Identifying prognostic biomarkers for palbociclib add-on therapy in fulvestrant-resistant breast cancer using cell-free DNA sequencing.

BACKGROUND: The FUTURE trial (UMIN000029294) demonstrated the safety and efficacy of adding palbociclib after fulvestrant resistance in patients with hormone receptor-positive (HR+)/human epidermal growth factor receptor 2-negative (HER2-) advanced and metastatic breast cancer (ABC/MBC). In this planned sub-study, cancer panel sequencing of cell-free DNA (cfDNA) was utilized to explore prognostic and predictive biomarkers for further palbociclib treatment following fulvestrant resistance. MATERIALS AND METHODS: Herein, 149 cfDNA samples from 65 patients with fulvestrant-resistant disease were analysed at the time of palbociclib addition after fulvestrant resistance (baseline), on day 15 of cycle 1, and at the end of treatment using the assay for identifying diverse mutations in 34 cancer-related genes. RESULTS: During the course of treatment, mutations in ESR1, PIK3CA, FOXA1, RUNX1, TBX3, and TP53 were the most common genomic alterations observed. Analysis of genomic mutations revealed that before fulvestrant introduction, baseline PIK3CA mutations were marginally lower in metastatic aromatase inhibitor (AI)-treated patients compared to adjuvant AI-treated patients (P = 0.063). Baseline PIK3CA mutations were associated with poorer progression-free survival [hazard ratio: 1.62, P = 0.04]. Comparative analysis between baseline and early-changing gene mutations identified poor prognostic factors including early-changing MAP3K1 mutations (hazard ratio: 4.66, P = 0.04), baseline AR mutations (hazard ratio: 3.53, P = 0.04), and baseline PIK3CA mutations (hazard ratio: 3.41, P = 0.02). Notably, the relationship between ESR1 mutations and mutations in PIK3CA, MAP3K1, and TP53 weakened as treatment progressed. Instead, PIK3CA mutations became correlated with TP53 and FOXA1 mutations. CONCLUSIONS: Cancer panel testing for cfDNA identified prognostic and predictive biomarkers for palbociclib add-on therapy after acquiring fulvestrant resistance in patients with HR+/HER2- ABC/MBC.

Investigation of in-line filter replacement intervals for infusion.

BACKGROUND: In-line filters in peripheral and central venous catheters are used to remove bacterial cells mechanically. A recent study indicated an extension of the use of infusion sets to 7 days. There is no evidence regarding replacement intervals for in-line filters. AIM: To test in-line filters that were used continuously for 7 days in order to investigate their ability to remove bacteria and assess the flow rate. METHODS: Three different in-line filters were attached to an ELNEOPA-NF No. 2 premixed infusion bag of intravenous hyperalimentation, into which Staphylococcus epidermidis ATCC12228 or Escherichia coli ATCC25922 was inoculated. These experiments were compared with a control infusion. The infusion was dropped at a flow rate of 40 mL/h and replaced at 24-h intervals for 7 days. Samples were collected 24 h after drop initiation. FINDINGS: S. epidermidis was not detected in droplets between Days 1 and 6, but In-line filters 1 and 2 showed droplets containing 6-10 colony-forming units/mL on Day 7. E. coli was not detected in any of the filters after 7 days of continuous use. Flow rates <40 mL/h were observed on Day 7 for In-line filter 3 in studies of S. epidermidis, and on Days 4 and 3 for In-line filters 2 and 3, respectively, in studies of E. coli. CONCLUSION: This study revealed differences in bacterial removal and flow rates under high inoculation between the three in-line filters tested. It is suggested that in-line filters can be used continuously for a maximum of 6 days, and reductions in flow rate after 48 h of continuous use should be noted carefully.

Revisiting the definition of estrogen receptor positivity in HER2-negative primary breast cancer.

