Lillian Young

Most early disseminated cancer cells detected in bone marrow of breast cancer patients have a putative breast cancer stem cell phenotype.

Purpose: The presence of disseminated tumor cells (DTC) in the bone marrow of breast cancer patients is an acknowledged independent prognostic factor. The biological metastatic potential of these cells has not yet been shown. The presence of putative breast cancer stem cells is shown both in primary tumors and distant metastases. These cells with a CD44+CD24−/low phenotype represent a minor population in primary breast cancer and are associated with self-renewal and tumorigenic potential. Recognizing the potential effect of prevalence of putative stem cells among DTC, we evaluated the bone marrow DTC. Experimental Design: We employed the double/triple-staining immunohistochemistry protocol and modified the established bone marrow cytokeratin (CK) staining protocol by adding steps for additional antigens, CD44 and/or CD24. We evaluated 50 bone marrow specimens, previously categorized as CK+ from early breast cancer patients. CK+ cells were examined for CD44 and CD24 expression by light microscopy, fluorescence microscopy, and spectral imaging. Results: We detected the putative stem cell–like phenotype in all CK+ specimens. The mean prevalence of putative stem/progenitor cells was 72% and median prevalence was 65% (range, 33-100%) among the overall DTC per patient, compared with primary tumors where this phenotype is reported in <10% of cells. Conclusions: This is the first evidence of the existence of the putative stem-like phenotype within the DTC in bone marrow in early breast cancer patients. All patients had a putative stem cell phenotype among the DTC and most individual DTC showed such phenotype. Future molecular characterization of these cells is warranted.

Antigen retrieval immunohistochemistry under the influence of pH using monoclonal antibodies.

Antigen retrieval (AR) incorporating high-temperature microwave (MW) heating of tissue sections before immunostaining is a revolutionary technique that can unmask the antigens in formalin-fixed tissue sections, thus making them available for immunohistochemical staining. Although high temperature is believed to be the primary mechanism in retrieval of antigens, a variety of chemical solutions have been tested to define an optimal AR solution. We tested the hypothesis that pH of the AR solution may influence the quality of immunostaining by using seven different AR buffer solutions at a series of different pH values ranging from 1 to 10. We evaluated the staining of monoclonal antibodies to cytoplasmic antigens (AE1, HMB45, NSE), nuclear antigens (MIB-1, PCNA, ER), and cell surface antigens (MT1, L26, EMA) on routinely formalin-fixed, paraffin-embedded sections under different pH conditions with MW heating for 10 min. The intensity of immunostaining was graded in a blinded fashion. The pH value of the AR buffer solution was carefully measured before, immediately after, and 15 min after the AR procedure. The influence of pH on AR immunohistochemical staining can be summarized into three patterns. Some antigens (L26, PCNA, AE1, EMA, and NSE) showed excellent retrieval throughout the pH range. Other antigens (MIB1 and ER) showed strong intensity of immunohistochemical staining at very low pH and at neutral to high pH, but a dramatic decrease in the intensity of the AR immunostaining at moderately acidic pH (pH 3-6). Still others (MT1 and HMB45) showed increasing intensity of the AR immunostaining with increasing pH, but only weak immunostaining at low pH. Among the seven buffer solutions at any given pH value, the intensity of AR immunostaining was very similar. However, Tris-HCl buffer tended to produce better results at higher pH, compared with other buffers. Although high-temperature heating is believed to be the most important factor for the AR technique, the pH value of the AR solution is an important co-factor for some antigens. Optimization of the AR system should therefore include optimization of the pH of the AR solution. Our results indicate that AR immunostaining of Tris-HCl or sodium acetate buffer at pH 8-9 may be suitable for most antigens, although certain nuclear antigens show optimal staining at low pH.

Antigen retrieval technique utilizing citrate buffer or urea solution for immunohistochemical demonstration of androgen receptor in formalin-fixed paraffin sections.

We developed a staining protocol for demonstration of androgen receptor (AR) in formalin-fixed, paraffin-embedded tissue sections. The method is based on the antigen retrieval microwave (MW) heating technique. Results are compared with different types of enzyme digestion pre-treatments. The strongest immunostaining signal and clearest background were obtained by MW heating of dewaxed paraffin sections in 5% urea or citrate buffer solution (pH 6); pure distilled water gave less consistent results. Enzymatic digestion with pepsin (0.05% in 2 N HCl) for 30 min at room temperature, or trypsin followed by pronase, or pronase digestion alone, also produced enhanced staining of AR in some cases, but there was more nonspecific background, and specific reactivity was less intense. The antigen retrieval MW method can be used to demonstrate AR epitope in prostate tissue after fixation in formalin for as long as 7 days. AR immunolocalization was also compared in frozen and paraffin sections processed from the same specimen of prostate carcinoma tissue and was found to be qualitatively and quantitatively similar. This study also provided new information concerning the basic principles of the antigen retrieval MW method that may be helpful in further development of this technique.

