College of American Pathologists
Printable Version

  No shortage of subtleties in CLL diagnoses


CAP Today




August 2011
Feature Story

Karen Titus

Chronic lymphocytic leukemia is not a diva disease —it ­doesn’t announce itself with exaggerated, self-important gestures. As many as 90 percent of patients present with asymptomatic disease.

But the low-key demeanor of CLL can trip up physicians who aren’t hematologists or hematopathologists, suggests Lynne Abruzzo, MD, PhD, since they “tend to think of CLL as an indolent disease of elderly patients.” Most patients (the thinking goes) will die of a heart attack or stroke before CLL starts causing trouble. “But this really ­isn’t the case,” she says.

In a June webinar on CLL, sponsored by the Association for Molecular Pathology, Dr. Abruzzo explored a much more nuanced portrayal of the disease, which is the most common leukemia in the Western hemisphere, comprising some 25 percent of all cases. Each year about 10,000 new patients in the United States are diagnosed with CLL. “As our population ages it will probably become even more common,” says Dr. Abruzzo, professor of hematopathology and medical director of the clinical cytogenetics laboratory, The University of Texas MD Anderson Cancer Center, Houston.

Age, kind to almost no one, is especially cruel in CLL. The median age of patients is about 65 years, she says, and the incidence increases with advancing age. About 30 percent of patients, however, are under age 60, and 15 percent are under 50. More than twice as many men than women are affected. Men also appear to have a worse prognosis, she says.

A typical case of CLL on morphology shows relatively small lymphocytes with a mature appearance. They have oval nuclei and dense chromatin, inconspicuous nucleoli, and scant cytoplasm. “Individually,” she says, “you really can’t tell that these cells are neoplastic. And occasional cells may have atypical cytologic features, such as nuclear clefts.”

In the bone marrow, the infiltrate is composed predominantly of small cells, and these infiltrates can be nodular, interstitial, diffuse, or a combination of these patterns. Pathologists might even see proliferation centers in the bone marrow—a useful clue for distinguishing between CLL and mantle cell lymphoma.

CLL has a characteristic immunophenotype. Cells express pan B-cell antigens (although CD20 expression tends, like the global economy, to be weak). They express CD5—rarely found on the vast majority of normal B cells—and are CD23 positive. They also express surface IgM and, frequently, IgD, though both these markers also list toward weak expression.

For now, the disease is treatable but not curable. Death occurs due to complications of immune dysfunction or myelosuppression.

“The hematologists who take care of these patients call this the disease of thirds,” Dr. Abruzzo says, a comparison that calls to mind photography’s so-called rule of thirds. In both cases, the result is a more balanced picture. For CLL, the breakdown looks like this:

  • One third of patients present with indolent disease, which remains indolent. These patients have a normal survival.
  • One third of patients present with indolent disease that progresses.
  • One third of patients start out with aggressive disease.

One of the major problems with CLL prognosis is that it’s hard to determine, when patients first present, which group they belong to, especially if they present with low-stage disease.

A typical survival curve for patients with CLL shows that when patients first present, most are treated with fludarabine chemoimmunotherapy and achieve a complete remission. Most of these patients eventually relapse, however. When this occurs, fewer of them are subsequently able to achieve a complete remission; those who do tend to relapse sooner. It’s a dark spiral. “The longer someone has the disease, and the more often they require treatment, the worse they do,” Dr. Abruzzo says.

Given this prognostic peculiarity, researchers have looked at a large number of potential clinical markers, many of which, happily enough, have proved useful. Among them:

  • clinical stage (Rai or Binet stage), patient’s age, patient’s gender.
  • histologic markers (such as the pattern and extent of marrow involvement).
  • serum markers (including β2-microglobulin—“a very useful marker,” says Dr. Abruzzo—lactate dehydrogenase, and thymidine kinase).
  • cellular markers (such as surface CD38 and intracellular ZAP70).
  • molecular markers (particularly cytogenetic abnormalities and the somatic mutation status of the immunoglobulin heavy chain variable region gene, or IGHV).

