Solving diagnostic dilemmas with Multiplex IHC: early changes and prognosis in breast cancer

Jan. 1, 2012

Differentiation of cancer precursors from benign disease presents several diagnostic challenges which result from the highly subjective diagnostic criteria and the sheer complexity of the disease states. The diagnosis can strongly affect the choice of treatment modalities. This article will deal with use of novel Multiplex IHC technologies which can increase diagnostic accuracy in three critical areas where differentiation of neoplastic breast disease can be challenging and have a substantial effect on the choice of treatment, patient recovery times, patient anxiety, drug side effects, and costs for the patient and the healthcare system.

“Lobular vs. ductal” affects prognosis

Approximately 5% to 15% of breast cancers are invasive lobular carcinomas. Differentiating lobular and ductal disease can be challenging. Since classic lobular disease shows a more indolent clinical course, treatment paradigms are different than for ductal disease. For example, treatment for lobular carcinoma in situ (LCIS) does not generally include surgical resection. This reflects a better prognosis compared to ductal carcinoma in situ (DCIS), where progression to invasive carcinoma occurs post-surgically in 8% to 10% of cases. Dr. David Hicks, in a recent interview conducted by the author, comments: “DCIS is a surgical disease where you want to clear the margins in a surgical setting. LCIS is considered a risk lesion and not an obligate precursor, so a watchful-waiting approach as well as close follow up is often appropriate for classic LCIS patients.”

Lobular disease is often detected by a loss of e-cadherin,1 a protein involved in cell adhesion, using immunohistochemical (IHC) methods. However, the use of a negative marker alone is problematic.

The LC / DC™ Multiplex IHC test (Biocare Medical) consists of an antibody against the cell junction protein p120 in combination with E-cadherin to provide increased confidence in the differentiation of lobular vs. ductal neoplastic disease. Expression of p120 in combination with the loss of E-cadherin expression favors lobular disease (Figure 1). Expression of E-cadherin alone, or with low levels of focal p120 expression, however, favors ductal disease—hyperplasia, carcinoma in situ, or invasive carcinoma. (Figure 2) This Multiplex IHC test helps to differentiate variants of ductal disease, since E-cadherin marks the myoepithelial layer surrounding breast ducts.

Figure 1. p120 Positive, a. Lobular Hyperplasia, and b. Invasive Lobular Carcinoma Figure 2. p120 Negative, E-cadherin Positive (Ductal Cell Proliferation)

Dr. Hicks says, “In our hands, the Multiplex IHC combination of E-cadherin and p120 provided additional information informing the differentiation of ductal vs. lobular disease, making tumor cells easier to find. In addition, this Multiplex IHC test is particularly useful to reduce false negatives and false positives for microinvasion; when there are overlapping features of lobular carcinoma in situ and ductal carcinoma in situ in a lesion; when there is necrosis potentially masking microinvasion; to differentiate higher nuclear grade LCIS patients such as pleomorphic lobular carcinoma in situ which may mimic DCIS; and in LCIS samples where LCIS cells grow into a duct, or the lobule becomes distended so that its appearance is similar to a duct, making the differentiation of LCIS and DCIS more challenging.”

Usual hyperplasia vs. atypical ductal hyperplasia

Early changes that can lead to ductal breast carcinoma include both usual ductal hyperplasia (UDH) and atypical ductal hyperplasia (ADH).2 UDH, a benign lesion, does not result in an increased probability of developing breast cancer, so its presence may result in a watchful-waiting treatment scenario. In contrast, ADH is associated with progression to invasive carcinoma in nearly 4% to 5% of cases post-surgery,3 with risk increasing if the patient is premenopausal or has a family history of breast cancer. Because of the increased risk of progressing to cancer seen in patients with ADH, they are followed more closely or considered for risk reducing cancer prevention therapy.

The ADH-5™ Multiplex IHC (Biocare Medical) supports the differentiation of UDH from ADH. According to Sunil Badve, MD FRCPath, Professor, Departments of Pathology and Internal Medicine at Indiana University, a polyclonal proliferation, where some luminal epithelial cells express cytokeratins 5 and 14 (CK5/14) while others express CK7/18, favors UDH. In cases where all luminal epithelial cells express CK7/18 only, this monoclonal proliferation favors ADH.

A study led by Dr. Badve demonstrated high variation in the diagnosis of ADH, UDH, and DCIS when patient slides were interpreted by nine different pathologists. However, when Badve’s team used a novel Multiplex IHC test containing high and low molecular weight cytokeratins and p63 (ADH-5, Biocare Medical), concordance among pathologists increased by 34%. Dr. Badve’s study reduces the incidence of ADH based on use of the ADH-5 Multiplex IHC test, which is consistent with the literature, as it is well known that ADH is overdiagnosed. Prior studies indicate that as many as 40% of lesions diagnosed as ADH on core biopsies were found to be benign lesions upon excision.4


Microinvasive carcinoma of the breast is among the most commonly misdiagnosed breast disease entities.5 Diagnostic results can be false positive due to misinterpretation of lobular cancerization and sclerosing adenosis. False negative results can be due to masking of the foci of disease by inflammatory cells or falsely identifying tumor cells as histiocytes. Says Dr. Hicks: “In my laboratory, I use ADH-5 if I am worried about microinvasion since it clarifies whether the myoepithelial layer is intact, resolving potential misdiagnosis due to lobular cancerization and sclerosing adenosis, and helps to visualize tumor cells masked by inflammatory processes.” He has also used the LC/DC Multiplex IHC since visualization of a small foci of p120 expression outside of the myoepithelial layer (marked by E-cadherin) favors microinvasion if consistent with the index of suspicion and morphologic differential.

Microinvasion, if properly diagnosed and treated, is associated with a cure rate approaching 100% with local treatment alone,5 so it is important to identify this early and accurately using tools such as Multiplex IHC.


Multiplex IHC techniques have broad utility in differentiation of benign lesions from cancer precursors in breast and other cancers.
For more information on use of Multiplex IHC techniques to aid in the diagnosis of early changes in breast, prostate or bladder cancer, please visit

Mark D. Cross is Senior Director of Sales and Marketing for California-based Biocare Medical.


  1. Rakha EA ,et al. Clinical and biological significance of e-cadherin protein expression, in invasive lobular carcinoma of the breast. Am J Surg Pathol. October 2010;34(10):1472-9.
  2. Jain RK, et al. Atypical ductal hyperplasia: interobserver and intraobserver variation. Mod Pathol. July 2011; 917-923.
  3. Dupont W, Page D. Risk factors for breast cancer in women with proliferative breast disease. N Engl J Med. 1985; 312:146-151.
  4. Gal-Gombos EC, et al. Large-needle core biopsy in atypical intraductal epithelial hyperplasia including IHC expression of HMW cytokeratin: analysis of results of a single institution. Breast J. 2002;8:269-274.
  5. Hoda RS, et al. Microinvasive carcinoma of breast, a commonly misdiagnosed entity. Arch Pathol Lab Med. Sept 2001; Vol.125.