Victor P. Andrade

Lobular Carcinoma in Situ: A 29-Year Longitudinal Experience Evaluating Clinicopathologic Features and Breast Cancer Risk

PURPOSE The increased breast cancer risk conferred by a diagnosis of lobular carcinoma in situ (LCIS) is poorly understood. Here, we review our 29-year longitudinal experience with LCIS to evaluate factors associated with breast cancer risk. PATIENTS AND METHODS Patients participating in surveillance after an LCIS diagnosis are observed in a prospectively maintained database. Comparisons were made among women choosing surveillance, with or without chemoprevention, and those undergoing bilateral prophylactic mastectomies between 1980 and 2009. RESULTS One thousand sixty patients with LCIS without concurrent breast cancer were identified. Median age at LCIS diagnosis was 50 years (range, 27 to 83 years). Fifty-six patients (5%) underwent bilateral prophylactic mastectomy; 1,004 chose surveillance with (n = 173) or without (n = 831) chemoprevention. At a median follow-up of 81 months (range, 6 to 368 months), 150 patients developed 168 breast cancers (63% ipsilateral, 25% contralateral, 12% bilateral), with no dominant histology (ductal carcinoma in situ, 35%; infiltrating ductal carcinoma, 29%; infiltrating lobular carcinoma, 27%; other, 9%). Breast cancer incidence was significantly reduced in women taking chemoprevention (10-year cumulative risk: 7% with chemoprevention; 21% with no chemoprevention; P < .001). In multivariable analysis, chemoprevention was the only clinical factor associated with breast cancer risk (hazard ratio, 0.27; 95% CI, 0.15 to 0.50). In a subgroup nested case-control analysis, volume of disease, which was defined as the ratio of slides with LCIS to total number of slides reviewed, was also associated with breast cancer development (P = .008). CONCLUSION We observed a 2% annual incidence of breast cancer among women with LCIS. Common clinical factors used for risk prediction, including age and family history, were not associated with breast cancer risk. The lower breast cancer incidence in women opting for chemoprevention highlights the potential for risk reduction in this population.

Protocol for PTEN Expression by Immunohistochemistry in Formalin-fixed Paraffin-embedded Human Breast Carcinoma

The PI3K/PTEN pathway plays a major role in carcinogenesis. Dysregulation of this pathway occurs frequently in breast cancer, and loss of PTEN expression is emerging as a potentially important mechanism of resistance to the widely used anti-HER2 therapy, trastuzumab. However, assays for loss of PTEN expression have suffered from lack of consistency. Here, we describe an automated and reliable protocol for PTEN protein expression by immunohistochemistry in formalin-fixed paraffin-embedded tissue that can be easily incorporated into clinical trials.

Temporal and spatial patterns of endogenous danger signal expression after wound healing and in response to lymphedema

While acute tissue injury potently induces endogenous danger signal expression, the role of these molecules in chronic wound healing and lymphedema is undefined. The purpose of this study was to determine the spatial and temporal expression patterns of the endogenous danger signals high-mobility group box 1 (HMGB1) and heat shock protein (HSP)70 during wound healing and chronic lymphatic fluid stasis. In a surgical mouse tail model of tissue injury and lymphedema, HMGB1 and HSP70 expression occurred along a spatial gradient relative to the site of injury, with peak expression at the wound and greater than twofold reduced expression within 5 mm (P < 0.05). Expression primarily occurred in cells native to injured tissue. In particular, HMGB1 was highly expressed by lymphatic endothelial cells (>40% positivity; twofold increase in chronic inflammation, P < 0.001). We found similar findings using a peritoneal inflammation model. Interestingly, upregulation of HMGB1 (2.2-fold), HSP70 (1.4-fold), and nuclear factor (NF)-κβ activation persisted at least 6 wk postoperatively only in lymphedematous tissues. Similarly, we found upregulation of endogenous danger signals in soft tissue of the arm after axillary lymphadenectomy in a mouse model and in matched biopsy samples obtained from patients with secondary lymphedema comparing normal to lymphedematous arms (2.4-fold increased HMGB1, 1.9-fold increased HSP70; P < 0.01). Finally, HMGB1 blockade significantly reduced inflammatory lymphangiogenesis within inflamed draining lymph nodes (35% reduction, P < 0.01). In conclusion, HMGB1 and HSP70 are expressed along spatial gradients and upregulated in chronic lymphatic fluid stasis. Furthermore, acute expression of endogenous danger signals may play a role in inflammatory lymphangiogenesis.