Strategies for improving the immunohistochemical staining of various intranuclear prognostic markers in formalin-paraffin sections: Androgen receptor, estrogen receptor, progesterone receptor, p53 protein, proliferating cell nuclear antigen, and Ki-67 antigen revealed by antigen retrieval techniques

Different variations of the antigen retrieval technique using different retrieval solutions have been evaluated for their effectiveness in restoring the antigenicity of six intranuclear antigens, each of which is a potentially valuable prognostic indicator in formalin-fixed, paraffin-embedded tissue sections. The results of immunohistochemical staining for estrogen receptor, progesterone receptor, androgen receptor, p53 protein, proliferating cell nuclear antigen, and Ki-67 antigen were compared following the different antigen retrieval approaches. The strongest immunostaining signal with the clearest background was obtained by microwave heating of dewaxed paraffin sections for 10 minutes in 0.05 mol/L glycine HCl (pH 3.5) or in citrate buffer solution (pH 6). Urea solution, distilled water, and lead thiocyanate solution yielded improvements with some antigens, but less consistently and less impressively than glycine HCl buffer or citrate buffer. Following antigen retrieval nuclear staining was sharply defined and could be achieved consistently in a variety of tissues after formalin fixation for as long as 7 days. The duration of fixation, however, was an important variable; generally, the longer the fixation time the more vigorous the retrieval procedure required. This study demonstrates the ability to stain a variety of intranuclear antigens, which are not readily demonstrable otherwise, in formalin-paraffin sections with a high degree of consistency and reproducibility. The availability of methods that are effective in paraffin sections may facilitate studies of the possible value of these markers as prognostic indicators for predicting the response of major tumors to different forms of therapy. This study also provided insight into the basic principles of the antigen retrieval method, which may be helpful in attempts to develop a more uniformly standardized technique applicable to many different antigen systems.

Combined Effects of p53, p21, and pRb Expression in the Progression of Bladder Transitional Cell Carcinoma

PURPOSE To determine the combined effects of p53, p21, and pRb alterations in predicting the progression of bladder transitional cell carcinoma. PATIENTS AND METHODS p53, p21, and pRb expression was examined immunohistochemically on archival radical cystectomy samples from 164 patients with invasive or high-grade recurrent superficial transitional cell carcinoma (TCC; lymph node-negative, 117 patients; lymph node-positive, 47 patients). Median follow-up was 8.6 years. Based on percentage of nuclear reactivity, p53 was considered as wild-type (0% to 10%) or altered (>10%); p21 was scored as wild-type (>10%) or altered (<10%); and pRb status was considered wild-type (1% to 50%) or altered (0% or >50%). RESULTS As individual determinants, the p53, p21, and pRb status were independent predictors of time to recurrence (P<.001, P<.001, and P<.001, respectively), and overall survival (P<.001, P=.002, and P=.001, respectively). By examining these determinants in combination, patients were categorized as group I (no alteration in any determinant, 47 patients), group II (any one determinant altered, 51 patients), group III (any two determinants altered, 42 patients), and group IV (all three determinants altered, 24 patients). The 5-year recurrence rates in these groups were 23%, 32%, 57%, and 93%, respectively (log-rank P<.001), and the 5-year survival rates were 70%, 58%, 33%, and 8%, respectively (log-rank P<.001). After stratifying by stage, the number of altered proteins remained significantly associated with time to recurrence and overall survival. CONCLUSION This study suggests that alterations in p53, p21, and pRb act in cooperative or synergistic ways to promote bladder cancer progression. Examining these determinants in combination provides additional information above the use of a single determinant alone.

INCIDENCE OF OCCULT LYMPH NODE METASTASES IN PATHOLOGICAL STAGE C (pT3NO) PROSTATE CANCER

PURPOSE To determine the incidence of occult lymph node metastases in patients with stage pT3N0 prostate cancer. MATERIALS AND METHODS Lymph nodes from 95 patients with stage pT3N0 prostate cancer were analyzed by immunohistochemistry for extrinsic epithelial cells using epithelial-specific monoclonal antibodies. The extrinsic epithelial cells were also tested for prostate specific antigen expression. RESULTS Occult lymph node metastases were identified in 15 cases (16%) and were more frequent in patients with high primary Gleason grade tumors and seminal vesicle invasion (p = 0.03). In all cases the extrinsic cells were of prostate origin based on prostate specific antigen expression. CONCLUSIONS Occult lymph node metastases can be detected in a substantial proportion of patients with stage pT3N0 prostate cancer, are associated with known predictors of disease progression, and may be useful in identifying patients at risk for recurrence and progression.

Multidrug resistance (MDR-1) expression in AIDS-related lymphomas.

