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Purpose: Claudin-6 (CLDN6) is expressed at elevated levels in multiple human cancers including ovarian and endometrial malignancies, with little or no detectable expression in normal adult tissue. This expression profile makes CLDN6 an ideal target for development of a potential therapeutic antibody–drug conjugate (ADC). This study describes the generation and preclinical characterization of CLDN6–23-ADC, an ADC consisting of a humanized anti-CLDN6 monoclonal antibody coupled to monomethyl auristatin E (MMAE) via a cleavable linker. Experimental Design: A fully humanized anti-CLDN6 antibody was conjugated to MMAE resulting in the potential therapeutic ADC, CLDN6–23-ADC. The antitumor efficacy of CLDN6–23-ADC was assessed for antitumor efficacy in CLDN6-positive (CLDN6+) and -negative (CLDN6−) xenografts and patient-derived xenograft (PDX) models of human cancers. Results: CLDN6–23-ADC selectively binds to CLDN6, versus other CLDN family members, inhibits the proliferation of CLDN6+ cancer cells in vitro, and is rapidly internalized in CLDN6+ cells. Robust tumor regressions were observed in multiple CLDN6+ xenograft models and tumor inhibition led to markedly enhanced survival of CLDN6+ PDX tumors following treatment with CLDN6–23-ADC. IHC assessment of cancer tissue microarrays demonstrate elevated levels of CLDN6 in 29% of ovarian epithelial carcinomas. Approximately 45% of high-grade serous ovarian carcinomas and 11% of endometrial carcinomas are positive for the target. Conclusions: We report the development of a novel ADC, CLDN6–23-ADC, that selectively targets CLDN6, a potential onco-fetal-antigen which is highly expressed in ovarian and endometrial cancers. CLDN6–23-ADC exhibits robust tumor regressions in mouse models of human ovarian and endometrial cancers and is currently undergoing phase I study.

Supplemental Figure S2: Amino acid sequence alignment of CLDN6 (P56747) and CLDN9 (O95484) with the extracellular loops highlighted

Methods for Supplemental Data

Purpose:Claudin-6 (CLDN6) is expressed at elevated levels in multiple human cancers including ovarian and endometrial malignancies, with little or no detectable expression in normal adult tissue. This expression profile makes CLDN6 an ideal target for development of a potential therapeutic antibody–drug conjugate (ADC). This study describes the generation and preclinical characterization of CLDN6–23-ADC, an ADC consisting of a humanized anti-CLDN6 monoclonal antibody coupled to monomethyl auristatin E (MMAE) via a cleavable linker.Experimental Design:A fully humanized anti-CLDN6 antibody was conjugated to MMAE resulting in the potential therapeutic ADC, CLDN6–23-ADC. The antitumor efficacy of CLDN6–23-ADC was assessed for antitumor efficacy in CLDN6-positive (CLDN6+) and -negative (CLDN6−) xenografts and patient-derived xenograft (PDX) models of human cancers.Results:CLDN6–23-ADC selectively binds to CLDN6, versus other CLDN family members, inhibits the proliferation of CLDN6+ cancer cells in vitro, and is rapidly internalized in CLDN6+ cells. Robust tumor regressions were observed in multiple CLDN6+ xenograft models and tumor inhibition led to markedly enhanced survival of CLDN6+ PDX tumors following treatment with CLDN6–23-ADC. IHC assessment of cancer tissue microarrays demonstrate elevated levels of CLDN6 in 29% of ovarian epithelial carcinomas. Approximately 45% of high-grade serous ovarian carcinomas and 11% of endometrial carcinomas are positive for the target.Conclusions:We report the development of a novel ADC, CLDN6–23-ADC, that selectively targets CLDN6, a potential onco-fetal-antigen which is highly expressed in ovarian and endometrial cancers. CLDN6–23-ADC exhibits robust tumor regressions in mouse models of human ovarian and endometrial cancers and is currently undergoing phase I study.

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Includes supplementary tables S1, S2, S5, S6, S7 and S9

File includes supplementary figures S1 to S5

Supplementary Data: List of TRIO investigators, Supplementary Materials and Methods, Supplementary Results, Supplementary Discussion, Supplementary References, Supplementary Tables, and Supplementary Figures.