Clonal relatedness between lobular carcinoma in situ and synchronous malignant lesions

IntroductionLobular carcinoma in situ (LCIS) has been accepted as a marker of risk for the development of invasive breast cancer, yet modern models of breast carcinogenesis include LCIS as a precursor of low-grade carcinomas. We provide evidence favoring a clonal origin for LCIS and synchronous estrogen receptor-positive malignant lesions of the ductal and lobular phenotype.MethodsPatients with prior LCIS undergoing mastectomy were identified preoperatively from 2003 to 2008. Specimens were widely sampled, and frozen blocks were screened for LCIS and co-existing malignant lesions, and were subject to microdissection. Samples from 65 patients were hybridized to the Affymetrix SNP 6.0 array platform. Cases with both an LCIS sample and an associated ductal carcinoma in situ (DCIS) or invasive tumor sample were evaluated for patterns of somatic copy number changes to assess evidence of clonal relatedness.ResultsLCIS was identified in 44 of the cases, and among these a DCIS and/or invasive lesion was also identified in 21 cases. A total of 17 tumor pairs had adequate DNA/array data for analysis, including nine pairs of LCIS/invasive lobular cancer, four pairs of LCIS/DCIS, and four pairs of LCIS/invasive ductal cancer. Overall, seven pairs (41%) were judged to be clonally related; in five (29%) evidence suggested clonality but was equivocal, and five (29%) were considered independent. Clonal pairs were observed with all matched lesion types and low and high histological grades. We also show anecdotal evidence of clonality between a patient-matched triplet of LCIS, DCIS, and invasive ductal cancer.ConclusionOur results support the role of LCIS as a precursor in the development of both high-grade and low-grade ductal and lobular cancers.

p21cip/WAF is a key regulator of long-term radiation damage in mesenchyme-derived tissues

This study aimed to determine the mechanisms responsible for long-term tissue damage following radiation injury. We irradiated p21-knockout (p21(-/-)) and wild-type (WT) mice and determined the long-term deleterious effects of this intervention on mesenchyme-derived tissues. In addition, we explored the mechanisms of radiation-induced mesenchymal stem cell (MSC) dysfunction in isolated bone marrow-derived cells. p21 expression was chronically elevated >200-fold in irradiated tissues. Loss of p21 function resulted in a >4-fold increase in the number of skin MSCs remaining after radiation. p21(-/-) mice had significantly less radiation damage, including 6-fold less scarring, 40% increased growth potential, and 4-fold more hypertrophic chondrocytes in the epiphyseal plate (P<0.01). Irradiated p21(-/-) MSCs had 4-fold increased potential for bone or fat differentiation, 4-fold greater proliferation rate, and nearly 7-fold lower senescence as compared to WT MSCs (P<0.01). Ectopic expression of p21 in knockout cells decreased proliferation and differentiation potential and recapitulated the WT phenotype. Loss of p21 function markedly decreases the deleterious effects of radiation injury in mesenchyme-derived tissues and preserves tissue-derived MSCs. In addition, p21 is a critical regulator of MSC proliferation, differentiation, and senescence both at baseline and in response to radiation.

PI3K pathway activation in high-grade ductal carcinoma in situ-implications for progression to invasive breast carcinoma

Purpose: To assess the prevalence of phosphoinositide 3-kinase (PI3K) pathway alterations in pure high-grade ductal carcinoma in situ (DCIS) and DCIS associated with invasive breast cancer (IBC), and to determine whether DCIS and adjacent IBCs harbor distinct PI3K pathway aberrations. Experimental Design: Eighty-nine cases of pure high-grade DCIS and 119 cases of high-grade DCIS associated with IBC were characterized according to estrogen receptor (ER) and HER2 status, subjected to immunohistochemical analysis of PTEN, INPP4B, phosphorylated (p)AKT and pS6 expression, and to microdissection followed by Sequenom genotyping of PIK3CA and AKT1 hotspot mutations. Results: Alterations affecting the PI3K pathway were found in a subset of pure DCIS and DCIS adjacent to IBC. A subtype-matched comparison of pure DCIS and DCIS adjacent to IBC revealed that PIK3CA hotspot mutations and pAKT expression were significantly more prevalent in ER-positive/HER2-negative DCIS adjacent to IBC (P values, 0.005 and 0.043, respectively), and that in ER-negative/HER2-positive cases INPP4B loss of expression was more frequently observed in pure DCIS (a P value of 0.013). No differences in the parameters analyzed were observed in a pairwise comparison of the in situ and invasive components of cases of DCIS and adjacent IBC. Analysis of the PIK3CA-mutant allelic frequencies in DCIS and synchronous IBC revealed cases in which PIK3CA mutations were either restricted to the DCIS or to the invasive components. Conclusion: Molecular aberrations affecting the PI3K pathway may play a role in the progression from high-grade DCIS to IBC in a subset of cases (e.g., a subgroup of ER-positive/HER2-negative lesions). Clin Cancer Res; 20(9); 2326–37. ©2014 AACR.

Gene expression profiling of lobular carcinoma in situ reveals candidate precursor genes for invasion

Lobular carcinoma in situ (LCIS) is both a risk indicator and non‐obligate precursor of invasive lobular carcinoma (ILC). We sought to characterize the transcriptomic features of LCIS and ILC, with a focus on the identification of intrinsic molecular subtypes of LCIS and the changes involved in the progression from normal breast epithelium to LCIS and ILC.

