Predicted sensitivity to endocrine therapy for stage II-III hormone receptor-positive and HER2-negative (HR+/HER2-) breast cancer before chemo-endocrine therapy.

BACKGROUND: We proposed that a test for sensitivity to the adjuvant endocrine therapy component of treatment for patients with stage II-III breast cancer (SET2,3) should measure transcription related to estrogen and progesterone receptors (SETER/PR index) adjusted for a baseline prognostic index (BPI) combining clinical tumor and nodal stage with molecular subtype by RNA4 (ESR1, PGR, ERBB2, and AURKA). PATIENTS AND METHODS: Patients with clinically high-risk, hormone receptor-positive (HR+), human epidermal growth factor receptor 2 (HER2)-negative (HR+/HER2-) breast cancer received neoadjuvant taxane-anthracycline chemotherapy, surgery with measurement of residual cancer burden (RCB), and then adjuvant endocrine therapy. SET2,3 was measured from pre-treatment tumor biopsies, evaluated first in an MD Anderson Cancer Center (MDACC) cohort (n = 307, 11 years' follow-up, U133A microarrays), cut point was determined, and then independent, blinded evaluation was carried out in the I-SPY2 trial (n = 268, high-risk MammaPrint result, 3.8 years' follow-up, Agilent-44K microarrays, NCI Clinical Trials ID: NCT01042379). Primary outcome measure was distant relapse-free survival. Multivariate Cox regression models tested prognostic independence of SET2,3 relative to RCB and other molecular prognostic signatures, and whether other prognostic signatures could substitute for SETER/PR or RNA4 components of SET2,3. RESULTS: SET2,3 added independent prognostic information to RCB in the MDACC cohort: SET2,3 [hazard ratio (HR) 0.23, P = 0.004] and RCB (HR 1.77, P < 0.001); and the I-SPY2 trial: SET2,3 (HR 0.27, P = 0.031) and RCB (HR 1.68, P = 0.008). SET2,3 provided similar prognostic information irrespective of whether RCB-II or RCB-III after chemotherapy, and in both luminal subtypes. Conversely, RCB was most strongly prognostic in cancers with low SET2,3 status (MDACC P < 0.001, I-SPY2 P < 0.001). Other molecular signatures were not independently prognostic; they could effectively substitute for RNA4 subtype within the BPI component of SET2,3, but they could not effectively substitute for SETER/PR index. CONCLUSIONS: SET2,3 added independent prognostic information to chemotherapy response (RCB) and baseline prognostic score or subtype. Approximately 40% of patients with clinically high-risk HR+/HER2- disease had high SET2,3 and could be considered for clinical trials of neoadjuvant endocrine-based treatment.

Circulating tumor DNA in neoadjuvant-treated breast cancer reflects response and survival.

BACKGROUND: Pathologic complete response (pCR) to neoadjuvant chemotherapy (NAC) is strongly associated with favorable outcome. We examined the utility of serial circulating tumor DNA (ctDNA) testing for predicting pCR and risk of metastatic recurrence. PATIENTS AND METHODS: Cell-free DNA (cfDNA) was isolated from 291 plasma samples of 84 high-risk early breast cancer patients treated in the neoadjuvant I-SPY 2 TRIAL with standard NAC alone or combined with MK-2206 (AKT inhibitor) treatment. Blood was collected at pretreatment (T0), 3 weeks after initiation of paclitaxel (T1), between paclitaxel and anthracycline regimens (T2), or prior to surgery (T3). A personalized ctDNA test was designed to detect up to 16 patient-specific mutations (from whole-exome sequencing of pretreatment tumor) in cfDNA by ultra-deep sequencing. The median follow-up time for survival analysis was 4.8 years. RESULTS: At T0, 61 of 84 (73%) patients were ctDNA positive, which decreased over time (T1: 35%; T2: 14%; and T3: 9%). Patients who remained ctDNA positive at T1 were significantly more likely to have residual disease after NAC (83% non-pCR) compared with those who cleared ctDNA (52% non-pCR; odds ratio 4.33, P = 0.012). After NAC, all patients who achieved pCR were ctDNA negative (n = 17, 100%). For those who did not achieve pCR (n = 43), ctDNA-positive patients (14%) had a significantly increased risk of metastatic recurrence [hazard ratio (HR) 10.4; 95% confidence interval (CI) 2.3-46.6]; interestingly, patients who did not achieve pCR but were ctDNA negative (86%) had excellent outcome, similar to those who achieved pCR (HR 1.4; 95% CI 0.15-13.5). CONCLUSIONS: Lack of ctDNA clearance was a significant predictor of poor response and metastatic recurrence, while clearance was associated with improved survival even in patients who did not achieve pCR. Personalized monitoring of ctDNA during NAC of high-risk early breast cancer may aid in real-time assessment of treatment response and help fine-tune pCR as a surrogate endpoint of survival.