List of upregulated genes in CTC-derived brain metastases compare to other metastases (lung, bone and ovary)

Hematogenous metastasis is initiated by a subset of circulating tumor cells (CTC) shed from primary or metastatic tumors into the blood circulation. Thus, CTCs provide a unique patient biopsy resource to decipher the cellular subpopulations that initiate metastasis and their molecular properties. However, one crucial question is whether CTCs derived and expanded ex vivo from patients recapitulate human metastatic disease in an animal model. Here, we show that CTC lines established from patients with breast cancer are capable of generating metastases in mice with a pattern recapitulating most major organs from corresponding patients. Genome-wide sequencing analyses of metastatic variants identified semaphorin 4D as a regulator of tumor cell transmigration through the blood–brain barrier and MYC as a crucial regulator for the adaptation of disseminated tumor cells to the activated brain microenvironment. These data provide the direct experimental evidence of the promising role of CTCs as a prognostic factor for site-specific metastasis.Significance:Interests abound in gaining new knowledge of the physiopathology of brain metastasis. In a direct metastatic tropism analysis, we demonstrated that ex vivo–cultured CTCs from 4 patients with breast cancer showed organotropism, revealing molecular features that allow a subset of CTCs to enter and grow in the brain.This article is highlighted in the In This Issue feature, p. 1

African American women with breast cancer present more commonly with aggressive tumors that do not express the estrogen receptor (ER) and progesterone receptor (PR) compared with European American women. Whether this disparity is the result of inherited factors has not been established. We did an admixture-based genome-wide scan to search for risk alleles for breast cancer that are highly differentiated in frequency between African American and European American women, and may contribute to specific breast cancer phenotypes, such as ER-negative (ER−) disease. African American women with invasive breast cancer (n = 1,484) were pooled from six population-based studies and typed at ∼1,500 ancestry-informative markers. We investigated global genetic ancestry and did a whole genome admixture scan searching for breast cancer–predisposing loci in association with disease phenotypes. We found a significant difference in ancestry between ER+PR+ and ER−PR− women, with higher European ancestry among ER+PR+ individuals, after controlling for possible confounders (odds ratios for a 0 to 1 change in European ancestry proportion, 2.84; 95% confidence interval, 1.13-7.14; P = 0.026). Women with localized tumors had higher European ancestry than women with non–localized tumors (odds ratios, 2.65; 95% confidence interval, 1.11-6.35; P = 0.029). No genome-wide statistically significant associations were observed between European or African ancestry at any specific locus and breast cancer, or in analyses stratified by ER/PR status, stage, or grade. In summary, in African American women, genetic ancestry is associated with ER/PR status and disease stage. However, we found little evidence that genetic ancestry at any one region contributes significantly to breast cancer risk or hormone receptor status. (Cancer Epidemiol Biomarkers Prev 2009;18(11):3110–7)

Purpose: Biomarkers from two randomized phase III trials were analyzed to optimize selection of patients for lapatinib therapy.Experimental Design: In available breast cancer tissue from EGF30001 (paclitaxel ± lapatinib in HER-2-negative/unknown metastatic breast cancer, n = 579) and EGF100151 (capecitabine ± lapatinib in HER-2-positive metastatic breast cancer, n = 399), HER-2 gene amplification by fluorescence in situ hybridization (FISH), HER-2 mRNA by reverse transcription-PCR (RT-PCR), HER-2 protein expression by HercepTest immunohistochemistry (IHC), epidermal growth factor receptor (EGFR) mRNA level by RT-PCR, and EGFR protein by IHC were analyzed and compared with clinical outcome. HER-2 was determined by FISH in an academic reference/research laboratory and in a large, high-volume commercial reference laboratory.Results: The HER-2 gene was amplified in 47% (344 of 733) and IHC was 3+ in 35% (279 of 798), with significant correlation (P < 0.01) between FISH and IHC. Positive EGFR immunostaining (IHC 1+, 2+, or 3+) in 28% (213 of 761) correlated with EGFR mRNA levels by RT-PCR (r = 0.59; P < 0.01). HER-2 gene amplification/overexpression was associated with improved clinical outcomes (progression-free survival; P < 0.001) in both trials. A significant improvement in outcome was seen in FISH-positive and IHC 0, 1+, or 2+ patients. HER-2 mRNA expression correlated with HER-2 FISH (r = 0.83) and IHC status (r = 0.72; n = 138). No correlation was found between EGFR expression (IHC or mRNA) and responsiveness to lapatinib regardless of HER-2 status. Although a significant correlation with lapatinib responsiveness was observed among “HER-2-negative” breast cancer patients in the large, high-volume commercial reference laboratory, this was not confirmed in the academic reference/research laboratory.Conclusions: Women with HER-2-positive metastatic breast cancer benefit from lapatinib, whereas women with HER-2-negative metastatic breast cancer derive no incremental benefit from lapatinib.

