Diagnostic performance of quantitative shear wave elastography in the evaluation of solid breast masses: determination of the most discriminatory parameter.

OBJECTIVE: The purpose of this article is to assess the diagnostic performance of quantitative shear wave elastography in the evaluation of solid breast masses and to determine the most discriminatory parameter. SUBJECTS AND METHODS: B-mode ultrasound and shear wave elastography were performed before core biopsy of 123 masses in 112 women. The diagnostic performance of ultrasound and quantitative shear wave elastography parameters (mean elasticity, maximum elasticity, and elasticity ratio) were compared. The added effect of shear wave elastography on the performance of ultrasound was determined. RESULTS: The mean elasticity, maximum elasticity, and elasticity ratio were 24.8 kPa, 30.3 kPa, and 1.90, respectively, for 79 benign masses and 130.7 kPa, 154.9 kPa, and 11.52, respectively, for 44 malignant masses (p < 0.001). The optimal cutoff value for each parameter was determined to be 42.5 kPa, 46.7 kPa, and 3.56, respectively. The AUC of each shear wave elastography parameter was higher than that of ultrasound (p < 0.001); the AUC value for the elasticity ratio (0.943) was the highest. By adding shear wave elastography parameters to the evaluation of BI-RADS category 4a masses, about 90% of masses could be downgraded to BI-RADS category 3. The numbers of downgraded masses were 40 of 44 (91%) for mean elasticity, 39 of 44 (89%) for maximum elasticity, and 42 of 44 (95%) for elasticity ratio. The numbers of correctly downgraded masses were 39 of 40 (98%) for mean elasticity, 38 of 39 (97%) for maximum elasticity, and 41 of 42 (98%) for elasticity ratio. There was improvement in the diagnostic performance of ultrasound of mass assessment with shear wave elastography parameters added to BI-RADS category 4a masses compared with ultrasound alone. Combined ultrasound and elasticity ratio had the highest improvement, from 35.44% to 87.34% for specificity, from 45.74% to 80.77% for positive predictive value, and from 57.72% to 90.24% for accuracy (p < 0.0001). The AUC of combined ultrasound and elasticity ratio (0.914) was the highest compared with the other combined parameters. CONCLUSION: There was a statistically significant difference in the values of the quantitative shear wave elastography parameters of benign and malignant solid breast masses. By adding shear wave elastography parameters to BI-RADS category 4a masses, we found that about 90% of them could be correctly downgraded to BI-RADS category 3, thereby avoiding biopsy. Elasticity ratio (cutoff, 3.56) appeared to be the most discriminatory parameter.

IGFBP7 reduces breast tumor growth by induction of senescence and apoptosis pathways.

Insulin-like growth factor binding protein 7 (IGFBP7) has been shown to be a tumor suppressor in a variety of cancers. We previously have shown that IGFBP7 expression is inversely correlated with disease progression and poor outcome in breast cancer. Overexpression of IGFBP7 in MDA-MB-468, a triple-negative breast cancer (TNBC) cell line, resulted in inhibition of growth and migration. Xenografted tumors bearing ectopic IGFBP7 expression were significantly growth-impaired compared to IGFBP7-negative controls, which suggested that IGFBP7 treatment could inhibit breast cancer cell growth. To confirm this notion, 14 human patient primary breast tumors were analyzed by qRTPCR for IGFBP7 expression. The TNBC tumors expressed the lowest levels of IGFBP7 expression, which also correlated with higher tumorigenicity in mice. Furthermore, when breast cancer cell lines were treated with IGFBP7, only the TNBC cell lines were growth inhibited. Treatment of NOD/SCID mice harboring xenografts of TNBC cells with IGFBP7 systemically every 3-4 days inhibited tumorigenesis, with associated anti-angiogenic effects, together with increased apoptosis. Upon examining the mechanism of IGFBP7-mediated growth inhibition in TNBC cells, we found that cells not only were arrested in G1 phase of the cell cycle but also underwent senescence as a result of treatment with IGFBP7. Interestingly, IGFBP7 treatment was also associated with strong activation of the stress-associated p38 MAPK pathway, together with upregulation of p53 and the cyclin-dependent protein kinase (CDK) inhibitor, p21(cip1). Prolonged treatment of cells with IGFBP7 resulted in increased cell death, marked by an increase in apoptotic cells and associated cleaved PARP. This is the first study showing that exogenous IGFBP7 inhibits TNBC cell growth both in vitro and in vivo. Taken together, these results suggest IGFBP7 treatment might have therapeutic potential for TNBC.

