Bone marrow tests

Myeloma usually grows inside the bone marrow. Bone marrow tests (aspirate and biopsy) are performed routinely to diagnose multiple myeloma and are also used for monitoring during the course of treatment. A bone marrow biopsy provides: 

• information about the amount of disease
• its aggressiveness
• molecular/genetic abnormalities that help predict the disease course

Bone marrow biopsies are necessary because they provide the only direct access to tumor cells for examination. Such biopsies do not always present an accurate sample of what is occurring elsewhere in the marrow; myeloma is patchy and is not distributed evenly throughout the bone marrow. 

Other tissue biopsies are performed less frequently to determine if myeloma is present outside the bone marrow. A biopsy may also be performed in early-stage disease on a solitary plasmacytoma (myeloma composed of plasma cells).

Bone Marrow Aspiration and Core Biopsy

Bone marrow aspiration and bone marrow biopsy are routinely performed at diagnosis. These tests are ordered at the doctor’s discretion at the following intervals: 

• after treatment 
• annually during periods of remission 
• at other points when the doctor deems it necessary to determine a patient’s status 

Bone marrow biopsy is also a reliable way to monitor the status of patients with oligosecretory, or non-secretory myeloma. Also, the Freelite test can be used in some such cases. 

The bone marrow is composed of both solid and liquid matter: 

• The solid part is a sponge-like structure consisting of a fibrous network filled with liquid. 
• The  liquid portion contains the following: 
  • blood-making (hematopoietic) stem cells
  • blood cells in various stages of maturation
  • raw materials required for cell production (i.e., iron, folate, and vitamin B12) 

Plasma cell percentage

The normal range is  1%–2% (Note: < 5% is complete response, or CR).

The pathologist will examine the bone marrow aspirate or core under the microscope and determine the percentage of plasma cells in the sample:

• Normal bone marrow has about 2% or fewer plasma cells.
• The presence of 60% or more plasma cells in the bone marrow is an independent myeloma-defining event.

Myeloma cells aren’t distributed evenly throughout the bone marrow in the skeleton. For that reason, the iliac crest is most often chosen as the biopsy site. It provides the best representative sample. 

Plasma cell morphology

The appearance of myeloma cells is distinct, with large nuclei that make the cells look like pimento-stuffed olives. The pathologist records the appearance and number of these cells. Words such as “mature,” “immature,” or “atypical” are used to describe the plasma cells. Generally, “mature” cells suggest a better prognosis than “immature” or “atypical” plasma cells.

Specimen quality 

By assessing the quality and condition of the sample under the microscope, your doctor can judge if the sample is representative of what is going on in the bone marrow.

Next, the samples collected through aspiration and core biopsy undergo the following assessments:

Immunohistochemistry (also known as Immunophenotyping) and Flow Cytometry

Immunohistochemistry (IHC), also called immunophenotyping, is the process of detecting antigens in tissue samples by introducing antibodies that bind to them.

IHC is one of the tests used to determine stringent complete response (sCR) to therapy as defined by the IMWG Uniform Response Criteria. In addition to the criteria for complete response (CR), the IMWG criteria for sCR include a normal free light chain ratio and the absence of clonal plasma cells in the bone marrow by immunohistochemistry or immunofluorescence.

Immunophenotypic analysis of a myeloma patient’s bone marrow identifies myeloma protein markers, if they are present. A fluorophore, or fluorescent marker, is attached to each antibody. It glows when it finds the correct antigen on the surface of the myeloma cells. Several antibodies are usually used simultaneously. The fluorophores are given different colors (fluorochrome) for each antibody. The bone marrow sample cells and selected antibodies are sent through a flow cytometer.

A flow cytometer is a laser-based instrument that reads the fluorophores and identifies and sorts the myeloma cells.

Next Generation Flow (NGF) cytometry is 

• a highly accurate way to detect minimal residual disease (MRD) after treatment
• uses many antibodies and myeloma-specific computer software to rapidly perform eight-color immunophenotyping of myeloma cells
• can detect 1 myeloma cell per approximately 1,000,000 bone marrow cells in a laboratory sample

Cytogenetics, (also known as Karyotyping)

Human beings have two copies of each of their 23 chromosomes in every cell in their bodies. Standard cytogenetics is the assessment of the chromosomes in dividing cells after a brief culture in the laboratory.

