Nils M. Diaz

Persistent Activation of Stat3 Signaling Induces Survivin Gene Expression and Confers Resistance to Apoptosis in Human Breast Cancer Cells

Purpose: Signal transducer and activator of transcription 3 (Stat3) protein is persistently activated in breast cancer and promotes tumor cell survival. To gain a better understanding of the role of constitutive Stat3 signaling in breast cancer progression, we evaluated the expression profile of potential Stat3-regulated genes that may confer resistance to apoptosis. Experimental Design: Stat3 signaling was blocked with antisense oligonucleotides in human MDA-MB-435s breast cancer cells and Affymetrix GeneChip microarray analysis was done. The candidate Stat3 target gene Survivin was further evaluated in molecular assays using cultured breast cancer cells and immunohistochemistry of breast tumor specimens. Results: Survivin, a member of the inhibitor of apoptosis protein family, was identified as a potential Stat3-regulated gene by microarray analysis. This was confirmed in Survivin gene promoter studies and chromatin immunoprecipitation assays showing that Stat3 directly binds to and regulates the Survivin promoter. Furthermore, direct inhibition of Stat3 signaling blocked the expression of Survivin protein and induced apoptosis in breast cancer cells. Direct inhibition of Survivin expression also induced apoptosis. Increased Survivin protein expression correlates significantly (P = 0.001) with elevated Stat3 activity in primary breast tumor specimens from high-risk patients who were resistant to chemotherapy treatment. Conclusions: We identify Survivin as a direct downstream target gene of Stat3 in human breast cancer cells that is critical for their survival in culture. Our findings suggest that activated Stat3 signaling contributes to breast cancer progression and resistance to chemotherapy by, at least in part, inducing expression of the antiapoptotic protein, Survivin.

Mapping Geographic Zones of Cancer Risk with Epigenetic Biomarkers in Normal Breast Tissue

Purpose: Genetic alterations were previously identified in normal epithelia adjacent to invasive cancers. The aim of this study was to determine DNA methylation in histologically normal tissues from multiple geographic zones adjacent to primary breast tumors. Experimental Design: First, methylation status of a 4-kb region of RASSF1A promoter was interrogated using oligonucleotide-based microarray in 144 samples (primary tumors, 47; adjacent normals, 69; reduction mammoplasty tissues, 28). Second, allelic imbalance (AI)/loss of heterozygosity (LOH) surrounding RASSF1A promoter were analyzed in 30 samples (tumors, 8; adjacent normals, 22). Third, global methylation screening of 49 samples (tumors, 12; adjacent normals, 25; reduction mammoplasty, 12) was done by differential methylation hybridization. Real-time quantitative methylation-specific PCR was used to validate the microarray findings. Results: DNA methylation in the core RASSF1A promoter was low in reduction mammoplasty tissues (P = 0.0001) when compared with primary tumors. The adjacent normals had an intermediate level of methylation. The regions surrounding the core were highly methylated in all sample types. Microsatellite markers showed AI/LOH in tumors and some of the adjacent normals. Concurrent AI/LOH and DNA methylation in RASSF1A promoter occurred in two of six tumors. Global methylation screening uncovered genes more methylated in adjacent normals than in reduction mammoplasty tissues. The methylation status of four genes was confirmed by quantitative methylation-specific PCR. Conclusions: Our findings suggest a field of methylation changes extending as far as 4 cm from primary tumors. These frequent alterations may explain why normal tissues are at risk for local recurrence and are useful in disease prognostication.

Significance of Sentinel Lymph Node Micrometastases in Human Breast Cancer

BACKGROUND The significance of micrometastatic disease in the sentinel lymph nodes (SLN) of patients with invasive breast cancer has been questioned. The objective of our study was to review the impact of micrometastatic carcinoma detected by SLN biopsy. STUDY DESIGN Between January 1997 and May 2004, 2,408 patients with invasive breast cancer and an SLN with micrometastatic (N0[i+], N1mi) or no metastatic (N0[i-]) disease were identified through our breast database. Slide review was performed and reclassified by the 6(th) edition of the American Joint Committee on Cancer Staging Manual. Of these, 27 were excluded from analysis because of evidence of macrometastatic disease on slide review or enrollment in the American College of Surgeons Oncology Group Z10 study. RESULTS Of 2,381 patients, 2,108 were N0(i-), 151 were N0(i+), and 122 were N1mi. Overall and disease-free survivals of patients with an N1mi SLN were substantially worse than those in patients with an N0(i-) SLN (p < 0.001 and p=0.006, respectively). Additional positive non-SLNs were identified in 15.5% (15 of 97) of N1mi patients and 9.3% (10 of 107) of N0(i+) patients undergoing completion axillary lymph node dissection. Overall survival of the N0(i+) SLN patients not undergoing axillary dissection was substantially less than those undergoing axillary dissection (p=0.02). CONCLUSIONS Detection of micrometastatic carcinoma (N1mi) in the SLNs of invasive breast cancer patients is a major indicator of poorer survival compared with N0(i-) patients. Although survival of patients with an N0(i+) SLN does not statistically differ from that of N0(i-) patients, 9.3% of these patients had additional axillary nodal disease on axillary dissection, and N0(i+) patients had a decreased survival when axillary dissection was omitted.