This latter is an important prognostic marker. In 1999, two groups showed, in back-to-back articles in Blood (Damle RN, et al. 94:1840–1847; Hamblin TJ, et al. 94:1848–1854), that there were significant differences in the prognosis for patients with CLL depending on their IGHV somatic mutation status. The median survival for patients with mutated variable region genes was about 25 years, while the median survival for patients with unmutated IGHV region genes was only about eight years. “In general, about half of patients have mutated IGHV genes in their CLL cells, and about half have unmutated genes,” she says.

It’s a loaded word, “mutation.” Dr. Abruzzo notes, however, that it refers to “a normal mechanism that generates antibody diversity in response to antigen.” When a B cell interacts with antigen, she explains, the process introduces point mutations into the IGHV and immunoglobulin light chain variable, or IGLV, region. Most of these mutations are placed in the complementary determining regions, which come in contact with, and bind to, the antigen. This, in turn, results in higher affinity antigen binding sites.

This normal process of somatic mutation—which requires antigen, T cell help, CD40/CD154 stimuli, and cytokines in the germinal center microenvironment—is generally confined to centroblasts within the germinal center, she says. The progeny of these cells do one of two things: 1) they die, or 2) they expand in response to the antigen presented on the follicular dendritic cells. The cells then differentiate into either plasma cells (secreting immunoglobulin) or memory B cells.

Until recently, researchers thought that unmutated cases arose from antigen-inexperienced B cells, that is, B cells that had yet to encounter antigen in the germinal center (and thus had not undergone somatic mutation). Mutated cases were thought to have arisen from a post-germinal center memory B cell that had undergone somatic mutation in the germinal center.

Poking a pin in the balloon, Dr. Abruzzo sums up the above scenario as “very appealing—but probably not the case.”

“There are several lines of evidence that argue against unmutated cases arising from naive B cells,” she says. Gene expression profiling studies show that mutated and unmutated cases express many genes in common. Moreover, the gene expression patterns of mutated and unmutated cases resemble each other, as well as resembling that of antigen-experienced memory B cells. “Also, if you look at the surface markers that are expressed by unmutated CLL cases,” she says, “they express markers that are associated with antigen experience—for instance, CD27.”

The new model for thinking about CLL origins reflects emerging knowledge of normal B cell development.

Mature B cells in peripheral lymphoid tissues can follow two different developmental pathways, Dr. Abruzzo says, depending on whether they respond to T cell-independent antigen or T cell-dependent antigen. The latter induce somatic mutation—B cells within germinal centers are now known to respond to antigens that require T cell help. In contrast, B cells that are generally confined to the marginal zone B cells respond to T-independent antigens, which tend to have repeating subunits, she says, such as bacterial polysaccharides. This response may or may not induce somatic mutations.

So, under the new model, researchers suspect that unmutated CLL cases develop in response to low-avidity antigens via a T cell-independent event that occurs outside the germinal center; mutated CLL cases, on the other hand, are now thought to arise from B cells that would normally have responded to a high-avidity antigen that drives T cell-dependent germinal center formation.

For routine diagnostic use, identifying somatic mutation was considered to be somewhat bunglesome—a bit like bringing declamatory acting back to the stage. But since it was such a good marker of prognosis, the hunt for surrogate markers of somatic mutation status was soon on. One of the first was ZAP70. This is a 70 kD cytoplasmic tyrosine phosphoprotein, normally expressed in T and NK cells (though not in B cells), that’s critical for T cell receptor signaling. Early studies (Crespo M, et al. N Engl J Med. 2003;348:1764–1775) showed a high concordance between ZAP70 and IGHV somatic mutation status: Unmutated cases usually were ZAP70 positive, and mutated cases usually ZAP70 negative. In addition to being a good marker of somatic mutation status, she says, ZAP70 was found to be a “very good” marker of prognosis in that tricky group of patients: those with early-stage disease.

