The following letter and reply have been jointly
published by consent in the Archives of Pathology & Laboratory Medicine
and CAP TODAY:
Although I agree with the majority of the American
Society of Clinical Oncology/College of American Pathologists Guideline
Recommendations for Human Epidermal Growth Factor Receptor 2 Testing in
Breast Cancer by Wolff, et al,1 I am
concerned with the sample exclusion criteria for human epidermal growth
factor receptor 2 (HER2) immunohistochemistry (Related Article: SRS:
Blood bank information systems, table 5), both as they relate to the
issue at hand and for the precedent this list of guidelines sets in an
area of pathology certain to see more regulation.
The American Society of Clinical Oncology/College of
American Pathologists panel delineated optimal formalin fixation time
for HER2 immunohistochemistry performed on excisional biopsies as six
to 48 hours. Furthermore, the panel mandates that a negative HER2 result
from suboptimal specimens (i.e., those fixed less than six hours or longer
than 48 hours) should be qualified in the report. It is intuitive that
grossly underfixed breast tissue is not an optimal specimen for HER2 testing,
but experience demonstrates that routine weekend fixation (up to 72 hours)
allows for good preservation of a wide range of antigens provided appropriate
heat-induced epitope retrieval is used. A study by Arber2
analyzing HER2 antigen preservation after prolonged formalin fixation
(seven-154 days) found no change in immunoreactivity for up to 20 days
in tissues from 33 different infiltrating breast carcinomas. This author
has evaluated HER2 antigen preservation in 20 infiltrating breast carcinomas
after seven to 14 days of formalin fixation and observed undiminished
strong membranous staining after heat-induced epitope retrieval (unpublished
data). Boenisch3 has shown convincingly
that heat-induced epitope retrieval can restore immunoreactivity after
prolonged formalin fixation. Twenty-nine of 30 antigens tested showed
equivalent staining intensity after four days of formalin fixation. A
recent review by Goldstein, et al,4
titled ''Recommendations for Improved Standardization of Immunohistochemistry,''
suggests a minimum formalin fixation time of eight hours (recommendation
one). No maximum fixation time is recommended, and it is stated that ''In
general, underfixation is a substantially larger problem than over-fixation''
Reading the Archives article in question I
failed to discover any referenced study supporting the sample exclusion
criteria. The authors simply allude to previous consensus conferences
dealing with HER2 testing (appendix E). Subsequently, Patrick Fitzgibbons,
MD, an author of the guideline article1 and chair of the CAP Immunohistochemistry
Committee, acknowledged to me in an e-mail (March 2007) that studies confirming
the hour-by-hour impact of formalin fixation on HER2 testing over the
range of fixation intervals seen in clinical practice have not been completed.
It is axiomatic that promulgated guidelines should be evidence based and
scientifically validated. In the absence of peer-reviewed study in its
support, adoption of the greater than 48-hour formalin fixation exclusion
criterion would be counterproductive. This is especially true in light
of the potential impact of said criterion on current laboratory practice
(i.e., necessitating staffing histology on the weekend to accommodate
Friday morning breast cancer surgeries). Based on the evidence cited herein,
a 72-hour maximum fixation exclusion criterion would be more appropriate,
allowing for routine weekend fixation while enabling accurate and reproducible
James F. Lombardo, MD
Ogden (Utah) Regional Medical Center
- Wolff AC, Hammond MEH, Schwartz JN, et al. American
Society of Clinical Oncology/ College of American Pathologists guideline
recommendations for human growth factor receptor 2 testing in breast
cancer. Arch Pathol Lab Med. 2007;131:18-43.
- Arber DA. Effect of prolonged formalin fixation
on the immunohistochemical reactivity of breast markers. Appl Immunohistochem
Mol Morphol. 2002;10:183-186.
- Boenisch T. Effect of heat-induced antigen retrieval
following inconsistent formalin fixation. Appl Immunohistochem Mol
- Goldstein NS. Recommendations for improved
standardization of immunohistochemistry. Appl Immunohistochem Mol
- M. Elizabeth H. Hammond, MD, of the Department
of Pathology, LDS Hospital, Intermountain Healthcare, Salt Lake City,
and co-chair of the ASCO/CAP HER2 Guideline Panel, replies (on behalf
of the panel): The American Society of Clinical Oncology/College
of American Pathologists panel appreciates thoughtful commentary that
has been provided to us by personal communication to various members
as well as to the CAP or as letters to the editor of Archives of
Pathology & Laboratory Medicine or CAP TODAY. These thoughtful
efforts help us to review problematic issues so that future revisions
can address them.