Forty years of cancer modelling in the mouse.

Mouse models of human cancer have played an important role in formulating modern concepts of multistage carcinogenesis, and are providing us with a new armoury of tools for the testing of novel therapeutic approaches to cancer treatment. The development of inducible and conditional technologies provide us with greater opportunity to generate mouse models which faithfully recapitulate human tumorigenesis, in terms of both the biology and the genetics of this disease. It is now feasible to control, in time and space, the development of tumours in almost any mouse tissue, such that we now have available mouse models of all major human cancers. Moreover, novel non-invasive approaches to tumour imaging will enable us to follow tumour development and metastasis in vivo, as well as the effects of candidate therapeutic drugs. Such new generation tumour models, which accurately emulate the disease state in situ, should provide a useful platform with which to experimentally test drugs targeted to specific gene products, or combinations of genes that control rate-limiting steps of tumour development.

Automated fluorescent detection of microsatellite instability.

Microsatellites are highly polymorphic repetitive DNA segments dispersed throughout the genome and have been widely used for genetic linkage analysis and allele loss. Instability of microsatellites sequences has been linked to deficiencies in DNA mismatch repair, and is observed in a number of different tumor types. Analysis of microsatellite instability is thought to be a useful clinical tool for cancer diagnosis. Fluorescent detection of microsatellite instability using an automated DNA sequencer holds several distinct advantages over traditional radioactive analysis and electrophoresis, allowing simultaneous analysis of a number of different markers for a large number of samples, high resolution, sensitivity, and clear interpretation of data. In this article we present an established protocol, which has been used successfully to detect microsatellite instability in DNA samples from human tumors and circulating tumor DNA in serum/plasma.

Detection of the Replication Error Phenotype in Ovarian Cancer-PCR Analysis of Microsatellite Instability.

Microsatellites are simple, tandemly repeated DNA sequences that are abundantly distributed throughout the human genome, and because of their polymorphic nature have been widely utilized as genetic markers (1). They consist of a repeating unit of 1 to 5 basepairs, averaging 25 to 60 bases in length, and are commonly found in the form d(CA)n: d(GT)n (2). It has been estimated that there are approximately 100,000 CA/GT repeat sequences in the human genome (3). Studies in patients with HNPCC (hereditary nonpolyposis colorectal cancer) first reported the appearance of instability at microsatellites sequences involving either an expansion or contraction of the repeat sequence (4,5). The suggestion that this might reflect a defect in DNA repair was vindicated when subsequent work demonstrated defects in one of four mismatch repair genes [reviewed in (6)]. Such microsatellite instability (MI) has now been reported in a variety of different tumor types including lung, breast, ovary, stomach, endometrium, and bladder [reviewed in (7)].

Dependence on RAD52 and RAD1 for anticancer drug resistance mediated by inactivation of mismatch repair genes.

Mismatch repair (MMR) proteins repair mispaired DNA bases and have an important role in maintaining the integrity of the genome [1]. Loss of MMR has been correlated with resistance to a variety of DNA-damaging agents, including many anticancer drugs [2]. How loss of MMR leads to resistance is not understood, but is proposed to be due to loss of futile MMR activity and/or replication stalling [3] [4]. We report that inactivation of MMR genes (MLH1, MLH2, MSH2, MSH3, MSH6, but not PMS1) in isogenic strains of Saccharomyces cerevisiae led to increased resistance to the anticancer drugs cisplatin, carboplatin and doxorubicin, but had no effect on sensitivity to ultraviolet C (UVC) radiation. Sensitivity to cisplatin and doxorubicin was increased in mlh1 mutant strains when the MLH1 gene was reintroduced, demonstrating a direct involvement of MMR proteins in sensitivity to these DNA-damaging agents. Inactivation of MLH1, MLH2 or MSH2 had no significant effect, however, on drug sensitivities in the rad52 or rad1 mutant strains that are defective in mitotic recombination and removing unpaired DNA single strands. We propose a model whereby MMR proteins - in addition to their role in DNA-damage recognition - decrease adduct tolerance during DNA replication by modulating the levels of recombination-dependent bypass. This hypothesis is supported by the finding that, in human ovarian tumour cells, loss of hMLH1 correlated with acquisition of cisplatin resistance and increased cisplatin-induced sister chromatid exchange, both of which were reversed by restoration of hMLH1 expression.

