Cellular signaling systems regulating the life cycles of cells
are so extremely complex that pathologist Christopher N. Otis, MD, of Tufts University, likens them to looking at “maps of stars where one attempts to trace what’s related to what.” And within that universe of communication is the oncogene, HER2, the target of breast cancer therapy designed to interrupt messages telling tumor cells to replicate.
HER2 has also been the target of escalating efforts by pathologists and assay vendors to ensure that testing is as accurate and efficient as possible in identifying patients who qualify for anti-HER2 therapy. In addition, ongoing research may be paving the way for a new paradigm for HER2 and related genetic testing that will allow oncologists to hone targeted treatments, which, to date, include Genentech’s humanized monoclonal antibody, trastuzumab, and GlaxoSmithKline’s lapatinib, a tyrosine kinase inhibitor. But many more drugs and drug combinations are in the pipeline.
In other words, when you ask experts what’s new with HER2 testing and its role in directing breast cancer therapy, be prepared to pull up a chair.
For one, the CAP is looking at the impact of genomic heterogeneity in invasive ductal breast carcinoma on HER2 testing accuracy. In rare cases, breast cancer specimens can show zones of amplification of the HER2 gene while other zones show normal numbers of the gene, explains M. Elizabeth H. Hammond, MD, who along with Gail H. Vance, MD, is heading up an ad hoc committee under the CAP Council on Scientific Affairs to explore the issue.
The interspersed areas of gene amplification and non-amplification can cause a pathologist performing fluorescence in situ hybridization, or FISH, to come up with a false-negative result, depending on what part of the tumor is analyzed, says Dr. Vance, professor of medical and molecular genetics at Indiana University, Indianapolis.
A tumor’s HER2 genomic heterogeneity can also affect the immunohistochemical HER2 result. But the best way to detect genomic heterogeneity in a sample, says Dr. Hammond, is to perform IHC on it first. “Areas of HER2 gene amplification will likely show increased protein expression. Using that information, one can then select those areas for FISH counting so that the amplified regions are included in the result,” says Dr. Hammond, professor of pathology at the University of Utah and former chair of pathology at LDS Hospital of Intermountain Healthcare.
The Council on Scientific Affairs ad hoc committee tackling genomic heterogeneity will first establish a consensual definition of it. From there, says Dr. Vance, “we will discuss the best way to identify genetic heterogeneity in HER2 specimens and how to avoid pitfalls in the current testing scheme [that may lead to false-negative results]. Once our discussions are collected in draft form, we plan to circulate them for commentary from the breast pathology community.”
Dr. Hammond, who was a co-chair of the HER2 guideline expert panel, says the recommendations may be included in an updated version of the ASCO/CAP HER2 testing guideline. Published in January 2007, the current guideline doesn’t address genomic heterogeneity because the expert panel that wrote it decided the issue required a more thorough investigation in order to generate a specific report, she says.
David J. Dabbs, MD, chief of pathology at Magee-Women’s Hospital at the University of Pittsburgh Medical Center, says silver in situ hybridization, or SISH, testing will be on the “forefront of helping to make a decision about how exactly to interpret HER2 heterogeneity.” That’s because when pathologists use SISH, they view the whole tissue slide rather than a “minute focus of the tumor with the fluorescence microscope,” as is done for FISH, he says. If pathologists were to widely adopt SISH testing, it would “afford an opportunity to do applied practical research about what these [genetically heterogeneous] patterns mean.”
The potential for more widespread use of SISH isn’t just pie in the sky. Some say Ventana Medical Systems’ automated Inform HER2 SISH DNA probe test, for which the company is seeking premarket approval from the Food and Drug Administration, would make it possible for labs to do the testing efficiently.
While some vendors now offer chromogenic probes for HER2 gene testing, the probes are all analyte-specific reagents and performed manually. Ventana’s HER2 SISH test is fully automated, requiring a six-hour run on its Benchmark series automated stainer, allowing a lab to do an overnight run, for example, and interpret the test the next day. That six-hour turnaround time compares to two to three days to do FISH, says Thomas Grogan, MD, founder and senior vice president of medical affairs for Ventana.
In Dr. Grogan’s view, SISH technology is a “transformational event,” one of the greatest he has witnessed in his 30 years of practice. “It’s exciting to think you can sit at your own microscope and sign out cases by looking at individual genes,” he says.