Association of low tumor RNA integrity with response to chemotherapy in breast cancer patients.

The CAN-NCIC-MA22 phase I/II clinical trial evaluated women with locally advanced or inflammatory breast cancer treated with epirubicin and docetaxel at 2 or 3 weekly intervals in sequential cohorts. The relationship between various biomarkers and treatment response was assessed. Breast biopsy cores were obtained from 50 patients pre-, mid-, and post-treatment. Immunohistochemical staining was performed to determine baseline levels of estrogen receptor (ER), progesterone receptor (PR), Her2/Neu protein (HER2), and topoisomerase II (Topo 2),expressed as percent positive stain. Tumor RNA integrity(RIN) and tumor cellularity were measured pre-, mid- and post-treatment by capillary electrophoresis and light microscopy after hematoxylin/eosin staining, respectively.Associations between 1) maximum RIN and 2) tumor cellularity at the three time points with baseline levels of ER,PR, Her2, and topo II were assessed using Spearman and Pearson correlation coefficients. Associations between RIN and tumor cellularity with chemotherapy dose level orpathologic response were assessed using one-way ANOVA.In this study, we observed that low mid-treatment maximum RIN (but not tumor cellularity) was associated with high chemotherapy drug dose level (P = 0.05) and eventual pathologic complete response (pCR) (P = 0.01). Posttreatment,low maximum RIN was found to be associated with low tumor cellularity (P = 0.004), and low tumor cellularity with pCR (P = 0.01). Post-treatment tumor cellularity was lowest in patients with tumors having high baseline PR levels (P = 0.05). The association of midtreatment RIN with drug dose level and with pCR suggests that tumor RIN may represent an important new biomarker for measuring response to chemotherapy in breast cancer patients.

A novel RING-type ubiquitin ligase breast cancer-associated gene 2 correlates with outcome in invasive breast cancer.

The RING finger family of proteins possess ubiquitin ligase activity and play pivotal roles in protein degradation and receptor-mediated endocytosis. In this study, we examined whether the breast cancer-associated gene 2 (BCA2), a novel RING domain protein, has E3 ubiquitin ligase activity and investigated its expression status in breast tumors. The full-length BCA2 gene was cloned from the human breast cancer cell line MDA-MB-468. It encodes an open reading frame of 304 amino acids and contains a RING-H2 domain. BCA2 maps to chromosome 1q21.1, a region known to harbor cytogenetic aberrations in breast cancers. We found that the BCA2 protein has an intrinsic autoubiquitination activity, the hallmark of E3 ligases, whereas mutant RING protein is not autoubiquitinated. This indicates that the BCA2 ubiquitin ligase activity is dependent on the RING-H2 domain. Using tissue microarrays and immunohistochemistry, we found strong to intermediate BCA2 staining in 56% of 945 invasive breast cancers cases, which was significantly correlated with positive estrogen receptor status [odds ratio (OR), 1.51; P = 0.004], negative lymph node status (OR, 0.73; P = 0.02), and an increase in disease-free survival for regional recurrence (OR, 0.45; P = 0.03). Overexpression of BCA2 increased proliferation and small interfering RNA inhibited growth of T47D human breast cancer cells and NIH3T3 mouse cells. The autoubiquitination activity of BCA2 indicates that it is a novel RING-type E3 ligase. Its association with clinical measures and its effects on cell growth indicate that BCA2 may be important for the ubiquitin modification of proteins crucial to breast carcinogenesis and growth.