PCR analysis of microsatellite instability.

Microsatellites are simple, tandemly repeated DNA sequences which are abundantly distributed throughout the human genome, and because of their polymorphic nature have been widely utilized as genetic markers (1). They consist of a repeating unit of 1-5 base pairs, averaging 25-60 bases in length, and are commonly found in the form d(CA)n:d(GT)n (2). It has been estimated that there are approximately 100,000 CA/GT repeat sequences in the human genome (3). Studies in colorectal tumors first reported the appearance of instability at microsatellites sequences involving either an expansion or contraction of the repeat sequence (4-6). Such microsatellite instability has now been reported in a variety of different tumor types including lung, breast, stomach, endometrium, and bladder (7-11, reviewed in 12). In addition, a number of other diseases are associated with instability in trinucleotide repeats, such as fragile X syndrome (13), myotonic dystrophy (14) and Huntington's Disease (15).

Estrogen regulation of transforming growth factor-alpha in ovarian cancer.

Transforming growth factor alpha (TGFalpha) may be induced by estrogen in estrogen responsive systems and can contribute to the growth-modulatory effects of this hormone. To test whether TGFalpha contributes to estrogen-regulated growth in ovarian cancers, we have compared the effects of 17beta-estradiol (E2) and TGFalpha in a range of ovarian carcinoma cell lines. Addition of E2 to the estrogen receptor (ER)-positive cell lines (PE01, PE04 and PE01CDDP) produced a 2-4 fold increase in TGFalpha protein concentrations in media conditioned by the cells. Both E2 and TGFalpha stimulated the growth of the PE01 and PE04 lines and inhibited the growth of the PE01CDDP line. Furthermore, the E2-mediated growth effects could be reversed by an epidermal growth factor (EGF) receptor-targeted antibody. E2 also down-regulated EGF receptor expression in ER-positive cell lines. In a series of primary ovarian tumors, higher concentrations of ER were associated with an increased percentage of tumors expressing TGFalpha mRNA and a decreased percentage expressing EGF receptor protein. All these data are consistent with E2 increasing production of TGFalpha in ER-positive ovarian cancer and this in turn acting through the EGF receptor to modulate growth in an autocrine manner.

hMLH1 expression and cellular responses of ovarian tumour cells to treatment with cytotoxic anticancer agents.

Loss of expression of the hMLH1 and hPMS2 subunits of the MutL alpha-mismatch repair complex is a frequent event (9/10) in independent cisplatin resistant derivatives of a human ovarian carcinoma cell line. However, only hMLH1 mRNA is decreased in these MutL alpha-deficient lines. No alterations in the levels of the hMSH2 and hMSH6 (GTBP) subunits of the MutS alpha-complex are observed. An increase in the proportion of ovarian tumours negative for the hMLH1 subunit is observed in samples taken at second look laparotomy after chemotherapy (36%: 4/11), compared to untreated tumours (10%: 4/39). No significant difference is observed for hMSH2, hMSH6 or hPMS2. Furthermore, cisplatin and doxorubicin-resistant ovarian lines deficient in hMLH1 expression are cross-resistant to 6-thioguanine and the methylating agent N-methyl-N-nitrosourea (MNU). Depletion of O6-alkylguanine-DNA-alkyltransferase (ATase) activity confers only limited increased sensitivity to MNU. Thus the mismatch repair deficient lines retain DNA damage tolerance even after ATase depletion. The hMLH1 deficient lines also lose ability to engage G1 and G2 cell cycle arrest after cisplatin damage. Together these data suggest that loss of hMLH1 expression may be a high frequency event following exposure of ovarian tumour cells to cisplatin and may be critically involved in the development of drug resistance. Thus, the hMLH1 status of these cells appears to be highly correlated with the ability to engage cell death and cell cycle arrest after DNA damage induced by cisplatin.

Expression of the heat shock protein HSP27 in human ovarian cancer.