Benign mechanical transport of breast epithelial cells to sentinel lymph nodes

The evaluation of sentinel lymph nodes (SLNs) for the presence of malignant epithelial cells is essential to the staging of breast cancer patients. Recently, increased attention has focused on the possibility that epithelial cells may reach SLNs by benign mechanical means, rather than by metastasis. The purpose of this study was to test the hypothesis that pre-SLN biopsy breast massage, which we currently use to facilitate the localization of SLNs, might represent a mode of benign mechanical transport. We studied 56 patients with invasive and/or in situ ductal carcinoma and axillary SLNs with only epithelial cells and/or cell clusters (≤0.2 mm in diameter and not associated with features of established metastases) detected predominantly in subcapsular sinuses of SLNs on hematoxylin and eosin- and/or anti-cytokeratin-stained sections. No patient had an SLN involved by either micro- or macro-metastatic carcinoma. Epithelial cells and cell clusters, ≤0.2 mm in size and without features of established metastases, occurred more frequently in the SLNs of patients who underwent pre-SLN biopsy breast massage (P < 0.001, χ2 test). The latter finding supports the hypothesis that pre-SLN biopsy breast massage is a mode of benign mechanical transport of epithelial cells to SLNs.

Radioactive seed localization breast biopsy and lumpectomy: can specimen radiographs be eliminated?

AbstractBackground: Wire localization (WL) is the current standard for surgical diagnosis of nonpalpable breast lesions. Many disadvantages inherent to WL are solved with radioactive seed localization (RSL). This trial investigated the ability of RSL to reduce the need for specimen radiographs and operating room delays associated with WL. Methods: A total of 134 women were entered onto an institutional review board–approved study. RSL was performed by placing a titanium seed containing .29 to 20 mCi of iodine-125 to within 1 cm of the suggestive breast lesion. The surgeon used a handheld gamma detector to locate and excise the iodine-125 seed and the lesion. Results: Specimen radiographs were eliminated in 98 (79%) of 124 patients. Surgical seed retrieval was 100% in 124 patients. No seed migration occurred after correct radiographical placement. A total of 26 (21%) of 124 patients required a specimen radiograph; 22 (85%) of these 26 were performed for microcalcifications. Conclusions: After surgical removal, RSL can eliminate specimen radiographs when the radiologist accurately places the seed and the pathologist grossly identifies the lesion. If small microcalcifications are noted before surgery, then specimen radiographs may be necessary. RSL reduced requirements for specimen radiographs, decreased OR time, improved incision placement, and improved resections to clear margins.

Completion axillary lymph node dissection minimizes the likelihood of false negatives for patients with invasive breast carcinoma and cytokeratin positive only sentinel lymph nodes

OBJECTIVE To document the incidence of metastatic disease in complete axillary lymph node dissections (CALND) of patients with invasive carcinoma after a sentinel lymph node (SLN) biopsy, positive only by immunohistochemical staining for cytokeratin (CK-IHC). METHODS Sections of all SLNs, negative by routine histology, were immunostained and examined for cytokeratin positive cells. Sections of lymph nodes from CALND specimens were interpreted using routine hematoxylin and eosin (H&E) staining. RESULTS A total of 409 patients (29.6%) had metastatic disease in at least one sentinel lymph node on H&E examination. Of 971 H&E negative patients, 78 (8.0%) were positive only by CK-IHC. Sixty-two of the CK-IHC positive only patients underwent CALND. Nine of these 62 patients (14.5%) had metastases identified in the CALND specimen. CONCLUSIONS Because 14.5% of patients with invasive breast cancer and SLNs positive only by CK-IHC were found to have H&E positive lymph nodes on CALND, we conclude first, that CK-IHC should be used to evaluate SLNs, and second, that CALND should be considered when SLNs are positive by CK-IHC only. This approach will result in an absolute reduction of the false negative rate (absolute false negative rate reduced by 2.6% in our series).