Analysis of the HER2/neu gene amplification in microdissected breast cancer tumour samples.

The HER2/neu oncogene has been reported to be amplified in > 20% of invasive ductal carcinomas. In order to investigate the HER2/neu status in pure populations of breast cancer cells, a laser capture microdissection (LCM) system was used. Formalin-fixed paraffin-embedded breast tissue areas corresponding to normal ducts, ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) were microdissected and genomic DNA was isolated by a modified proteinase K- phenol extraction method and subjected to PCR for HER2/neu analysis. One hundred % concordance for detection of the HER2/neu gene amplification was found between immunohistochemistry and PCR used in combination with LCM. Our results indicated that LCM is a powerful technique for isolating pure populations of cells from paraffin-embedded tissue sections and that these cells can be used to study genomic alterations at the DNA level.

Characterization of a novel human breast cancer associated gene (BCA3) encoding an alternatively spliced proline-rich protein.

As part of an integrated study of breast cancer gene expression, partial cDNAs were cloned from normal and tumor breast cells by subtractive-hybridization and differential display cloning. The DNA sequence for one of these breast cancer associated genes was used to construct the larger 1319 bp BCA3 cDNA sequence using ESTs without assigned names or functions. High-level BCA3 mRNA expression was found in breast and prostate tumor cell lines whereas normal breast and prostate tissues have low-level expression. Further analysis revealed possible functional domains and alternative splicing of BCA3 that we confirmed by RT-PCR analysis. Immunohistochemistry revealed that the protein is expressed in breast tumor cells in vivo, and not in surrounding stromal tissue.

Detection of lymphatic invasion in primary melanoma with monoclonal antibody D2-40: a new selective immunohistochemical marker of lymphatic endothelium.

OBJECTIVES: To identify the presence of lymphatic invasion in primary cutaneous melanoma using monoclonal antibody D2-40, a marker of lymphatic endothelium, and to correlate the presence of lymphatic invasion with other clinicopathologic characteristics of the tumors. DESIGN: Retrospective melanoma case series study comparing conventional hematoxylin-eosin staining with D2-40 immunostaining for detection of lymphatic invasion. SETTING: Departments of Pathology and Dermatology, Sunnybrook and Women's College Health Sciences Center, University of Toronto, Toronto, Ontario. Patients Forty-four consecutive cases of primary cutaneous melanoma with a tumor thickness greater than 0.75 mm were examined for presence of lymphatic invasion. RESULTS: Seven (16%) of 44 melanomas showed the presence of lymphatic invasion under immunostaining with D2-40. In 2 cases, subepidermal lymphatic involvement was present; in 5 cases lymphatic invasion was noted within the tumor, including 1 case of additional lymphatic invasion at the invasive edge of the tumor. Lymphatic invasion was not detected on routine hematoxylin-eosin staining. We observed a trend in the association between lymphatic invasion and 2 markers of tumor aggressiveness, namely, a deeper Clark level and increased frequency of ulceration, which suggests that lymphatic invasion detected with D2-40 may indicate a poor prognosis. CONCLUSIONS: Immunostaining with D2-40 increases the frequency of detection of lymphatic invasion relative to conventional hematoxylin-eosin staining in primary melanoma. Future outcome data will determine the prognostic significance of lymphatic invasion detected by D2-40 immunostaining.

Imaging of HER2/neu-positive BT-474 human breast cancer xenografts in athymic mice using (111)In-trastuzumab (Herceptin) Fab fragments.

