July 2011

Editor:
Fredrick L. Kiechle, MD, PhD

Q. What method other than gas chromatography/ mass spectrometry can be used to test methylmalonic acid, and do you know of any vendors that support the method?

A. Methylmalonic acid, or MMA, is important for normal fatty acid synthesis (and, notably, maintenance of the myelin sheath that surrounds nerves in the white matter of the central nervous system as well as sensory and motor nerves in the peripheral nervous system). The important reaction is the formation of succinyl Co-enzyme A (CoA) from methylmalonyl CoA. This step is catalyzed by the enzyme methylmalonyl CoA mutase; vitamin B12 is a cofactor. Measuring elevated levels of MMA helps identify the abnormality in pediatric patients with methylmalonic acidemia due to enzyme deficiency, as well as adult patients with vitamin B12 deficiency.

Tina Cowan, PhD, my colleague at Stanford who is director of biochemical genetics, reports that MMA testing for suspected enzyme deficiency is roughly equally divided between gas chromatography/mass spectrometry and tandem MS. It is likely that testing for suspected B12 deficiency is similarly divided between these two methods. We are not aware of any (potentially simpler) commercially available assays. If MMA testing for vitamin B12 deficiency becomes more widespread, this might change. But, for the moment, there is still significant controversy about this use of MMA.¹

Reference

1. Solomon LR. Cobalamin-responsive dis­orders in the ambulatory care setting: unreliability of cobalamin, methylmalonic acid, and homocysteine testing. Blood. 2005;105:978–985.

James D. Faix, MD
Stanford Clinical Labs at Hillview
Stanford University School of Medicine
Palo Alto, Calif.

Member, CAP Chemistry Resource
Committee, Standards Committee,
Council on Scientific Affairs

Q. Our hematology/oncology clinicians have begun requesting that CD68 be performed on all cases of newly diagnosed Hodgkin lymphoma. These requests stem from a March 11, 2010 article in the New England Journal of Medicine, “Tumor-associated macrophages and survival in classic Hodgkin’s lymphoma.” Though photos with the article show CD68 immunohistochemical analyses of samples obtained from a patient in each of the treatment-success and -failure groups, there is uncertainty about how to interpret the CD68 stain, specifically in cases with intermediate scores. Do you have suggestions or comments?

A. The article by Steidl and colleagues demonstrated that an increased number of CD68+ macrophages correlated with a shortened progression-free survival and greater likelihood of relapse after autologous stem cell transplantation, resulting in shortened disease-specific survival. In multivariate analysis, this adverse prognostic factor outperformed the international prognostic score for disease-specific survival. The 10-year disease-specific survival rate was significantly lower among subjects with a CD68 immunohistochemistry score of three (59.6 percent) than among those with a score of two (67.4 percent) or one (88.6 percent). Secondary treatment administered with curative intent failed in only 12.5 percent of subjects with a CD68 IHC score of one, compared with a failure rate of 51.7 percent in subjects with a score of two and 62.5 percent in subjects with a score of three. In particular, there was a significant correlation between the failure of autologous stem cell transplantation and a score of more than one. The absence of CD68+ macrophages in patients with limited-stage disease defined a subgroup of patients with a long-term disease-specific survival of 100 percent with the use of current treatment strategies.

According to the supplementary appendix the authors of the article provided, the scoring of CD68 was performed as follows:

• 1.5-mm duplicate tissue microarray cores were obtained from representative areas containing Hodgkin Reed-Sternberg cells in 166 diagnostic biopsies evaluated by both morphology and immunohistochemistry.
• For CD68 staining, scoring was performed in three representative high-power fields, and the relative percentage of CD68+ macrophages in relation to overall cellularity was reported as an average of both duplicate cores according to three groups: <five percent, IHC score one; five to 25 percent, IHC score two; >25 percent, IHC score three. Elevated numbers of CD68+ cells were defined as IHC scores of two or three.
  The results of the study were that increased tumor-associated macrophages identified by CD68 immunostain on diagnostic specimens correlated with a worse disease-specific survival in both univariate and multivariate analyses. Let us consider this topic further, because the study did not address some of the issues that hematopathologists face when performing the CD68 stain routinely on cases of classic Hodgkin lymphoma. These issues are as follows:
• The study was performed on tissue microarray cores, and the authors did not address the variability of CD68 staining that can be seen in intact tissue sections. Should the area of highest macrophage density be scored, or should we average the number of macrophages in multiple representative areas?
• Are the numbers of CD68+ macrophages best quantified using visual estimation or manual counting? The latter is labor-intensive, of course, and not practical in daily practice.

The study by Steidl, et al., is promising in that it recognizes the role of the microenvironment, and specifically macrophages, in Hodgkin lymphoma prognosis. The issues regarding interpretation of CD68 staining in Hodgkin lymphoma are valid, however, and have yet to be rigorously tested in the literature. Until such time that they are tested, there are limitations on the application of this stain on routine cases. If clinicians insist on obtaining this information, it seems reasonable to provide it to them with the understanding that scoring is not straightforward, or even reproducible, in many cases. It is expected that recognition of cases with very low (<five percent) and very high (>50 percent) numbers of macrophages, as shown in the article, will be straightforward, while scoring of the intermediate cases will be problematic. On a practical level, recognition of score one, which identifies a group of patients with excellent prognosis, might be more important than making the distinction between scores two and three.

This is clearly an area that warrants further investigation. At the moment, CD68 staining in Hodgkin lymphoma should be interpreted with caution, especially in cases with intermediate (five to 50 percent) numbers of macrophages

Reference

1. Steidl CF, Lee T, Shah SP, et al. Tumor-associated macrophages and survival in classic Hodgkin’s lymphoma. N Engl J Med. 2010;362:875–885.

Randa Alsabeh, MD
Director, Immunopathology Laboratory
Director, Hematopathology Fellowship
Associate Professor,
Cedars-Sinai Medical Center
Associate Clinical Professor,
University of California, Los Angeles

Member, CAP Immunohistochemistry Committee