The relationship of the heat shock protein HSP27 in ovarian cancer to several biological and clinical parameters was investigated in a series of primary tumors and cell lines. Analysis of 72 primary tumors (54 malignant, 5 borderline, and 13 benign neoplasms) indicated that malignant tumors expressed higher HSP27 concentrations than benign tumors (median values, 0.56 versus 0.25 ng/microgram cytosolic protein; P = 0.032). Tumors from patients with advanced stage (stages II, III, or IV) disease contained significantly higher HSP27 concentrations than tumors from stage I patients (P = 0.018), and an HSP27 content >2.0 ng/microgram cytosolic protein was associated with reduced survival (P = 0.03). Tumors that had demonstrated progressive growth after chemotherapy had a significantly higher HSP27 content than tumors that were static or responsive (P = 0.022). These data indicate that HSP27 is associated with more aggressive malignant ovarian disease and with inherent resistance to chemotherapy. Concentrations of HSP27 were also correlated with indicators of estrogen sensitivity. Therefore, the HSP27 concentration correlated with the estrogen receptor (all tumors, P = 0.0014; malignant tumors only, P = 0.047) but not with the progesterone receptor concentration. Analysis of ovarian cancer cell lines in vitro and in vivo indicated that the HSP27 content was higher in cell lines that were estrogen receptor rich and whose growth was modulated by estrogen as compared with those that were not. Additionally, two estrogen receptor-rich ovarian carcinoma lines demonstrated a small but significant decrease in HSP27 levels in response to 17beta-estradiol in culture. These results suggest that HSP27 may help identify tumors responsive to estrogens.

Growth-control of human ovarian-carcinoma cells by insulin-like growth-factors.

The role of the insulin-like growth factors (IGFs) in 3 cultured human ovarian cancer cell lines (PEO1, PEO4, PEO14) was investigated. All three cell lines express mRNA for IGF-I and the PEO14 cell line expresses mRNA for IGF-II. Protein expression of IGF-II was demonstrated in the PEO14 and PEO4 cell lines. All 3 cell lines expressed mRNA for the IGF type I, IGF type II and insulin receptors; the presence of type I IGF receptors was confirmed by immuno-cytochemistry. IGF-I and insulin markedly stimulated the proliferation of PEO1 and PEO4 but not PEO14 cells while all 3 lines were insensitive to the addition of exogenous IGF-II.

The regulation of growth and protein expression by estrogen in vitro: a study of 8 human ovarian carcinoma cell lines.

The effects of 17 beta-estradiol (E2) on the growth and the levels of estrogen receptor (ER), progesterone receptor (PR) and pS2 protein were examined in a range of 8 ovarian carcinoma cell lines. E2 stimulated growth of the 3 cell lines with an ER content of 80-220 fmol/mg protein but not the 5 cell lines with ER concentrations less than 20 fmol/mg protein. After exposure to E2, ER concentration in 2 of the 3 responsive cell lines was decreased relative to untreated cells and in 2 lines, progesterone receptors were increased. No change in steroid receptor levels was observed in cell lines with low or negligible levels of receptors. The pS2 protein was not induced by E2 in the 5 ovarian carcinoma cell lines examined. These results indicate that E2 can stimulate the growth of some ER-positive ovarian carcinoma cells and that these effects may be associated with changes in the cellular levels of steroid hormone receptors.

Identification of M cells and their distribution in rabbit intestinal Peyer's patches and appendix.

The distribution of intestinal membranous (M) cells has been studied within the follicle-associated epithelium of rabbit Peyer's patches and appendix. Vimentin expression has been assessed as a primary criterion to identify rabbit M cells in tissue sections and in whole tissue preparations. This criterion has been compared to the use of the absence of alkaline phosphatase which, due to its heterogeneous distribution within the enterocyte population, is less reliable than vimentin expression as a marker for rabbit M cells. The pattern of vimentin immunostaining revealed that the majority of M cells are located in the periphery of the follicle-associated epithelium, the dome apex being largely free of M cells. This distribution was confirmed by scanning electron microscopy. Vimentin is also expressed by follicle-associated epithelial cells in the vicinity of crypts which lack the typical lymphocyte-containing pocket of M cells. Cytoplasmic peanut agglutinin binding coincides with vimentin-expression throughout the follicle-associated epithelium but is absent from vimentin-negative enterocytes. The co-localisation of these two phenotypic markers in both M cells and epithelial cells adjacent to crypts, which lack the typical morphology of fully developed rabbit M cells, suggests that they correspond to immature M cells which by their location appear to derive directly from undifferentiated crypt stem cells and not from mature columnar enterocytes.