Trastuzumab (Herceptin) Fab were prepared by digestion of intact IgG with immobilized papain, derivatized with diethylenetriaminepentaacetic acid (DTPA) and radiolabeled with (111)In. The dissociation constant (Kd) for binding of Fab to HER2/neu-positive SK-BR-3 human breast cancer cells was two- to threefold higher than for intact IgG (14-36 vs. 8-14 nM). The binding affinity was not significantly decreased after DTPA derivatization (Kd=47 nM). (111)In-trastuzumab Fab localized specifically in HER2/neu-positive BT-474 human breast cancer xenografts in athymic mice with tumor uptake of 7.8+/-0.7% injected dose (ID)/g and tumor/blood ratio of 25.2+/-1.6 at 72 h postinjection compared with 2.7+/-0.7% ID/g and 7.0+/-0.9 for (111)In-HuM195 anti-CD33 Fab (significantly different, P<.001). Small (3-5 mm in diameter) BT-474 tumors were imaged with (111)In-trastuzumab Fab as early as 24 h postinjection.

A phase I/II trial of epirubicin and docetaxel in locally advanced breast cancer (LABC) on 2-weekly or 3-weekly schedules: NCIC CTG MA.22.

This phase I/II neoadjuvant trial (ClinicalTrials.gov identifier NCT00066443) determined maximally-tolerated doses (MTD), dose-limiting toxicities, response-to-therapy, and explored the role of novel response biomarkers. MA.22 accrued T3N0, any N2 or N3, and T4 breast cancer patients. Treatment was 6 cycles of 3-weekly (Schedule A; N = 47) or 8 cycles of 2-weekly (Schedule B; N = 46) epirubicin/docetaxel chemotherapy in sequential phase I/II studies, with growth factor support. In phase I of each schedule, MTDs were based on DLT. In phase II, clinical responses (CR/PR) and pathologic complete responses (pCR) were assessed. Tumor biopsy cores were obtained pre-, mid-, and post-treatment: 3 for pathologic assessment; 3 for microarray studies. DLT for Schedule A was febrile neutropenia at 105 mg/m(2) epirubicin and 75 mg/m(2) docetaxel; for schedule B, it was fatigue at 75 mg/m(2) for both agents. Phase II doses were 90 mg/m(2) epirubicin/75 mg/m(2) docetaxel for Schedule A and 60 mg/m(2) (both agents) for Schedule B. Schedule A CR/PR and pCR rates were 90 and 10 %, with large reductions in tumor RNA content and integrity following treatment; Schedule B results were 93 and 0 %, with smaller reductions in RNA quality. Pre-treatment expression of several genes was associated with clinical response, including those within a likely amplicon at 17q12 (ERBB2, TCAP, GSDMB, and PNMT). The combination regimens had acceptable toxicity, good clinical response, induction of changes in tumor RNA content and integrity. Pre-treatment expression of particular genes was associated with clinical responses, including several near 17q12, which with ERBB2, may better identify chemoresponsiveness.

Pathologic Characteristics of Breast Cancer that Predict for Local Recurrence After Lumpectomy Alone.

Breast conservation surgery (BCS) plus irradiation has been shown to be equivalent to mastectomy in controlling ipsilateral breast cancer recurrence. The purpose of this study is to evaluate the factors that determine the rate of local recurrence in a group of patients treated with partial mastectomy without postoperative radiation, adjuvant hormonal therapy, or chemotherapy. We also assess the role of standard pathologic features, specifically lymphovascular invasion (LVI) in identifying high- and low-risk subsets of patients. We have a cohort of 293 patients treated with partial mastectomy followed prospectively for a median of 8 years. Data collected included patient's age, tumor size, tumor morphology, tumor grade, the extent of ductal carcinoma in situ (DCIS), the presence of LVI, lymph node status, and hormone receptors. Statistical analyses carried out were Kaplan-Meier plots with Wilcoxon (Peto-Prentice) test statistics for univariate analysis and Cox stepwise regression for multivariate analysis; the end point was local recurrence. The relapse rate in this cohort was 26%. In univariate analysis the significant factors associated with prolonged disease-free survival included older age, negative nodes, positive estrogen receptor (ER) status, and absence of LVI. Small tumor size was significant only in the univariate analysis. In the multivariate analysis, absence of comedocarcinoma entered the model in addition to the other variables. If the variables are stratified, a group of 66 patients with 6% local recurrence rate was identified. These were node-negative women >/=50 years of age with no LVI, no comedo DCIS, and ER-positive tumors. This study clearly indicates the important role of pathologic parameters in assessing the risk of recurrence.