To date, most concerns relate to specimen handling
and its effect on human epidermal growth factor receptor 2 (HER2) test
results. Understandably, there is consternation expressed about the lack
of scientific evidence related to fixation types and fixation times. We
share that consternation. The panel reviewed existing scant information
and depended also on the extensive experience of many of the panel members
and experts from large commercial laboratories who provided us with their
input. All guidelines created by the American Society of Clinical Oncology
or the College of American Pathologists in the past have depended on both
scientific evidence and expert opinion, especially when existing evidence
was scant or nonexistent.
It was the considered and unanimous opinion of panel
experts that defining the guideline fixative should be based on the requirements
of the existing Food and Drug Administration-approved testing methods
for HER2. All of those methods specify that tissue must be fixed in buffered
formalin, although the length of fixation time is not described. The panel
and these experts also felt strongly that the guideline needed to define
standardized fixation intervals and then modify those requirements if
necessary when and if data about optimal intervals became available. The
expert panel members felt that intervals of six to 48 hours were most
commonly recommended for breast cancer predictive tests. I presented published
data from Intermountain Healthcare, which clearly demonstrated the adverse
impact of prolonged fixation on estrogen receptor results in our health
care system.1 Because breast specimens
are submitted for estrogen receptor assays as well as for HER2 assays,
these intervals seemed the most appropriate recommendation at this time.
The adverse effects of underfixation are well known, as is pointed out
by the letter writer and documented in the literature.2-4
It is likely that the recommendation for fixation of core biopsies will
also be revised in the future to be a minimum of six hours, based on recent
published work.2 We look forward to
other data validating or suggesting modification of fixation intervals
during the next year. The panel is committed to modifying the document
as needed based on input from the pathology and oncology community.
Our recommendation to provide information in any negative
HER2 report about the potential impact of prolonged or foreshortened fixation
time was included to guide oncologists in clinical decisions. Oncologists
usually assume that any test result is accurate. Providing information
about mitigating circumstances will help them to understand the complexities
of these test interpretations so that they can make better informed decisions
for their patients. The intent of all pathologists and oncologists is
to provide the most appropriate treatment to patients based on accurate
testing data. It is fervently hoped that the current guideline will provide
us with strategies to make such accurate testing more widespread. The
panel is committed to changing the guideline when and if data are provided
to recommend those changes.
- Nkoy F, Hammond EH, Rees WL, et al. Day of surgery
affects estrogen receptor test results in women with breast cancer.
The 28th San Antonio Breast Cancer Symposium; December 2005. Abstract
- Goldstein NS, Ferkowicz M, Odish E, et al. Minimum
formalin fixation time for consistent estrogen receptor immunohistochemical
staining of invasive breast carcinoma. Am J Clin Pathol. 2003;120:86-92.
- Nadji M, Gomez-Fernandez C, Ganjei-Azar P, et al.
Immunohistochemistry of estrogen and progesterone receptors reconsidered:
experience with 5,993 breast cancers. Am J Clin Pathol. 2005;123:21-27.
- Yaziji H, Goldstein LC, Barry TS, et al.
HER-2 testing in breast cancer using parallel tissue-based methods.
I concur with Gregory Tetrault, MD (Letter, August
2007, page 6). The adage "garbage in, garbage out" certainly applies to
results reported out on unacceptable specimens.
I have been a medical technologist for almost 40 years,
the last 17 in management but I still work the bench. Though preanalytical
factors are now buzzwords, they have been important to most laboratorians
for years. Standard laboratory practice includes not running tests on
hemolyzed specimens, unfilled coagulation tubes, specimens drawn above
an IV, clotted CBCs, and so on. In my opinion, if you run tests on these
specimens, it is comparable to running tests on the wrong patient.
We spend a lot of time in the laboratory ensuring the
integrity of the patient samples. We then run quality control, look at
Levey-Jennings charts, follow Westgard rules, etc., to give clinicians
the most accurate laboratory test results possible so they can best treat
If you establish a reputation as a quality-driven,
reliable laboratory, clinicians will call you when they question a result,
and this is the best relationship you can establish for the care of patients.
On the other hand, if you report out inaccurate results, or physiologically
impossible results, even with a disclaimer, without trying to obtain a
new specimen, you are not only doing a disservice to the laboratory but
also to the clinician and, most important, to the patient.
Most laboratorians I have come across over the years
consider themselves professionals, and, much like the medical doctor oath,
we strive to "do no harm."
Marilyn C. Kenyon,
Director of Laboratory Services
St. Joseph Hospital