: General Considerations 1711

p18 INK4a and p18 INK4c can inhibit the regulatory effects of RB and result Cytogenetic Microarrays
in cell-cycle release. Decreased expression of the two proteins is consid- Fluorescence in situ hybridization (FISH) has become the gold standard
ered to be a late disease progression event. 76 for the detection and classification of myeloma. However, FISH can-
not provide information about chromosomal abnormalities without the
Genetic Expression Changes in Myeloma use of large scale panels of probes. Comparative genomic hybridization
Myeloma is genetically heterogeneous and more closely resembles solid (CGH) arrays overcome some of the short falls of FISH technology by
tumors than other hematologic malignancies. Because of its high degree providing a genome-wide view of chromosomal changes, but do so at a
of tumor heterogeneity, myeloma gene-expression microarrays have reduced resolution of 10 to 20 megabases. To combat the low resolution
proven invaluable to our understanding and treatment of myeloma. of CGH arrays, small nucleotide polymorphism (SNP)-based technol-
Four stratification models exist designed to classify myeloma based on ogy has been employed. SNP arrays can detect copy number changes
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the risk profile as determined by gene-expression profiles. Of the four, and have improved the resolution of CGH arrays to a submegabase
one model has proven most reliable, as it retains its prognostic relevance level. An early report validated the use of SNP arrays by comparing
with newer therapeutic regimens. This model compares the expression its results to those obtained by FISH analysis using identical samples.
of 70 different genes to devise a scoring system that ranks a sample as Furthermore, uniparental disomy (UPD) was identified as prevalent in
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high risk (13 percent of patients) or low risk. Abnormalities of expres- myeloma samples and may occur through several mechanisms, includ-
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sion that map to chromosome 1 are disproportionately represented ing mitotic nondisjunction and mitotic recombination. A technical
within the model, with an impressive number of upregulated genes advance was the introduction of the Affymetrix Cyto Scan HD arrays.
mapping to 1q and a high percentage of downregulated genes mapping The Cyto Scan arrays incorporate the most up-to-date SNP library to
to 1p. The 70-gene model offers a reliable method for the detection of generate a 2.6-million probe library across the human genome, allow-
high-risk myeloma. However, it does not take into account all important ing for resolution up to 50 kb. The technology has not been published
genes related to myeloma. One important gene not found in this model in a myeloma study yet, but the resolution matches that of FISH and
is MYC, which is a transcription factor that influences the expression can provide a wealth of knowledge related to changes in copy number,
of many genes through the binding of consensus sequences within the mosaic chromosomes and loss of heterozygosity. Using these technolo-
noncoding region of genes. It is thought to regulate the expression of 15 gies numerous cytogenetic changes were identified that may be impor-
percent of all genes. MYC expression within myeloma and monoclonal tant for the development and progression of monoclonal gammopathy
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gammopathy varies depending on the state of the disease. In high-risk to myeloma.
monoclonal gammopathy and early stages of myeloma, the increase in
MYC expression is primarily a result of a decrease in transcriptional TECHNIQUES TO ASSESS CYTOGENETIC
regulation, whereas in late disease states, the 8q region encoding
MYC translocates to the immunoglobulin loci, resulting in dysregula- INFORMATION
tion of expression because of the influence of neighboring regulatory
elements. 32 FLUORESCENCE IN SITU HYBRIDIZATION
In PCNs, FISH has emerged as one of the most useful and reliable meth-
Drug Resistance ods for the detection of chromosomal abnormalities and performing
Refractory myeloma has several causes, with dysregulation of gene risk assessment on newly diagnosed patients. G-banding cytogenetic
expression contributing significantly to the overall development of drug analysis requires actively dividing cells for identification of chromoso-
resistance. The serine/threonine kinase NEK2 induces drug resistance mal changes, not commonly seen in plasma cells, whereas FISH anal-
in myeloma through the activation of efflux drug pumps. Overexpres- ysis can overcome this limitation to an extent and detect structural
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sion of NEK2 results in the activation of the AKT pathway and subse- abnormalities in non-dividing plasma cells. A panel of FISH probes is
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quently of NF-κB, which results in the upregulation of ABC drug efflux used to query for commonly identified cytogenetic changes, including
transporters. Overexpression of the antiapoptotic molecule MCL-1 t(4;14), t(11;14), t(14;16), t(14;20), t(6;14), del(13q14), and del(17p13),
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induces drug resistance through the inhibition of apoptosis. More spe- and probes for hyperdiploidy (5, 7, 9, 11, 15, and 17). Cytogenetic abnor-
cifically, MCL-1 overexpression results in the inhibition of the proapop- malities, using FISH, are observed in more than 90 percent of myeloma
totic BCL-2 family members, which results in blocking apoptosis. patients. Panels like the one described here provide essential informa-
tion and paired with gene-expression profiles provides clinicians with
Next-Generation Sequencing essential data for diagnosis and the development of treatment regimens.
Next-generation sequencing or deep sequencing relies on the ability to
sequence large amounts of small DNA fragments quickly and assemble
the output into a complete coherent data set. This technique is providing METAPHASE CYTOGENETICS
new insights into our understanding of existing neoplastic mechanisms Approximately 30 percent of patients with newly diagnosed myeloma
and identifying novel mechanisms and genes that had evaded detection present with chromosomal abnormalities by metaphase cytogenetics,
thus far. One of the first reports to use next-generation sequencing was and this percentage increases to 50 percent in patients with relapsed
from a study that compared 38 whole-tumor genomes from patients myeloma. Cytogenetic changes broadly divide patients into one of two
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with myeloma to matched normal DNA. Novel genes involved in categories: patients with hyperdiploid chromosomal profile and non-
histone methylation, protein translation and blood coagulation were hyperdiploid, encompassing hypodiploid and hypotetraploid cases.
identified. Mutations to the toll-like receptor (TLR) 4, the adaptor mol- Hyperdiploid cytogenetic profiles are characterized by trisomies of
ecule TRAF3, and to the kinases CYLD, RIPK4, and BTRC also were many odd-numbered chromosomes, namely 3, 5, 7, 9, 15, 19, and 21
discovered. A greater-than-anticipated change in NF-κB was observed, and are associated with a favorable outcome. Nonhyperdiploid profiles
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including identifying 11 mutated genes involved in the NF-κB pathway. are highly enriched with translocations at the IGH loci (14q32) with
Furthermore, the prognostic value of next-generation sequencing was partner chromosomes, most importantly resulting in t(4;14), t(6;14),
tested by using the sequencing technology to detect minimal residual t(11;14), t(14;16), and t(14;20). However, many of the translocations
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disease (MRD) in myeloma patient samples. 95 result in the activation of cell-cycle regulators and oncogenes like cyclin

Kaushansky_chapter 105_p1707-1720.indd 1711 9/18/15 9:44 AM