Phase I trial of intraoperative detection of tumor margins in patients with HER2-positive carcinoma of the breast following administration of 111In-DTPA-trastuzumab Fab fragments.

INTRODUCTION: Our aim was to conduct a Phase I clinical trial to determine the feasibility of intraoperative detection of tumor margins in HER2 positive breast carcinoma using a hand-held γ-probe following administration of (111)In-DTPA-trastuzumab Fab fragments. Accurate delineation of tumor margins is important for preventing local recurrence. METHODS: Six patients with HER2-positive in situ or invasive ductal carcinoma were administered 74MBq (0.5mg) of (111)In-DTPA-trastuzumab Fab fragments and counts in the tumor, surgical cavity wall and en face margins were measured intraoperatively at 72h post-injection using the Navigator or C-Trak γ-probes. Margins were evaluated histologically. Quantitative whole body planar imaging was performed to estimate radiation absorbed doses using OLINDA/EXM software. SPECT imaging of the thorax was performed to evaluate tumor uptake. The pharmacokinetics of elimination from the blood and plasma were determined over 72h. RESULTS: There were no acute adverse reactions from (111)In-DTPA-trastuzumab Fab fragments and no changes in hematological or biochemical indices were found over a 3month period. (111)In-DTPA-trastuzumab Fab fragments exhibited a biphasic elimination from the blood and plasma with t1/2α=11.9h and 7.5h, respectively, and t1/2ß=26.6 and 20.7h, respectively. The radiopharmaceutical accumulated in the liver, spleen and kidneys. SPECT imaging did not reveal tumor in any patient. The mean effective dose was 0.146mSv/MBq (10.8mSv for 74MBq). Counts in excised tumors were low but were higher than in margins. Margins in two patients harboured tumor but this was not correlated with counts obtained using the γ-probes. Surgical cavity counts were high and likely due to detection of γ-photons outside the surgical field. CONCLUSION: We conclude that it was not feasible, at least at the administered amount of radioactivity used in this study, to reliably detect the margins of disease in patients with in situ or invasive ductal carcinoma intraoperatively using a hand-held γ-probe and (111)In-DTPA-trastuzumab Fab fragments due to low uptake in the tumor and involved margins.

D2-40 is expressed on the luminal surface of pulmonary airspaces in normal developing and adult lung but is lost in conditions associated with intra-alveolar infiltrates.

The D2-40 antigen is a glycosylated sialomucin that is strongly expressed by lymphatic endothelial cells. Recently we observed the expression of D2-40 on the luminal surface of pulmonary airspaces in lung sections. The aim of the study was to assess the expression of D2-40 antigen in normal lung development and in various pathologic conditions in which abnormal alveolar infiltrates were present. Formalin-fixed lung tissue was selected from 42 fetal/neonatal autopsy cases ranging in gestational age from 12 to 41 weeks and from 10 adult lungs. In the fetal/neonatal group, 22 cases were histologically normal, whereas 20 were abnormal (including cases of pneumonia, alveolar hemorrhage, meconium aspiration, pulmonary hypoplasia, and pulmonary interstitial emphysema). In the adult group, 5 cases were histologically normal and 5 had pneumonia. Immunohistochemical staining was performed on all cases using antibody to D2-40. All cases of normal fetal/neonatal lung and normal adult lung showed diffuse strong expression of D2-40 on the luminal surface of the alveolar lining cells. D2-40 expression was also noted on the bronchiolar lining cells of normal fetal/neonatal lung. In all cases in which there was an abnormal infiltrate or foreign material within the airspaces, expression of D2-40 was lost in the alveolar lining. The production of the D2-40 antigen in the alveolar lining occurs as early as 12 weeks gestation and continues to be present throughout all other stages of lung development, as well as in adult lung. These results suggest that D2-40 may have a cell membrane protective function.