P-glycoprotein is a product of the multidrug resistance (MDR-1) gene. In non-Hodgkins lymphoma, less than 20% of untreated de novo lymphomas express MDR-1 compared with approximately 50% after failure of chemotherapy. We wished to study the expression of MDR-1 in AIDS-related non-Hodgkins lymphoma (AIDS-NHL). Tissue biopsies from 50 patients with newly diagnosed AIDS-NHL were studied by immunohistochemical analysis using C494, a monoclonal antibody specific for the MDR-1 isoform of P-gp. MDR-1 expression was correlated with patient demographics, lymphoma characteristics, response to chemotherapy, and survival. Forty-six males and four females with a median age of 38 years (range 26-63) were studied. A prior AIDS-defining opportunistic infection was reported in 35 patients (70%). The median CD4+ lymphocyte count was 69/mm(3) (range 0-920). Thirty-two patients (63%) had received prior anti-HIV therapy, including a protease inhibitor in five (10%). Pathologic types consisted of diffuse large cell in 13 (26%), immunoblastic in 13 (26%), small non-cleaved in 22 (44%), and high grade not otherwise specified in two (4%). The majority of patients (76%) had stage III/IV disease. Pre-treatment lymphoma tissues from 33 patients (66%) stained positively for MDR-1. MDR-1 positive patients had a significantly lower complete remission rate compared to MDR-1 negative patients (33 versus 65%, P=0.042). Duration of complete response was significantly longer in MDR-1 negative patients compared with MDR-1 positive patients (not reached versus 9.9 months, P=0.003). Strategies to overcome MDR-1 expression may be important for initial treatment in patients with AIDS-NHL.

Calcium-induced Modification of Protein Conformation Demonstrated by Immunohistochemistry: What Is the Signal?

A recent study by Morgan et al. on the mechanism of the heating antigen retrieval (AR) has raised an interesting issue concerning calcium-induced modification of protein conformation demonstrated by immunohistochemistry (IHC). The current study is based on calcium-induced modification of thrombospondin (TSP) and Ki-67, as demonstrated by IHC using seven monoclonal antibodies (MAbs) to TSP and an MAb MIB1. Experiments were carried out on frozen tissue sections of bladder carcinoma and lymph node. Frozen sections were incubated with solutions of 50 mM CaCl2 and/or 10 mM EDTA at 4C overnight before formalin or acetone fixation for TSP and Ki-67, respectively. Sections were then fixed in 10% neutral buffered formalin or acetone before immunostaining. Seven MAbs to TSP, named Ab1 to 7 representing clone numbers of A4.1, D4.6, C6.7, A6.1, B5.2, A2.5, and HB8432, respectively, and MIB1 were utilized as primary antibodies. ABC was used as the detection system and AEC as the chromogen for immunohistochemical staining. An extracellular immunostaining pattern represented a positive result for TSP, and nuclear staining for MIB1. Frozen sections preincubated in 50 mM CaCl2 overnight at 4C showed significant loss of staining and/or altered staining pattern for six of the seven antibodies to TSP and MIB1 compared to positive controls not exposed to CaCl2. Lack of immunostaining of TSP and MIB1 attributable to exposure to CaCl2 could be partially recovered by incubating the frozen sections in EDTA. Calcium-induced modification of protein structure was demonstrated more than 10 years ago on the basis of immunochemical techniques. In this study, similar calcium-induced modification of protein was detectable by IHC in frozen tissue sections, suggesting that calcium-induced modification of protein structure may occur independently of fixation-induced modification. The fact that calcium binding may affect IHC staining is not surprising in view of the fact that antibody/antigen interactions are protein structure-dependent. However, in this experiment the change occurred before and independent of formalin fixation and does not necessarily imply a role for calcium in AR. There may be a valuable role for the use of chemical modification in visualization of protein structure changes in tissue sections by IHC.

Antigen Retrieval Using pH 3.5 Glycine-HCI Buffer or Urea Solution for Immunohistochemical Localization of Ki-67

A new antibody (MIB-1) has been described, permitting the demonstration of Ki-67 proliferation antigen in paraffin sections. However, satisfactory results were obtained only after subjecting tissue sections to microwave based antigen retrieval in citrate buffer solution. Other buffer solutions produce equivalent or better results and also permit use of the original Ki-67 antibody, which hitherto has been considered ineffective for paraffin sections.

Human Breast Cancer Histoid An In Vitro 3-Dimensional Co-culture Model That Mimics Breast Cancer Tissue

Progress in our understanding of heterotypic cellular interaction in the tumor microenvironment, which is recognized to play major roles in cancer progression, has been hampered due to unavailability of an appropriate in vitro co-culture model. The aim of this study was to generate an in vitro 3-dimensional human breast cancer model, which consists of cancer cells and fibroblasts. Breast cancer cells (UACC-893) and fibroblasts at various densities were co-cultured in a rotating suspension culture system to establish co-culture parameters. Subsequently, UACC-893, BT.20, or MDA.MB.453 were co-cultured with fibroblasts for 9 days. Co-cultures resulted in the generation of breast cancer histoid (BCH) with cancer cells showing the invasion of fibroblast spheroids, which were visualized by immunohistochemical (IHC) staining of sections (4 µm thick) of BCH. A reproducible quantitative expression of C-erbB.2 was detected in UACC-893 cancer cells in BCH sections by IHC staining and the Automated Cellular Imaging System. BCH sections also consistently exhibited qualitative expression of pancytokeratins, p53, Ki-67, or E-cadherin in cancer cells and that of vimentin or GSTPi in fibroblasts, fibronectin in the basement membrane and collagen IV in the extracellular matrix. The expression of the protein analytes and cellular architecture of BCH were markedly similar to those of breast cancer tissue.