Molecular subtypes at baseline.

Supplementary Table S1. Associations of Overall Breast Cancer Risk with 74 Breast Cancer Risk Variants in African Americans with Study-specific Results.

Supplementary Figure S1, Supplementary Figure S1. Regional plots of 3p24, 11q13, 12p11, 14q13, 16q12/TOX3, 16q12/FTO, 16q23, and 19p13.

Purpose: To investigate the clinical relevance of PTEN in HER2-amplified and HER2-nonamplified disease.Experimental Design: We assessed PTEN status in two large adjuvant breast cancer trials (BCIRG-006 and BCIRG-005) using a PTEN immunohistochemical (IHC) assay that was previously validated in a panel of 33 breast cancer cell lines and prostate cancer tissues with known PTEN gene deletion.Results: In the HER2-positive patient population, absence of tumor cell PTEN staining occurred at a rate of 5.4% and was independent of ER/PR status. In contrast, 15.9% of HER2-negative patients exhibited absence of PTEN staining with the highest frequency seen in triple-negative breast cancer (TNBC) subgroup versus ER/PR-positive patients (35.1% vs. 10.9%). Complete absence of PTEN staining in tumor cells was associated with poor clinical outcome in HER2-positive disease. Those patients whose cancers demonstrated absent PTEN staining had a significant decrease in disease-free survival (DFS) and overall survival (OS) compared with patients with tumors exhibiting any PTEN staining patterns (low, moderate, or high). Trastuzumab appeared to provide clinical benefit even for patients lacking PTEN staining. In the HER2-negative population, there were no statistically significant differences in clinical outcome based on PTEN status.Conclusions: This study is the largest to date examining PTEN status in breast cancer and the data suggest that the rate and significance of PTEN status differ between HER2-positive and HER2-negative disease. Furthermore, the data clearly suggest that HER2-positive patients with PTEN loss still benefit from trastuzumab. Clin Cancer Res; 21(9); 2065–74. ©2015 AACR.

Supplementary Table 2 from An Admixture Scan in 1,484 African American Women with Breast Cancer

Purpose: Lapatinib, a dual epidermal growth factor receptor (EGFR) and HER2 inhibitor, remains unproven in non–HER2-amplified metastatic breast cancer (MBC). EGF30008, a phase III trial of letrozole and lapatinib versus letrozole and placebo, demonstrated that lapatinib significantly improves outcome for postmenopausal women with HER2-amplified, but not HER2-negative, MBC. The hypothesis that low hormone receptor status is associated with benefit in this HER2-negative cohort was tested.Experimental Design: A blinded retrospective biomarker evaluation used immunohistochemistry (IHC) to semiquantify estrogen receptor (ER) and progesterone receptor (PgR) expression (n = 821/952). HER2 status was determined by IHC and confirmed by FISH (n = 326). Effects of these biomarkers on progression-free survival (PFS) were examined in patients with available tissue.Results: In HER2-negative, ER-positive MBC, median PFS was analyzed by ER and PgR expression (H-score) by quartile (Q). There was significant improvement in patients with low ER expression (Q1, H-score P = 0.01). No benefit was associated with stronger ER expression (Q2/3, H-score ≥160 and Conclusion: In postmenopausal patients with advanced hormone receptor–positive disease, weak ER expression is associated with worse outcome with letrozole treatment compared with the combination. The addition of lapatinib significantly improved PFS for this patient subgroup and augments data supporting interaction between steroid hormone and peptide hormone signaling. A prospective study validating this hypothesis is required. Clin Cancer Res; 20(3); 736–43. ©2013 AACR.

A. Subgroup analyses of percent change in Ki67 from baseline to week 2 were performed on KE population based on progesterone receptor status, tumor size, baseline disease stage, tumor grade, baseline lymph node involvement, and baseline Ki67 (index 20%). B. Ki67 changes at baseline, 2 weeks, and end of treatment. C. Geometric mean percentage suppression of Ki67 from baseline to end of treatment by treatment arm. D. Mean percentage rate of response as determined by complete cell cycle arrest (responder: Ki76

PDF - 66K, This table contains the clinical characterisitcs of patients enrolled in the EGF 30008 trial.