A human bone NOD/SCID mouse model to distinguish metastatic potential in primary breast cancers.

In this study, we created a clinically relevant model using NOD/SCID mice engrafted with human bone fragments in both right and left flanks, and show that the human bone implants are viable and functional for more than 6 mo. To investigate the growth and metastatic behavior of breast cancer, human primary breast tumor specimens were transplanted into human bone grafts under only the right flanks of the human-bone NOD/SCID mice. Some of the engrafted tumors proliferated extensively with massive neo-vascularization and also metastasized to the initially tumor-free left flank bone grafts. We show for the first time that this mouse model can be used to distinguish between primary breast tumors that do or do not proliferate and metastasize to contralateral human bone implants that were initially tumor-free.

Breast metastasis by medullary thyroid carcinoma detected by FDG positron emission tomography.

Medullary thyroid carcinoma (MTC) is an uncommon thyroid cancer comprising 5% to 8% of thyroid neoplasms. In contrast to common thyroid tumors, this tumor originates from the calcitonin-producing C cells. Regional metastases to cervical lymph nodes occur early in the disease, whereas distant metastasis occurs late. Common metastatic sites include the liver, bone, brain, and adrenal medulla. We present a case of MTC metastatic to the breast. We report on this case for the following reasons: (1) metastasis to the breast is an extremely rare occurrence and could be easily confused clinically and pathologically with a primary breast neoplasm and (2) this is the first reported case of detection of breast metastasis by an MTC using FDG ((18)F-fluoro-2-deoxy-D-glucose) positron emission tomography with an accompanying histologic description.

Lymphatic invasion identified by monoclonal antibody D2-40, younger age, and ulceration: predictors of sentinel lymph node involvement in primary cutaneous melanoma.

OBJECTIVES: To assess whether lymphatic invasion identified by immunostaining with monoclonal antibody (Mab) D2-40 in primary cutaneous melanomas correlates with other clinicopathologic factors and to assess whether lymphatic invasion is a potential predictor of sentinel lymph node (SLN) status. DESIGN: Retrospective case-series study. SETTING: Academic referral center. Patients Ninety-six consecutive patients with primary cutaneous melanomas 1 mm thick or greater with adequate pathologic material available for immunohistochemical studies and SLN biopsy. MAIN OUTCOME MEASURES: Association between lymphatic invasion identified by immunostaining with Mab D2-40 in primary cutaneous melanoma and correlation with the clinicopathologic features and the association of all of the factors with SLN status. RESULTS: Lymphatic invasion identified by immunostaining with Mab D2-40 was significantly associated with deeper Clark level of invasion (P < .001), and greater Breslow tumor thickness (P = .01) SLN positivity was identified in 23 of 96 cases (24%). At univariate analysis, younger age (P = .03), ulceration (P < .006), lymphatic invasion (P < .02), deeper Clark level of invasion (P < .008), Breslow tumor thickness (P = .008), and tumor site on the trunk (P = .02) were significantly associated with SLN metastases. At multivariate analysis, only younger age (P = .04), ulceration (P = .03), and lymphatic invasion detected by immunostaining with Mab D2-40 (P = .01) were significantly associated with SLN positivity. The probability of SLN positivity was 13% when all 3 independent prognostic factors yielded negative findings and increased to 61% when all 3 variables yielded positive findings. CONCLUSIONS: Breslow tumor thickness, Clark level of invasion, and tumor site on the trunk predicted SLN status at univariate analysis. Multivariate regression analysis showed that lymphatic invasion identified by immunostaining with Mab D2-40, younger age, and ulceration were the only independent prognostic factors. The most significant predictor of SLN metastasis was the positivity of all 3 independent prognostic factors (61%). Findings of this study suggest that assessment of lymphatic invasion by immunostaining with Mab D2-40 with other clinicopathologic factors can be used to identify patients who could be spared the need for SLN biopsy.