Of course, a lab can also run FISH for HER2 testing on the Benchmark stainer, so one might ask, why bother switching to SISH? Pathologists and technicians performing FISH have to go in the dark and count individual fluorescent signals in a particular cell and may have trouble identifying individual cells that are part of the invasive part of the cancer. They take a picture of the fluorescent slide and the slide fades over time. They come out of the dark and compare the H&E of what they looked at. But how can one know if pictures were taken of the invasive carcinoma area? With SISH, the case can be archived, reviewed, and viewed for HER2 gene status in a morphological context.
For the SISH test, Ventana is using a patented nanotechnology that gives a high signal-to-noise ratio. The principle is similar to high-quality silver photography. Dr. Grogan, in fact, credits the discovery of Pluto a century ago to the advent of fine-grain silver photography that allowed someone using a telescope to identify the planet among all the other “small dots in the sky.” In an analogous way, nanotechnology, he says, allows elemental silver to undergo self-assembly, “so you get a magnified signal and visualize individual genes with a standard light microscope.”
Ventana’s HER2 SISH test will also have an image-analysis application from BD Diagnostics-TriPath for use on the Ventana Image Analysis System. BD-TriPath will submit a companion 510(k) application to the FDA for the image analysis.
If the Ventana application pans out, making SISH routinely available to pathologists, could it end up supplanting some FISH or even IHC testing?
Raymond Tubbs, DO, section head of molecular genetic pathology at the Cleveland Clinic, predicts that “when pathologists see how easy it is to objectively visualize HER2 gene copy signals directly using a conventional light microscope, many will prefer to do SISH rather than IHC. But that doesn’t mean IHC will go away,” he says, “[because] some pathologists will still prefer the ease of use of IHC despite its inherent interpretative subjectivity.”
“Many pathologists will continue to do IHC and, if a case falls in the equivocal zone, they may do their own SISH testing in house rather than sending the case out to a reference laboratory for FISH testing,” Dr. Tubbs predicts.
Michael Press, MD, professor of pathology at the University of Southern California, points out, however, that FISH has a proven track record for assessing HER2 gene amplification. And while “SISH has the advantage of not requiring fluorescence microscopy,” he says, “SISH would have to demonstrate a similar high level of accuracy and interlaboratory reproducibility in order to compete effectively with FISH as a diagnostic assay.”
On the IHC testing front, a recent pilot study conducted by pathologist Keith J. Kaplan, MD, suggests a role for HER2 image analysis to help avoid unnecessary in situ hybridization testing in IHC cases overscored by pathologists as a 2+.
In the study, Dr. Kaplan used Aperio’s ScanScope and computerized imaging algorithm on 81 breast cancer specimens previously tested by IHC and FISH in 2006 at Northwestern University when he was a pathologist there. Image analysis appeared in the study to have much less tendency to “falsely overcall” a sample as being 2+ by immunohistochemistry, which some pathologists may do in order to reflex to FISH and thus ensure they don’t miss HER2-positive tumors, says Dr. Kaplan, who is now a senior associate consultant in the Department of Laboratory Medicine and Pathology, Division of Anatomic Pathology, Mayo Clinic, Rochester, Minn.
Thus, the imaging algorithm provides a more objective test to say a case is 1+. Using it, he says, “you may be able to avoid doing unnecessary FISH testing as a quality measure because the quality measure is the algorithm.”
Immunohistochemical and in situ hybridization methods provide semiquantitative, binary (yes, no) results for HER2 testing. But Monogram Biosciences’ new CLIA-validated HERmark assay run on its VeraTag technology platform uses neither method. Instead, the assay measures total HER2 protein and HER2:HER2 dimerization, which the company hopes will lead to more accurate predictions of which patients will respond to anti-HER2 therapies.
Monogram will not offer the HERmark test, which will be performed in its reference lab, until it completes studies determining the assay’s clinical utility. The company presented data at the December 2007 San Antonio Breast Cancer Symposium showing the assay predicts survival in patients with advanced breast cancer treated with trastuzumab. Additional studies are underway.
The HERmark assay uses “small molecular tags” that selectively bind to specific protein analytes, explains Alfred Merriweather, chief financial officer for Monogram Biosciences, San Francisco. Through a “molecular scissors” mechanism in the assay, these tags are released when they come in very close proximity to the scissors. “We can then measure the released tags through capillary electrophoresis to quantitatively ... measure the quantity of the analyte to which the tag was formerly bound,” Merriweather says. Because the tags can be measured precisely and quantitatively, Monogram can measure both the level of the HER2 protein and the HER2 dimers, he explains.