At MD Anderson, ZAP70 immunohistochemistry is routinely done on all new cases of CLL. Bear in mind, Dr. Abruzzo says, that normal T cells stain positive—but they tend to be a little darker. “So you have to make sure, when you call a case positive, that you’re actually examining the neoplastic cells.” The small T cells do serve as a good built-in control. “If your stain is completely negative, then the chances are that your stain didn’t work,” she says.

Interestingly, ZAP70 may be a better predictor of time to first therapy than immunoglobulin heavy chain status (Admirand J, et al. Mod Pathol. 2004;17:954–961), though “This is still somewhat controversial,” says Dr. Abruzzo.

ZAP70 can be assessed by flow cytometry as well as by IHC, though this is technically difficult, and not all of the antibodies are standardized, says Dr. Abruzzo. Laboratories are reporting success with its use nonetheless. She remains a fan of IHC, which she says is a robust and easy method. It can also be done retrospectively.

CD38 is the other strong contender for a surrogate marker. Since it’s found on the cell surface, it can be easily measured by flow cytometry. Initially, it was reported that positivity (greater than 30 percent positive cells) was correlated with unmutated somatic mutation status, while cases that had mutated IGHV genes expressed low levels of CD38. Today, however, it’s known that unmutated cases actually show a wide range of CD38 expression. Moreover, levels do not appear to be stable over time and may change with disease progression. “However, we still measure CD38, because it does appear to have independent prognostic value.”

Cytogenetic findings in CLL have also benefited from a recent face-lift. According to older literature, says Dr. Abruzzo, it appeared that only about 20 percent of CLL cases had cytogenetic abnormalities. “This is because the CLL cells actually don’t divide well in culture, and die in culture,” she says, adding that conventional karyotyping requires dividing cells.

When FISH is used, about 80 percent of cases show an abnormality, such as deletions at 13q14 (found in 50 percent of patients), trisomy 12 (20 percent), deletions at 11q22-23 (20 percent), deletions at 17p13 (five percent), or deletions at 6q21 (five percent). Interestingly, she notes, by conventional cytogenetic analysis, trisomy 12 is the most common CLL abnormality. A paper by Dohner H, et al (N Engl J Med. 2000;343:1910– 1916) established the prognostic importance of FISH in CLL patients, findings since replicated in subsequent studies. “They found that patients with deletions in 17p had an absolutely dismal prognosis.” Those with deletions in 11q fared poorly, while those with a trisomy 12 or no abnormality had an intermediate prognosis. Patients with deletions in the long arm of chromosome 13 had the best prognosis of all. Other papers have since shown that patients with deletions at 17p are resistant to standard therapies, such as fludarabine, cyclophosphamide, and rituximab (FCR), and will likely benefit from more aggressive therapies, including, possibly, bone marrow transplantation.

The Dohner study included previously treated patients. Deletion 17p can be acquired as a secondary abnormality. In a study (Tam CS, et al. Blood. 2009;114:957–964) performed at the Mayo Clinic and MD Anderson, researchers looked at the outcome of asymptomatic, early stage patients (Rai stage zero to two) with a de novo deletion of 17p, that is, a deletion identified at the time of diagnosis, rather than one acquired during the course of disease. Such patients, it turns out, have a good overall survival at three years. “So in contrast to some of the earlier studies, our data does not support the idea that patients with 17p deletions necessarily have aggressive disease and need aggressive treatment up front.”

Translocations are rare in CLL, making it difficult to know their prognostic significance when they do occur (though there’s some evidence to suggest that certain translocations are associated with more aggressive disease).

CLL cases with the translocation 14;19 tend to show atypical morphology. They’re cytologically heterogeneous and frequently have indented nuclei, says Dr. Abruzzo. These cases also have an atypical immunophenotype that’s quite similar to cases of mantle cell lymphoma, that is, they strongly express CD20 and surface immunoglobulin; they’re CD23 negative; and they’re FMC7 positive. Unlike mantle cell lymphoma, however, these cases lack the 11;14 translocation. They frequently have trisomy 12, as well as other abnormalities.