Survival Outcomes in Node-Negative Breast Cancer Patients Evaluated With Complete Axillary Node Dissection Versus Sentinel Lymph Node Biopsy

BackgroundSentinel lymph node (SLN) biopsy combined with microstaging-associated immunohistochemical staining for cytokeratin more accurately assigns patients to their corresponding diagnostic stage. The purpose of this study was to compare the survival outcomes of node-negative patients who received an SLN biopsy with historical control data of node-negative patients who received routine complete axillary lymph node dissection (CALND) in the pre-SLN biopsy era.MethodsUnder institutional review board approval, 2458 node-negative invasive breast cancer patients between the ages of 25 and 94 years (mean, 60 years) were treated at our institution from January 1986 to May 2004. Of these 2458 patients, 604 (25%) were evaluated with CALND, whereas 1854 (75%) were evaluated with SLN biopsy. All were treated according to the current stage-specific guidelines. Kaplan-Meier graphs of overall survival and disease-free survival were constructed for each group of patients, and the two groups were compared by using the log-rank test.ResultsOverall survival and disease-free survival for the CALND and SLN biopsy groups did not differ significantly (P = .98). The average number of lymph nodes extracted in the pre-SLN biopsy group was 18, whereas the average number of SLNs extracted in the post-SLN biopsy group was 3.ConclusionsThe survival rate among node-negative breast cancer patients who received an SLN biopsy alone has proven to have no significant difference (P = .98) from the survival rate among node-negative patients who received a CALND. SLN biopsy alone should replace CALND as the primary tool for axillary staging of breast cancer in node-negative patients.

Modes of benign mechanical transport of breast epithelial cells to axillary lymph nodes.

The status of axillary lymph nodes is a key prognostic indicator available for the management of patients with breast cancer. Sentinel lymph node (SLN) evaluation as a predictor of lymph node status has led to increased use of ancillary methods, principally immunohistochemistry, to increase the sensitivity of the SLN biopsy. So-called “occult” micrometastases detected by such methods have led to speculation that some may have reached the SLNs by benign mechanical transport (BMT) rather than a metastatic process. We review evidence suggesting two potential modes of BMT: lymphatic transport of epithelial cells displaced by biopsy of the primary breast tumor and by breast massage-assisted SLN localization. The biopsy techniques under most scrutiny include fine needle aspiration and large-gauge core biopsy. The evidence implicating breast massage prior to SLN biopsy as a mode of BMT has been supported by statistical analysis; however, no method of distinguishing massage-associated cells in SLNs from true occult micrometastases is available. The significance of small epithelial clusters in SLNs is currently unknown. Thus, deviation from current biopsy and SLN-localizing practices is unwarranted.

Effect of Lymphatic Mapping on Diagnosis and Treatment of Patients with T1a, T1b Favorable Breast Cancer

ObjectiveTo investigate the incidence of nodal metastasis in a consecutive series of patients treated at the authors’ institution with highly selective criteria, and to determine the impact that lymphatic mapping and sentinel node biopsy have on the detection of nodal metastases in this carefully selected patient population. MethodsStudy patients were selected from the 7,750 breast cancer patients entered into the authors’ database from April 1989 to August 2001, based on the following criteria: nonpalpable, T1a and T1b, non-high nuclear grade tumors, without lymphovascular invasion. ResultsOf the 7,750 patients in the database 1,327 (17%) were found to have T1a and T1b lesions. Three hundred eighty-nine patients were confirmed to meet all four selection criteria. This represents 5% (389/7,750) of the authors’ breast cancer patients and 29.3% (389/1,327) of the authors’ T1a/T1b tumors. One hundred sixty patients were diagnosed before routine use of lymphatic mapping, and only one patient had a positive axillary lymph node. Two hundred twenty-nine patients underwent lymphatic mapping and sentinel lymph node biopsy, and 10 had a positive axillary lymph node. The difference in proportions of nodal positivity between the mapped and unmapped patients was significant. ConclusionsThis study clearly demonstrates the ability of lymphatic mapping and a more detailed examination of the sentinel node to increase the accuracy of axillary staging. It has been argued that this highly selected group of breast cancer patients possessing retrospectively identified “favorable” characteristics does not require axillary staging. This select population represents only 5% of breast cancer patients in this series, and the authors do not believe they can be accurately identified preoperatively. Therefore, the authors strongly argue for evaluation of the axillary nodal status by lymphatic mapping.

Problems with the use of breast conservation therapy for breast cancer in a patient with neurofibromatosis type 1: a case report.

Abstract:  Patients with Neurofibromatosis type I and breast cancer represent a subset of people who may be considered at high risk for secondary cancers after conventional whole breast radiation therapy and breast conservation surgery. A case of a 49‐year‐old woman with neurofibromatosis type I is presented. She was diagnosed with a 1.1‐cm right breast infiltrating ductal carcinoma. Clinical, diagnostic imaging, and pathologic features are discussed. Her initial treatment plan of breast conserving therapy was thwarted when her sentinel node biopsy was positive for micrometastatic disease in 1/14 lymph nodes. She elected to have a bilateral simple mastectomy. This case addresses the rare dilemma of offering breast conservation therapy as a viable option for patients with neurofibromatosis type I. Current data on radiation‐induced secondary cancers such as sarcoma after treatment for breast and other cancers are reviewed.