Supplement Text AND ALL Supplement Figures

Supplementary Fig Leg

Objective: Large body size has been associated with decreased risk of breast cancer in premenopausal women but with increased risk in postmenopausal women. Limited information is available about African-American women and differences by estrogen and progesterone receptor status.Methods: We analyzed data from the Women's Contraceptive and Reproductive Experiences Study among 3,997 white and African-American breast cancer case patients diagnosed in 1994 to 1998 and 4,041 control participants ages 35 to 64 years. We calculated multivariate odds ratios (OR) as measures of relative risk of breast cancer associated with self-reported body mass index (BMI) at age 18 and 5 years before diagnosis (recent BMI).Results: Risk tended to decrease with increasing BMI at age 18 years in all women [ORBMI ≥ 25 kg/m2 versus < 20 kg/m2 = 0.76; 95% confidence interval (CI), 0.63-0.90; Ptrend = 0.005] and with recent BMI in premenopausal women (ORBMI ≥ 35 kg/m2 versus < 25 kg/m2 = 0.81; 95% CI, 0.61-1.06; Ptrend = 0.05), unmodified by race. Among postmenopausal white but not African-American women, there was an inverse relation between recent BMI and risk. High recent BMI was associated with increased risk of estrogen receptor– and progesterone receptor–positive tumors among postmenopausal African-American women (ORBMI ≥ 35 kg/m2 versus < 25 kg/m2 = 1.83; 95% CI, 1.08-3.09; Ptrend = 0.03).Conclusion: Among women at age 35 to 64 years, BMI at age 18 years is inversely associated with risk of breast cancer, but association with recent BMI varies by menopause status, race, and hormone receptor status.Impact: Our findings indicate that studies of BMI and breast cancer should consider breast cancer subtypes. Cancer Epidemiol Biomarkers Prev; 19(6); 1532–44. ©2010 AACR.

Supplementary Tables 1-5 from A Case-Control Study of Body Mass Index and Breast Cancer Risk in White and African-American Women

Purpose:neoMONARCH assessed the biological effects of abemaciclib in combination with anastrozole in the neoadjuvant setting.Patients and Methods:Postmenopausal women with stage I–IIIB HR+/HER2− breast cancer were randomized to a 2-week lead-in of abemaciclib, anastrozole, or abemaciclib plus anastrozole followed by 14 weeks of the combination. The primary objective evaluated change in Ki67 from baseline to 2 weeks of treatment. Additional objectives included clinical, radiologic, and pathologic responses, safety, as well as gene expression changes related to cell proliferation and immune response.Results:Abemaciclib, alone or in combination with anastrozole, achieved a significant decrease in Ki67 expression and led to potent cell-cycle arrest after 2 weeks of treatment compared with anastrozole alone. More patients in the abemaciclib-containing arms versus anastrozole alone achieved complete cell-cycle arrest (58%/68% vs. 14%, P < 0.001). At the end of treatment, following 2 weeks lead-in and 14 weeks of combination therapy, 46% of intent-to-treat patients achieved a radiologic response, with pathologic complete response observed in 4%. The most common all-grade adverse events were diarrhea (62%), constipation (44%), and nausea (42%). Abemaciclib, anastrozole, and the combination inhibited cell-cycle processes and estrogen signaling; however, combination therapy resulted in increased cytokine signaling and adaptive immune response indicative of enhanced antigen presentation and activated T-cell phenotypes.Conclusions:Abemaciclib plus anastrozole demonstrated biological and clinical activity with generally manageable toxicities in patients with HR+/HER2− early breast cancer. Abemaciclib led to potent cell-cycle arrest, and in combination with anastrozole, enhanced immune activation.

Materials and methods.

Supplementary Data from HER-2 Gene Amplification, HER-2 and Epidermal Growth Factor Receptor mRNA and Protein Expression, and Lapatinib Efficacy in Women with Metastatic Breast Cancer

Supplemental Figure 2. Clinical outcome based on PTEN data availability. Kaplan Meier analysis was performed for patients with PTEN data available compared to patients without PTEN data available. Disease free survival (A, C) and overall survival (B, D) are shown for BCIRG 006 (HER2-amplified) (A, B) and BCIRG 005 (HER2-non-amplified) (C, D).