A HER2 dimer occurs when the HER2 receptor has combined with another HER receptor in the HER family, which includes four members: HER1, HER2, HER3, and HER4. When a HER2 receptor combines with another HER2 receptor, it creates what’s known as a HER2 homodimer, which is what the HERmark assay measures. HER2 can also pair up with other members of the HER family (HER 1, 3, and 4), creating heterodimers. (Eventually, Monogram plans to be able to measure HER2 heterodimers as well.)
What triggers dimerization, and why might measuring HER2 dimer levels be important in predicting a patient’s response to anti-HER2 drug therapy?
Jodi Weidler, PharmD, senior director of clinical and research collaborations for Monogram Biosciences, explains: “HER2 as a receptor exists in an open conformation or position” on the cell membrane. And “theoretically, as the concentration of other HER2 monomers increases, those molecules can more easily homodimerize.” When ligands or growth factors activate HER1, 3, and 4, those can also dimerize with HER2 and trigger downstream signaling pathways.
Dimerization of the HER2 receptors leads to auto-phosphorylation, explains Tufts University’s Dr. Otis, director of surgical pathology at Baystate Medical Center, Springfield, Mass. And that’s “key to activation of the typical tyrosine kinase cascade,” he says. Dr. Otis compares the cascade to one person telling two other people something, who tell many others until the message is widely disseminated—the message being one instructing cells to proliferate.
Daniel F. Hayes, MD, clinical director of the breast oncology program at the University of Michigan Comprehensive Cancer Center, Ann Arbor, puts it another way: “Dimerized receptors imply you have an activated system.”
“It’s like a stoplight with the light on versus a stoplight that’s not turned on—the latter isn’t going to affect traffic flow.” Thus, the theory is that rather than measuring HER2, you measure activation of HER2 to identify whether therapy is going to work, he says.
The National Surgical Adjuvant Breast and Bowel Project is working with Monogram Biosciences scientists, using the company’s HERmark assay, to test the hypothesis that trastuzumab benefits only patients with breast cancer tumors that have HER2 homodimers, says Soonmyung Paik, MD, director of the Division of Pathology at the NSABP.
GlaxoSmithKline is also looking at HER2 dimerization to see if it could potentially predict whether someone will benefit from its drug lapatinib, an intracellular tyrosine kinase inhibitor of HER1 and HER2, says Steve Stein, MD, group director of GlaxoSmithKline’s oncology-medicine development center. The FDA approved the drug last year for use in combination with capecitabine to treat patients with HER2-positive advanced breast cancer who have received previous chemotherapy (a taxane and anthracycline) and trastuzumab.
“Theoretically [lapatinib] should inhibit HER1:HER2 heterodimers,” says Dr. Stein. “But we have to prove that by measuring dimerization, and also show that potentially in instances where the anti-HER2 antibody [trastuzumab] may not work, lapatinib could potentially have a competitive advantage by inhibiting both HER1 and HER2.”
Dr. Hayes says clinical trials now underway will determine if lapatinib can complement or replace trastuzumab.
Genentech’s investigational drug, pertuzumab, a humanized monoclonal antibody, is designed to inhibit HER2 from dimerizing with all of the HER family members on the surface of cancer cells, says Genentech spokesperson Charlotte Arnold. She says Genentech and Roche are initiating a phase III trial of pertuzumab and trastuzumab for the first-line treatment of HER2-positive metastatic breast cancer.
HER2 testing by IHC and FISH has been shown clearly in clinical trials to identify patients with stage 4 metastatic breast cancer who will benefit from trastuzumab. Yet recent preliminary research by the NSABP has raised an intriguing question: Do patients with HER2-negative breast cancer also benefit from the drug in the adjuvant setting?
That hypothesis raised its controversial head when the NSABP’s Dr. Paik and his team retested breast cancer tumors that had been confirmed initially as HER2 positive by community labs in the trastuzumab clinical trial (B31) for early breast cancer. They discovered that about 10 percent of the tumors were HER2 negative by both IHC and FISH. And that 10 percent of patients responded to trastuzumab in a way that’s similar to response in patients who had HER2-positive tumors in the clinical trial.