Since the active growth rate of myeloma cells is usually fewer than 3%, this provides an incomplete assessment of chromosomal changes.

If abnormalities are noted, they are important. These abnormalities appear in the few cells that are actually growing.

When can cytogenetic testing be performed?

• This test may be performed on the bone marrow of newly diagnosed myeloma patients.
• It can be performed again after treatment to see if the therapy has eliminated all the cells with chromosomal abnormalities.
• It may also be performed at relapse to help determine
  • if it is time to resume therapy, and
  • if one therapy might be preferable to another.

In the laboratory, after the bone marrow specimen is allowed to grow in a special dish, the cells are stained and photographed to provide a karyotype that shows the number and arrangement of the chromosomes.

The chromosomes can be karyotyped only if the cells are undergoing division. Karyotyping is particularly valuable for

• identifying higher-than average-risk myeloma in patients with fewer than two copies of each chromosome (hypodiploidy)
• in those whose 13th chromosome is partially deleted during cell division (called “del 13” or “13q-“).

Fluorescence In-Situ Hybridization (FISH)

Fluorescence in situ hybridization (FISH) is a test used to assess genetic risk based on chromosomal abnormalities.

• It is not a substitute for karyotyping but is complementary to it.
• It is the assessment of the chromosomes of all myeloma cells in a bone marrow sample.
• It allows detection of changes whether myeloma cells are growing or not.
• It can detect numerical and structural chromosomal abnormalities.

For FISH:

1. The cells are fixed in paraffin wax.
2. Fluorescent probes that bind to certain sequences of the chromosome are attached.
3. Finally, each chromosome can be identified by a different color.

Chromosomes are made up of two chromatids paired in an X form, with the X shorter at the top and longer on the bottom. The “short arms” at the top half of the X are labeled “p” and the “long arms” at the bottom are labeled “q.” During normal cell division, the chromosomes divide in two, each single chromatid forming a duplicate of its genetic material in a new cell.

FISH is capable of detecting chromosomal translocations that can occur when gene sequences of one chromatid get shifted over to another chromatid during cell division, and the colors of the fluorescent probes from one chromosome appear in a different chromosome. Chromosomal deletions can be detected when a fluorophore color is absent.

What do FISH test results determine?

FISH results have been incorporated into the revised International Staging System (R-ISS) for myeloma because they provide a powerful tool for predicting risk and survival in myeloma.

The following cytogenetic abnormalities are considered to show high risk:

• t(4;14) (translocation of gene segments from chromosome 4 to 14)
• 17p–, del 17p (deletion of the short arm of chromosome 17)
• t(14;16) (translocation of gene segments from chromosome 14 to 16)
• 1q+ (gain of an additional long arm on chromosome 1)

Some chromosomal abnormalities signal aggressive myeloma. Other such abnormalities have no negative prognostic impact.

Nearly all myeloma patients demonstrate deletion of all or parts of chromosome 13 by FISH analysis. This is why deletion 13 is better determined by standard cytogenetics
than by FISH.

How are therapy choices related to chromosomal status?

The loss of the short arm of chromosome 17 by FISH analysis shows especially high risk because of an important tumor suppressor gene, p53, is located there. Tumor suppressor genes, also known as anti-oncogenes, control cell division and help prevent cancer cells from developing.

Therapy choices are often related to chromosomal status. For example:

• Regimens with Velcade® (bortezomib) for induction and maintenance therapy are preferred for patients with t(4;14) myeloma.
• Pomalyst® (pomalidomide) is less effective than Velcade for t(4;14),
• Yet, in studies thus far, Pomalyst is more effective than Velcade for overcoming the negative impact of del 17p.

Gene Expression Profiling (GEP)

GEP is performed on RNA extracted from myeloma cells. The genes present in the RNA are then probed on a special computer chip to provide a detailed picture of disease biology. GEP can:

• identify the dominant clone at any particular time in a patient’s disease course
• classify myeloma into different molecular subgroups
• identify the gene expression profile of patients with high-risk myeloma

GEP does not provide information about:

• non-dominant disease clones
• “driver” genetic mutations that allow the myeloma to grow and develop in new areas of the body

Various institutions have developed different GEP high-risk expression profiles that have not yet been standardized. GEP is not widely available.

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