Biological Markers Predictive of Invasive Recurrence in DCIS.

DCIS is a heterogeneous group of non-invasive cancers of the breast characterized by various degrees of differentiation and unpredictable propensity for transformation into invasive carcinoma. We examined the expression and prognostic value of 9 biological markers with a potential role in tumor progression in 133 patients with pure DCIS treated with breast conserving surgery alone, between 1982-2000. Histology was reviewed and immunohistochemical staining was performed. Pearson correlation coefficient was used to determine the associations between markers and histopathological features. Univariate and multivariate analysis examined associations between time to recurrence and clinicopathologic features and biological markers.Median age at diagnosis was 55 years (25-85). With a median follow up of 8.91 years, 41/133 patients recurred (21 as invasive recurrence). In this cohort 13.5% had low, 43% intermediate and 42% high nuclear grade. Comedo necrosis was found in 65% of cases. Expression of ER (62.4%), PR (55.6%), HER2/neu (31.6%), MIB1 (39.8%), p53 (22.6%), p21 (39.8%), Cyclin D1 (95.5%) calgranulin (20.5%), psoriasin (12%), was found in DCIS. HER2/neu was overexpressed in 45% that recurred as DCIS and 42.9% that recurred as invasive cancer, and only in 26.1% in cases that never recurred. On univariate analysis, HER2/neu overexpression was the only marker associated with an increased risk for any recurrence (p = 0.044). The hazard ratio for recurrence for HER2/neu positive DCIS was 1.927 (confidence interval 1.016-3.653) compared to HER2 negative DCIS. On multivariate analysis, HER2/neu overexpression remained the only independent variable significantly associated with any recurrence (p = 0.014) and with invasive recurrence (p = 0.044).This data suggest that HER2/neu testing may become an important parameter in the management of DCIS and the treatment of cases with positive HER2/neu status could be modified accordingly, similar to the current approach for HER2/neu positive invasive disease.

Intratumoral heterogeneity of HER2/neu in breast cancer--a rare event.

HER2/neu is overexpressed in about 20% of invasive breast carcinomas. Numerous studies have shown that there is high level of concordance between the HER2/neu status of the primary breast cancer and the metastases of a given patient. Recently, changes in HER2/neu status with tumor progression have been reported, suggesting the possibility of an emerging different tumor clone. Little is known about intratumoral heterogeneity with regard to HER2/neu oncoprotein overexpression. We identified nine cases of invasive ductal carcinoma that showed intratumoral variation in HER2/neu oncoprotein expression by immunohistochemistry. This was confirmed by the intratumoral variation in the amplification status of the HER2/neu gene by fluorescence in situ hybridization and by chromogenic in situ hybridization. The results of this study suggest that some cases of primary breast carcinoma are heterogeneous in regard to HER2/neu gene amplification or protein overexpression. Heterogeneity of HER2/neu status in a tumor may be a rare event or underestimated. This phenomenon should be examined as it may contribute to a better understanding of the variation in therapeutic responses and the conflicting data in studies about the prognostic and predictive role of HER2/neu status in subsets of breast cancer patients.

Significance of lymph vessel invasion identified by the endothelial lymphatic marker D2-40 in node negative breast cancer.