Supplementary Table 1 from An Admixture Scan in 1,484 African American Women with Breast Cancer

Supplemental Figure 3a. PTEN IHC assay validation and scoring. PTEN IHC was performed on a panel of FFPE breast cancer cell pellets of known PTEN status confirmed by western immunoblot. (A.) Western immunoblot analyses for MDA-MB-468, CAL-148, MDA-MB-361, MT-3, and MDA-MB-436 human breast cancer cell lines are illustrated above with PTEN IHC illustrated below.

CCR Translation for This Article from Quantitative ER and PgR Assessment as Predictors of Benefit from Lapatinib in Postmenopausal Women with Hormone Receptor–Positive, HER2-Negative Metastatic Breast Cancer

Supplemental Figure 4b. A serial section shows the same microscopic field after processing for PTEN localization by immunohistochemistry. Note the lack of immunostaining in the tumor cells.

Background: Genome-wide association studies have identified approximately 100 common genetic variants associated with breast cancer risk, the majority of which were discovered in women of European ancestry. Because of different patterns of linkage disequilibrium, many of these genetic markers may not represent signals in populations of African ancestry.Methods: We tested 74 breast cancer risk variants and conducted fine-mapping of these susceptibility regions in 6,522 breast cancer cases and 7,643 controls of African ancestry from three genetic consortia (AABC, AMBER, and ROOT).Results: Fifty-four of the 74 variants (73%) were found to have ORs that were directionally consistent with those previously reported, of which 12 were nominally statistically significant (P < 0.05). Through fine-mapping, in six regions (3p24, 12p11, 14q13, 16q12/FTO, 16q23, 19p13), we observed seven markers that better represent the underlying risk variant for overall breast cancer or breast cancer subtypes, whereas in another two regions (11q13, 16q12/TOX3), we identified suggestive evidence of signals that are independent of the reported index variant. Overlapping chromatin features and regulatory elements suggest that many of the risk alleles lie in regions with biological functionality.Conclusions: Through fine-mapping of known susceptibility regions, we have revealed alleles that better characterize breast cancer risk in women of African ancestry.Impact: The risk alleles identified represent genetic markers for modeling and stratifying breast cancer risk in women of African ancestry. Cancer Epidemiol Biomarkers Prev; 26(7); 1016–26. ©2017 AACR.

Supplemental Figure 1. Clinical trial schemata. Diagrammatic representation of the BCIRG 005 (A) and BCIRG 006 (B) adjuvant breast cancer clinical trials

Supporting Information from Optimizing Radiolabeled Engineered Anti-p185HER2 Antibody Fragments for In vivo Imaging

Circulating tumor cells (CTCs) captured from the blood of cancer patients may serve as a surrogate source of tumor material that can be obtained via a venipuncture (also known as a liquid biopsy) and used to better understand tumor characteristics. However, the only FDA-cleared CTC assay has been limited to the enumeration of surface marker–defined cells and not further characterization of the CTCs. In this study, we tested the ability of a semi-automated device capable of capturing and harvesting CTCs from peripheral blood based on cell size and deformability, agnostic of cell-surface markers (the Parsortix® PC1 System), to yield CTCs for evaluation by downstream techniques commonly available in clinical laboratories. The data generated from this study were used to support a De Novo request (DEN200062) for the classification of this device, which the FDA recently granted. As part of a multicenter clinical trial, peripheral blood samples from 216 patients with metastatic breast cancer (MBC) and 205 healthy volunteers were subjected to CTC enrichment. A board-certified pathologist enumerated the CTCs from each participant by cytologic evaluation of Wright-Giemsa-stained slides. As proof of principle, cells harvested from a concurrent parallel sample provided by each participant were evaluated using one of three additional evaluation techniques: molecular profiling by qRT-PCR, RNA sequencing, or cytogenetic analysis of HER2 amplification by FISH. The study demonstrated that the Parsortix® PC1 System can effectively capture and harvest CTCs from the peripheral blood of MBC patients and that the harvested cells can be evaluated using orthogonal methodologies such as gene expression and/or Fluorescence In Situ Hybridization (FISH).

PurposeTo evaluate the association between lifetime personal cigarette smoking and young-onset breast cancer (YOBC; diagnosed