The t(14;19) juxtaposes the BCL3 gene on chromosome 19 and the IGH gene on chromosome 14, resulting in overexpression of the BCL3 protein, Dr. Abruzzo explains. Such cases typically have unmutated IGHV region genes.

In rare cases, she says, it’s possible to see MYC rearrangements. “Cytogenetically, these are indistinguishable from those that we see in Burkitt’s lymphoma,” she says. These cases also have atypical morphology, in the guise of increased prolymphocytes, but have typical immunophenotypes.

Another rare translocation involves 2;14. The atypical morphology in these cases is characterized by plasmacytoid differentiation, nuclear indentations, and increased prolymphocytes; again, the immunotype is typical.

Several newish technologies are having a go at CLL diagnosis and prognosis, including array comparative genomic hybridization. Unlike conventional karyotypic analysis, but similar to FISH, array CGH does not require dividing cells, says Dr. Abruzzo. It measures copy number variation between test and reference DNA samples.

Tumor DNA is measured with one fluorochrome, the reference control DNA with another. Equal amounts of both DNAs are mixed together, then hybridized to the array. Genomic imbalances—either a gain or a loss of chromosomal material—results in an imbalance in the signal from one of the fluoro­chromes. While the arrays can be printed with BAC clones, Dr. Abruzzo says that oligonucleotide arrays are becoming more common in clinical use. “They’re easier to standardize. You don’t have to maintain BAC libraries.”

Array CGH is not perfect. Several entities pass under its radar: balanced rearrangements, clones that comprise less than 20 percent of total cells, and what’s called copy number neutral loss of heterozygosity (for example, uniparental disomy).

“But because CLL cells divide so poorly in culture, and because the disease is characterized by gains and losses, rather than translocations, array CGH is very well suited to CLL diagnosis,” Dr. Abruzzo says. “We use a targeted array to the regions that we know are important in CLL,” including deletions in the long arm of chromosome 13, gains in trisomy 12, loss in the long arm of chromosome 11, and loss in the short arm of chromosome 17p.

Also new on the CLL scene (as well as other diseases) are microRNAs, or miRNAs (see MicroRNA—is the contender a champ?). “You’ll be hearing more and more about them,” Dr. Abruzzo promises. These small (18 to 24 nucleotides long), noncoding RNAs are a new class of gene regulators, affecting cell cycle, differentiation, and apoptosis. As of June, some 1,400 miRNAs had been identified in the human genome, “and the number is increasing constantly.” They bind to the 3’ (3 prime) untranslated region of their mRNA target. But because they do so with incomplete complementarity, a single miRNA can regulate dozens of genes.

Dysregulated miRNA expression can lead to malignant transformation, Dr. Abruzzo says. She points to six miRNAs—admittedly an incomplete list—that are associated with lymphoid leukemias and lymphomas: miR-15a, miR-16-1, miR-155, miR-17~92 cluster, miR-29b, and miR-181a.

The first two belong to the critical 13q14 region. The protein coding gene for tumor genesis remained elusive, however, until the discovery that miR-15a and miR-16-1 were shown to be deleted in the vast majority of CLL cases with 13q deletions. When they function normally, miR-15a and miR-16-1 downregulate BCL2 protein, which is normally antiapoptotic.

It’s likely that specific patterns of miRNA expression will prove to have prognostic value in a variety of tumors, Dr. Abruzzo says, with miRNA signatures being developed into tests that await clinical validation.

Even before this happens, pathologists would do well to stay abreast of new developments in CLL. Dr. Abruzzo says that when she’s attended clinic sessions with her clinical collaborator, newly diagnosed patients ask about their somatic mutation status and cytogenetic abnormalities. “Patients have gotten very sophisticated,” she says. Even if prognostic markers don’t necessarily affect management right now, “it does give them an idea of how well they are likely to do.”

That makes perfect sense to Dr. Abruzzo. “If I had CLL,” she says, “I would want to know if I had mutated or unmutated immunoglobulin heavy chain variable region gene, and if I had a 13q deletion, or a 17p deletion.”

Karen Titus is CAP TODAY contributing editor and co-managing editor.

Related Links Related Links