Monoclonal antibody D2-40, a marker of lymphatic endothelium, identifies tumor emboli in lymph vessels. The aim of the study was to assess whether D2-40+ lymph vessel invasion (LVI) correlates with clinicopathologic factors including lymphovascular invasion (LVI) as assessed by haematoxylin and eosin-stained sections (H&E+ or H&E-) and to assess the prognostic significance in node-negative breast cancer. The study group consisted of 303 node-negative breast cancer patients that had a median follow-up of 7.6 years. Clinical and pathological data were retrieved from the Henrietta Banting database. Immunohistochemical staining was performed on formalin-fixed, paraffin-embedded tissue sections of the primary invasive carcinoma using D2-40. Immunostaining with CD31 was performed on the discordant cases that were H&E+/D2-40-. D2-40+ lymph vessel invasion was detected in 82/303 (27%) cases. The foci of lymphatic invasion occurred predominantly at the invasive front of the tumor. The absence of D2-40 and CD31 in 13/17 discordant cases was suggestive of retraction artefact. D2-40+ lymph vessel invasion correlated significantly with age (P=0.0003), tumor size (P=0.005), histological grade (P=0.0001), H&E+ (P=<0.0001) and estrogen receptor status (P=0.005) but not with histological type or progesterone receptor status. Multivariate analysis revealed that D2-40+ lymph vessel invasion was the only significant predictor of distant recurrence. There was no significant association between D2-40 status and local recurrence (P=0.752) or regional recurrence (P=0.13). Both D2-40+lymph vessel invasion (P=0.009) and H&E+LVI cases (P=0.02) were associated with overall shorter survival in univariate analysis. These data indicate that D2-40 identifies lymphatic invasion in breast tumors and is a significant predictor of outcome in breast cancer.

Breast cancer metastasis in a human bone NOD/SCID mouse model.

A major dilemma facing patients with breast cancer is how to decide between over treating indolent tumors and failing to adequately treat aggressive, potentially lethal cancers. Determination of the metastatic potential of a patient's breast cancer would clearly help guide those treatment decisions. Breast cancer commonly spreads to bone in 70% of women with advanced disease. However, the mechanism of bone metastasis is not well understood. One possibility is that the microenvironment within bone marrow, highly rich in growth factors and cytokines, is suitable for the proliferation of breast cancer cells. In this study, we developed a method for implanting human bone in NOD/SCID mice and show that the human bone implants are viable for more than 20 weeks. This human bone NOD/SCID mouse model provides an opportunity to functionally characterize human breast cancer cell behavior in an in vivo human microenvironment. Several breast tumor cell lines have been shown to grow in the human-bone-NOD/SCID model system, however each line has a different functional profile. Here we show that cotransplantation of GFP-MDA-MB-231 breast cancer cells with morcellized human bone allows for tissue specific metastasis to an initially tumor free bone implant. Furthermore, metastasis of breast tumor cells to implanted tumor-free human bone was seen when patient bone containing a metastatic breast tumor was implanted in the host mouse. With this model, we can distinguish between primary invasive breast tumors with and without bone metastatic potential.

p27 phosphorylation by Src regulates inhibition of cyclin E-Cdk2.

The kinase inhibitor p27Kip1 regulates the G1 cell cycle phase. Here, we present data indicating that the oncogenic kinase Src regulates p27 stability through phosphorylation of p27 at tyrosine 74 and tyrosine 88. Src inhibitors increase cellular p27 stability, and Src overexpression accelerates p27 proteolysis. Src-phosphorylated p27 is shown to inhibit cyclin E-Cdk2 poorly in vitro, and Src transfection reduces p27-cyclin E-Cdk2 complexes. Our data indicate that phosphorylation by Src impairs the Cdk2 inhibitory action of p27 and reduces its steady-state binding to cyclin E-Cdk2 to facilitate cyclin E-Cdk2-dependent p27 proteolysis. Furthermore, we find that Src-activated breast cancer lines show reduced p27 and observe a correlation between Src activation and reduced nuclear p27 in 482 primary human breast cancers. Importantly, we report that in tamoxifen-resistant breast cancer cell lines, Src inhibition can increase p27 levels and restore tamoxifen sensitivity. These data provide a new rationale for Src inhibitors in cancer therapy.