Clonal relationships between lobular carcinoma in situ and other breast malignancies

BackgroundRecent evidence suggests that lobular carcinoma in situ (LCIS) can be a clonal precursor of invasive breast cancers of both the ductal and lobular phenotypes. We sought to confirm these findings with an extensive study of fresh frozen breast specimens from women undergoing mastectomy.MethodsPatients with a history of LCIS presenting for therapeutic mastectomy were identified prospectively. Frozen tissue blocks were collected, screened for lesions of interest, and subjected to microdissection and DNA extraction. Copy number profiling, whole-exome sequencing, or both were performed. Clonal relatedness was assessed using specialized statistical techniques developed for this purpose.ResultsAfter exclusions for genotyping failure, a total of 84 lesions from 30 patients were evaluated successfully. Strong evidence of clonal relatedness was observed between an LCIS lesion and the invasive cancer for the preponderance of cases with lobular carcinoma. Anatomically distinct in situ lesions of both ductal and lobular histology were also shown to be frequently clonally related.ConclusionsThese data derived from women with LCIS with or without invasive cancer confirm that LCIS is commonly the clonal precursor of invasive lobular carcinoma and that distinct foci of LCIS frequently share a clonal origin, as do foci of LCIS and ductal carcinoma in situ.

Lymphovascular invasion and histologic grade are associated with specific genomic profiles in invasive carcinomas of the breast

Lymphovascular invasion (LVI) and histologic grade are clinical parameters of high prognostic value in breast cancer and indicate the level of tumor aggressiveness. Many studies have focused on the association of breast cancer subtypes with gene expression and chromosomal profiles, but considerably less genomic information is available regarding traditional prognostic factors such as histologic grade and LVI. We studied by array-CGH a group of 57 invasive ductal carcinomas of the breast to outline the DNA copy number aberration (CNA) profile linked to high histologic grades and LVI. Selected CNAs were validated using real-time quantitative PCR (qPCR). Furthermore, gene expression analysis was performed in a subset of 32 of these tumors, and findings were integrated with array-CGH data. Our findings indicated an accumulation of genomic alterations in high-grade breast tumors compared to low-grade samples. Grade III tumors showed higher number of CNAs and larger aberrations than low-grade tumors and displayed a wide range of chromosomal aberrations, which were mainly 5p, 8q, 10p, 17q12, and 19 gains, and 3p, 4, 5q proximal, 9p, 11p, 18q, and 21 losses. The presence of LVI, a well-established prognostic marker, was not significantly associated with increased genomic instability in comparison to breast tumors negative for LVI, considering the total number of chromosomal alterations. However, a slightly increase in the frequency of specific alterations could be detected in LVI-positive group, such as gains at 5p, 16p, 17q12, and 19, and losses at 8p, 11q, 18q, and 21. Three newly reported small-scale rearrangements were detected in high-risk tumors (LVI-positive grade III) harboring putative breast cancer genes (amplicons at 4q13.3 and 11p11.2, and a deletion at 12p12.3). Furthermore, gene expression analysis uncovered networks highlighting S100A8, MMP1, and MED1 as promising candidate genes involved in high-grade and LVI-positive tumors. In summary, a group of genomic regions could be associated with high-risk tumors, and expression analysis pinpointed candidate genes deserving further investigation. The data has shed some light on the molecular players involved in two highly relevant prognostic factors and may further add to the understanding of the mechanisms of breast cancer aggressiveness.

Differentially Expressed Genes in Window Trials are Influenced by the Wound-Healing Process: Lessons Learned from a Pilot Study with Anastrozole

BACKGROUND Perioperative window trials provide an opportunity to obtain intact tumor samples at two different time-points for evaluation of potential surrogate biomarkers. We report results of a pilot trial designed to determine if treatment-mediated changes in gene expression can be detected in formalin-fixed paraffin-embedded (FFPE) samples after 10-d exposure to anastrozole in estrogen receptor (ER)-positive breast cancer compared with untreated controls. METHODS Paired tumor samples (biopsy, surgical) were obtained from 26 postmenopausal women with ER-positive breast cancer. Patients were assigned anastrozole (1 mg/d) for 10 d immediately prior to surgery (13 cases) or no treatment (13 controls). Five hundred two cancer-related genes were examined by the Illumina cDNA-mediated annealing, selection, extension, and ligation, FFPE cDNA array (moderated t-test, P ≤ 0.005). Surrogate biomarkers reflecting changes in gene expression were examined by immunohistochemistry (Wilcoxon rank-based test, P < 0.05). RESULTS Sufficient RNA was available from 19 paired samples (8 controls, 11 cases). Frozen tissue and FFPE showed good correlation (r = 0.82). Within each group, 18 genes, reflecting roles in proliferation, angiogenesis, and apoptosis, showed differential expression from biopsy to surgery (P < 0.005). Estrogen-related genes were dysregulated in the treated group only. A reduction in Ki-67 was observed in 7 (54%) treated cases and in 1 (7.7%) control patient. CONCLUSIONS 10-d exposure to anastrozole resulted in dysregulation of 18/502 cancer-related genes, and Ki-67 was reduced in 54% of cases. FFPE samples demonstrated good correlation with frozen samples. However, changes in gene expression and increased Ki-67 in the control group suggest local effects of wound healing may represent a confounding factor in the interpretation of perioperative window trials.