Dr. Paik presented that initial observation in an abstract at the June 2007 ASCO meeting. Since then, he and his colleagues at the NSABP have validated their results by testing the tumors’ HER2 RNA levels, which were also very low. Thus, the data suggest patients benefited from trastuzumab even when they were HER2 negative, Dr. Paik says. “It’s very confusing,” he adds.
Though not statistically significant, a similar trend emerged from another adjuvant trial, North Central Cancer Treatment Group (NCCTG) N9831, says Anne Blackwood-Chirchir, MD, Genentech’s group medical director for bio-oncology.
The NSABP is designing a clinical trial now to test whether trastuzumab works in HER2-negative patients. The National Cancer Institute is reviewing the concept and, once approved, the NSABP could start the study late this year, Dr. Paik says.
What might be going on in the B31 study where women with tumors retested by the NSABP as HER2 normal or low responded to trastuzumab?
The most straightforward hypothesis, says Dr. Blackwood-Chirchir, is that “there is some variability in testing and that, in fact, the tumors, if you had frozen ones, would show increased levels of HER2.” Were that the case, she says, there would be “no need for a new hypothesis to explain the response to trastuzumab.”
Dennis Slamon, MD, PhD, director of clinical/ translational research at UCLA’s Jonsson Comprehensive Cancer Center, whose research led to the development of trastuzumab, says there are “a lot of open questions about whether these HER2-negative patients are indeed HER2 negative. Remember,” he says, “they never would have been in the [B31] trial unless someone somewhere called them HER2 positive.” Dr. Slamon says the issue over testing accuracy can be easily worked out by having NSABP submit the tumor specimens for open “round robin testing,” which he says has been proposed to the NSABP.
Another plausible explanation for Dr. Paik’s findings is tumor genomic heterogeneity. In that scenario, says Dr. Blackwood-Chirchir, “a small clone of HER2-positive cells in an otherwise HER2-negative tumor would be the cells that would go on to develop micrometastasis and, later, clinically detectable metastatic disease. In such cases, a patient with a tumor that tested HER2 negative might benefit from trastuzumab. The challenge would be in identifying such patients prospectively.”
A third hypothesis might be that trastuzumab has a different mechanism of action on breast cancer in the adjuvant setting than in stage 4 metastatic disease. In the adjuvant setting, Dr. Paik points out, the main tumor has been removed and any potential micrometastatic sites are being treated. And “those tiny tumor sites may respond very well to trastuzumab even when their HER2 levels are quite low....” He says the drug might work in that setting not only by simply inhibiting the HER2 signaling pathway but also through an antibody response that the drug is engineered to produce. Thus, the “response could be an indirect one where the trastuzumab causes the body to mount an immune attack.”
Tufts’ Dr. Otis poses another possible explanation. He says the level of expression of a protein similar to HER2—epidermal growth factor receptor—“turned out to have little bearing on the patient’s response to therapy in patients with colonic adenocarcinoma.” Thus, he suspects that trastuzumab could be blocking other membrane receptor tyrosine kinases in patients responding to the drug whose tumors are HER2 negative by IHC, FISH, and RNA testing.
“These proteins [HER2 and epidermal growth factor] are membrane receptor tyrosine kinases which act, though not exclusively, through a central pathway involving a protein called Akt. Think of a central cog in a wheel with spokes coming from it connecting to the HER2 pathway, the epidermal growth factor pathway, and still others,” he says. “Even if HER2 is not amplified, trastuzumab may still block other pathways acting through Akt.”
Dr. Paik says it’s still too early to figure out who really benefits from trastuzumab in the adjuvant setting. “In a few years,” he says, “we will have more complementary assays in addition to FISH and IHC that will better categorize the patient [in terms of individualized treatment needs and prognosis].”
David Hicks, MD, director of surgical pathology at the University of Rochester (NY) Medical Center, predicts there will someday be HER2 profiles similar perhaps to the Oncotype Dx test, which takes ER-positive tumors and divides them into low, intermediate, and high-risk for recurrence. In fact, that type of risk-stratification approach is more than on the drawing board for Monogram Biosciences.
Ventana’s Dr. Grogan agrees the field has a lot of research potential and “room for tests to be combined in a complementary way.”
The “gene test for HER2 is powerful ...,” he says. But ultimately, “I believe we will use a matrix of tests—it won’t be a single gene.”
Karen Lusky is a writer in Brentwood, Tenn.