Prognostic and predictive impact of gene expression in node-positive early breast cancer patients receiving dose-dense versus standard-dose adjuvant chemotherapy.

The utility of multigene expression assays in advanced (≥ 4 positive lymph nodes) early breast cancer (EBC) is limited. We conducted exploratory transcriptomic analysis of 758 genes (Breast Cancer 360 panel, nCounter® platform; NanoString) in primary tumor samples collected during a phase 3 trial comparing adjuvant taxane-containing dose-dense chemotherapy (ddCTX) versus standard-dosed chemotherapy (stCTX) in resected EBC with ≥ 4 positive lymph nodes. Prognostic and predictive associations with disease-free survival (DFS) and overall survival (OS) were evaluated by Cox regression with false discovery rate (FDR) adjustment. Data were available from tumor samples of 141/226 patients (median follow-up: 14 years). Several genes/signatures, including immune markers, showed prognostic relevance in unadjusted analyses. Of these, two remained significant after multiplicity adjustment: a positive effect on DFS of programmed cell death 1 ligand-2 (PD-L2) in the ddCTX arm (univariate HR: 0.53, FDR-adjusted P = 0.036) and a negative effect on OS of HER2-enriched (HER2-E) signature in the stCTX arm (univariate HR: 5.40, FDR-adjusted P = 0.036). Predictive analyses showed greater DFS benefit of ddCTX in tumors with high antigen processing machinery (APM) expression (multivariate interaction P = 0.024). Multigene expression assays have a prognostic and predictive potential in advanced EBC, and further investigation is warranted in order to identify candidates for de-escalated treatment. In addition, intrinsic subtype and immune gene expression have predictive potential.

Gene signatures in patients with early breast cancer and relapse despite pathologic complete response.

A substantial minority of early breast cancer (EBC) patients relapse despite their tumors achieving pathologic complete response (pCR) after neoadjuvant therapy. We compared gene expression (BC360; nCounter® platform; NanoString) between primary tumors of patients with post-pCR relapse (N = 14) with: (i) matched recurrent tumors from same patient (intraindividual analysis); and (ii) primary tumors from matched controls with pCR and no relapse (N = 41; interindividual analysis). Intraindividual analysis showed lower estrogen receptor signaling signature expression in recurrent tumors versus primaries (logFC = -0.595; P = 0.022). Recurrent tumors in patients with distant metastases also exhibited reduced expression of immune-related expression parameters. In interindividual analyses, primary tumor major histocompatibility complex class II expression was lower versus controls in patients with any relapse (logFC = -0.819; P = 0.030) or distant relapse (logFC = -1.151; P = 0.013). Primaries with later distant relapse also had greater homologous recombination deficiency than controls (logFC = 0.649; P = 0.026). Although no associations remained statistically significant following adjustment for false discovery rate, our results show that transcriptomic analyses have potential for prognostic value and may help in selecting optimal treatment regimens for EBC at risk of relapse and warrant further investigation.

Olaparib for metastatic breast cancer in a patient with a germline PALB2 variant.

There is a strong biologic rationale that poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitors may benefit a broader range of metastatic breast cancer (MBC) patients than covered by current approvals, which require a germline BRCA1/2 sequence variant affecting function. We report a patient with germline/somatic BRCA1/2 wild-type MBC, who had a dramatic response to the PARP inhibitor olaparib of at least 8 months' duration. The patient is a 37-year-old woman with recurrent, hormone receptor-positive, HER2-negative MBC that had progressed despite hormonal therapy and palbociclib. Sensitivity to olaparib was likely conferred by a germline sequence variant affecting function in PALB2 (exon 1, c.18G>T, p.(=)). This case documenting activity of olaparib monotherapy in germline/somatic BRCA1/2 wild-type MBC illustrates that the clinical potential of PARP inhibition in MBC extends beyond currently approved indications to additional patients whose tumors have (epi)genetic changes affecting homologous recombination repair.

MAP kinase-mediated phosphorylation of distinct pools of histone H3 at S10 or S28 via mitogen- and stress-activated kinase 1/2.

ERK and p38 MAP kinases, acting through the downstream mitogen- and stress-activated kinase 1/2 (MSK1/2), elicit histone H3 phosphorylation on a subfraction of nucleosomes--including those at Fos and Jun--concomitant with gene induction. S10 and S28 on the H3 tail have both been shown to be phospho-acceptors in vivo. Both phospho-epitopes appear with similar time-courses and both occur on H3 tails that are highly sensitive to TSA-induced hyperacetylation, similarities which might suggest that MSK1/2 phosphorylates both sites on the same H3 tails. Indeed, on recombinant histone octamers in vitro, MSK1 efficiently phosphorylates both sites on the same H3 tail. However, sequential immunoprecipitation studies show that antibodies against phosphorylated S10-H3 recover virtually all this epitope without depletion of phosphorylated S28-H3, and vice versa, indicating that the two phospho-epitopes are not located on the same H3 tail in vivo. Confocal immunocytochemistry confirms the clear physical separation of the two phospho-epitopes in the intact mouse nucleus. Finally, we used transfection-based experiments to test models that might explain such differential targeting. Overexpression and delocalisation of MSK1 does not result in the breakdown of targeting in vivo despite the fact that the ectopic kinase is fully activated by external stimuli. These studies reveal a remarkable level of targeting of S10 and S28 phosphorylation to distinct H3 tails within chromatin in the interphase mouse nucleus. Possible models for such exquisite targeting are discussed.

Heat shock, histone H3 phosphorylation and the cell cycle.

Genome-wide and gene-specific changes in histone H3 phosphorylation during heat shock have recently been described using two well-established experimental models, the "puffing" of heat shock loci in Drosophila polytene chromosomes and the induction of hsp70 mRNA transcripts in cultured mouse cells. Despite conservation of the molecular participants and overall stress response in these two organisms, some striking differences have emerged. Here, we summarize accounts of heat shock-modulated histone phosphorylation in Drosophila and mouse cells highlighting these differences. In addition, we describe a further complexity of this response in cultured mouse cells that becomes apparent when the nucleosomal response, referring to histone H3 and HMGN1 phosphorylation, is monitored through the cell cycle. This suggests that some heat shock-induced effects in mouse cells may be indirect and arise as a secondary consequence of the effect of heat shock on the cell cycle, complicating comparisons between the fly and mouse systems.

MSK2 and MSK1 mediate the mitogen- and stress-induced phosphorylation of histone H3 and HMG-14.

Cells respond to mitogenic or stress stimuli by the rapid induction of immediate-early (IE) genes, which occurs concomitantly with the phosphorylation of histone H3 and the high-mobility-group protein HMG-14. In mammalian cells this response is mediated via ERK and p38 MAP kinase pathways, but the identity of the downstream kinase that phosphorylates histone H3 has been contentious. One study, based on Coffin- Lowry cells defective in RSK2, reported that RSK2 was the histone H3 kinase, while a second study, based on the efficiency of RSKs and MSKs as in vitro histone H3 kinases, and their relative susceptibility to kinase inhibitors, suggested that MSKs were responsible. We show here that the histone H3 phosphorylation response is normal in Coffin-Lowry cells. Further more, we show that histone H3 and HMG-14 phosphorylation is severely reduced or abolished in mice lacking MSK1 and MSK2. We also show that, despite this, histone H3 acetylation is unimpaired in these cells and that IE genes can be induced, although at a reduced efficiency. We conclude that MSKs are the major kinases for histone H3 and HMG-14 in response to mitogenic and stress stimuli in fibroblasts.