Q&A column, 4/17

Editor: Frederick L. Kiechle, MD, PhD

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Q. Our laboratory receives requests for breast predictive marker testing (estrogen receptor, progesterone receptor, HER2, Ki-67) on biopsies of bone metastases. Is it appropriate to perform this testing on decalcified tissue?

A. Various decalcification methods are available that employ different chemical solutions (acid based, EDTA based, for example) and have variable effects on different epitopes, antibodies, and clones, with stronger acids—hydrochloric and nitric—generally more deleterious to immunostaining.^1,2 Given the increasing availability of, and need for, immunohistochemical, FISH, and molecular studies on metastatic tumors of all sites, we find it helpful to try to separate non-bony fragments from bony fragments at the time of grossing on all cases in which metastasis is clinically suspected. The bony and non-bony fragments are then submitted in separate cassettes, only one of which requires decalcification. This potentially yields a cassette of non-decalcified tissue for these important ancillary studies, even with small core bone biopsies. An alternative consideration for biopsies with relatively low content of bone spicules is to forgo decalcification solutions before tissue processing and employ a validated “surface decalcification” of the paraffin block in histology, as necessary, as is done for bone marrow biopsies in some laboratories.

However, there will inevitably be decalcified specimens that require special studies and predictive markers. For breast marker assessment, full validation of the immunostains with any alternative fixative (other than neutral-buffered formalin) and processing conditions is proscribed by American Society of Clinical Oncology/CAP guidelines and CAP checklists.^3-7 The most rigorous interpretation of these guidelines implies that validation testing on tissue decalcified with the laboratory’s own decalcification procedure using 40 positive and 40 negative cases is necessary.^3-7 However, the CAP anatomic pathology checklist (ANP.22985) acknowledges that full validation for decalcified specimens is not feasible for many laboratories, and therefore it recommends including a disclaimer within the patient report that the assays have not been validated on decalcified tissues along with a cautionary note for result interpretation.

Published data on the impact of decalcification on immunohistochemistry for breast predictive markers ER, PgR, HER2, and Ki-67 are relatively sparse (Table 1).^1,2,8-10 Schrijver and colleagues conducted in 2016 a detailed study comparing four different overnight decalcification solutions and procedures (1. Christensen’s buffer [formic acid and sodium formate based] with microwaving; 2. Christensen’s buffer without microwaving; 3. EDTA; 4. Formical-4 [formic acid, formaldehyde, methanol]) to control non-decalcified samples from up to 23 breast cancers, each then stained for ER, PgR, and HER2.² They also tested HER2 FISH and DNA/RNA yield for molecular studies.² They found slight decreases in percentage of positive cells in some cases, with biomarker discordance that would have an impact on patient treatment in only one to two cases per antibody (Table 1), and concluded overall that the EDTA protocol performed best in their hands.² Other smaller studies revealing the same trend in decreased intensity and/or percentage of tumor cells staining with relatively little effect on overall interpretation after decalcification are listed in Table1. ^1,8-10 Given the clinical necessity of performing breast predictive testing, in establishing their protocols laboratories should consider the results of these small studies, their local decalcification protocols, and the availability of decalcified material on which to perform full predictive marker validation.

1. Arber JM, Arber DA, Jenkins KA, Battifora H. Effect of decalcification and fixation in paraffin-section immunohistochemistry. Appl Immunohistochem. 1996;4(4):241–248.
2. Schrijver WA, van der Groep P, Hoefnagel LD, et al. Influence of decalcification procedures on immunohistochemistry and molecular pathology in breast cancer. Mod Pathol. 2016;29(12):1460–1470.
3. Wolff AC, Hammond ME, Schwartz JN, et al. American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. Arch Pathol Lab Med. 2007;131(1):18–43.
4. Hammond ME, Hayes DF, Dowsett M, et al. American Society of Clinical Oncology/College of American Pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer (unabridged version). Arch Pathol Lab Med. 2010;134(7):e48–72.
5. Fitzgibbons PL, Murphy DA, Hammond ME, Allred DC, Valenstein PN. Recommendations for validating estrogen and progesterone receptor immunohistochemistry assays. Arch Pathol Lab Med. 2010;134(6):930–935.
6. Fitzgibbons PL, Bradley LA, Fatheree LA, et al. Principles of analytic validation of immunohistochemical assays: Guideline from the College of American Pathologists Pathology and Laboratory Quality Center. Arch Pathol Lab Med. 2014;138(11):1432–1443.
7. Wolff AC, Hammond ME, Hicks DG, et al. Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline update. Arch Pathol Lab Med. 2014;138(2):241–256.
8. Darvishian F, Singh B, Krauter S, Chiriboga L, Gangi MD, Melamed J. Impact of decalcification on receptor status in breast cancer. Breast J. 2011;17(6):689–691.
9. Gertych A, Mohan S, Maclary S, et al. Effects of tissue decalcification on the quantification of breast cancer biomarkers by digital image analysis. Diagn Pathol. 2014;9:213.
10. Gruchy JR, Barnes PJ, Dakin Haché KA.
    CytoLyt fixation and decalcification pretreatments alter antigenicity in normal tissues compared with standard formalin fixation. Appl Immunohistochem Mol Morphol. 2015;23(4):297–302.

Megan Troxell, MD, PhD, Professor, Department of Pathology, Stanford University School of Medicine

Chair, CAP Immunohistochemistry Committee

Kristin Jensen, MD, Associate Professor, Department of Pathology, Stanford University School of Medicine

Chief, Pathology and Laboratory Medicine Service, Department of Pathology, Veterans Affairs Medical Center, Palo Alto, Calif.
Member, CAP Immunohistochemistry Committee

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Q. Is there a regulatory speed limit—whether a per day or a per hour “at the microscope” workload limit—on surgical pathology slide interpretations, similar to workload limits for cytology screening?

A. -“Speed limit” will imply turnaround time, for which most programs and practices have standard requirements, and it should be documented, adhered to, and periodically assessed.

However, the pathologist in her question raises the issue of “workload limit,” which is distinct. The CAP and other accrediting bodies and individual departments and programs have not defined workload criteria for practicing pathologists in the United States. Trainee (resident) work-hour limits and, as the pathologist points out, cytotechnologist workload limits are monitored, but not those of pathologists. The Accreditation Council for Graduate Medical Education limits residents to 80 hours or fewer a week.

That said, increasing attention is being paid to practicing physician workload because overwork leads to fatigue, errors, and group attrition. A timely article was published in the September issue of Archives of Pathology & Laboratory Medicine.1 Personally, I am unsure if workload restriction for pathologists will catch on in the United States, since it may be seen as limiting individual capacity, “productivity,” and in turn the profitability of private practices. Academic programs and hospital-based practices, on the other hand, have to be viewed distinctly and may have their own additional workloads (teaching, research, and administration).

1. Schrijver I. Pathology in the medical profession? Taking the pulse of physician wellness and burnout. Arch Pathol Lab Med. 2016;140(9):976–982.

Rajan Dewar, MD, PhD, Associate Professor, Institute for Social Research, University of Michigan, Ann Arbor

Member, CAP Surgical Pathology Committee

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Dr. Kiechle is a consultant, clinical pathology, Cooper City, Fla. Use the reader service card to submit your inquiries, or address them to Sherrie Rice, CAP TODAY, 325 Wau­ke­gan Road, Northfield, IL 60093; srice@cap.org. Those questions that are of general interest will be answered.