BACKGROUND: Although 1% has been used as cut-off for estrogen receptor (ER) positivity, several studies have reported that tumors with ER < 1% have characteristics similar to those with 1% ≤ ER < 10%. We hypothesized that in patients with human epidermal growth factor 2 (HER2)-negative breast cancer, a cut-off of 10% is more useful than one of 1% in discriminating for both a better pathological complete response (pCR) rate to neoadjuvant chemotherapy and a better long-term outcome with adjuvant hormonal therapy. Our objectives were to identify a percentage of ER expression below which pCR was likely and to determine whether this cut-off value can identify patients who would benefit from adjuvant hormonal therapy. PATIENTS AND METHODS: Patients with stage II or III HER2-negative primary breast cancer who received neoadjuvant chemotherapy followed by definitive surgery between June 1982 and June 2013 were included. Logistic regression models were used to assess the association between each variable and pCR. Cox models were used to analyze time to recurrence and overall survival. The recursive partitioning and regression trees method was used to calculate the cut-off value of ER expression. RESULTS: A total of 3055 patients were analyzed. Low percentage of ER was significantly associated with high pCR rate (OR = 0.99, 95% CI = 0.986-0.994, P < 0.001). The recommended cut-off of ER expression below which pCR was likely was 9.5%. Among patients with ER ≥ 10% tumors, but not those with 1%≤ER < 10% tumors, adjuvant hormonal therapy was significantly associated with long time to recurrence (HR = 0.24, 95% CI = 0.16-0.36, P < 0.001) and overall survival (HR = 0.32, 95% CI = 0.2-0.5, P < 0.001). CONCLUSION: Stage II or III HER2-negative primary breast cancer with ER < 10% behaves clinically like triple-negative breast cancer in terms of pCR and survival outcomes and patients with such tumors may have a limited benefit from adjuvant hormonal therapy. It may be more clinically relevant to define triple-negative breast cancer as HER2-negative breast cancer with <10%, rather than <1%, of ER and/or progesterone receptor expression.

Medium- to long-term outcomes of anatomical reconstruction of the radioulnar ligament to the ulnar fovea.

We examined the medium- to long-term outcome of the original reconstruction technique of the radioulnar ligament using a half-slip of the extensor carpi ulnaris tendon for chronic foveal avulsion of the triangular fibrocartilage complex. We treated 37 wrists in 36 patients with a minimum follow-up of 5 years. Of 36 patients, 12 had more than 10 years follow-up. At final follow-up, 30 patients (31 wrists) reported no pain, five patients (five wrists) reported mild occasional pain and one patient reported severe persistent pain. Two patients had lost 30° of supination. Complete stability of the distal radio-ulnar joint was achieved in 31 wrists, moderate instability was noted in five and severe distal radio-ulnar joint instability remained in one. The size of the bone tunnel was assessed radiologically: in two it had enlarged; in 23 it was unchanged; and in 12 it appeared to have closed over the tendon graft. The overall clinical outcomes were 28 excellent, four good, four fair and one poor. The 12 wrists with more than 10 years follow-up had seven excellent, three good and two fair clinical results. This reattachment technique appears to work and last over the medium- to long-term. LEVEL OF EVIDENCE: IV.

Contactless electrical conductivity measurement of metallic submicron-grain material: Application to the study of aluminum with severe plastic deformation.

We measured the electrical conductivity σ of aluminum specimen consisting of submicron-grains by observing the AC magnetic susceptibility resulting from the eddy current. By using a commercial platform for magnetic measurement, contactless measurement of the relative electrical conductivity σn of a nonmagnetic metal is possible over a wide temperature (T) range. By referring to σ at room temperature, obtained by the four-terminal method, σn(T) was transformed into σ(T). This approach is useful for cylinder specimens, in which the estimation of the radius and/or volume is difficult. An experiment in which aluminum underwent accumulative roll bonding, which is a severe plastic deformation process, validated this method of evaluating σ as a function of the fraction of high-angle grain boundaries.

Changes in tumor expression of HER2 and hormone receptors status after neoadjuvant chemotherapy in 21,755 patients from the Japanese breast cancer registry.

BACKGROUND: We investigate rates of pathologic complete response (pCR) and tumor expression of ER, PgR, HER2 discordance after neoadjuvant chemotherapy using Japanese breast cancer registry data. PATIENTS AND METHODS: Records of more than 300,000 breast cancer cases treated at 800 hospitals from 2004 to 2013 were retrieved from the breast cancer registry. After data cleanup, we included 21,755 patients who received neoadjuvant chemotherapy and had no distant metastases. pCR was defined as no invasive tumor in the breast detected during surgery after neoadjuvant chemotherapy. HER2 overexpression was determined immunohistochemically and/or using fluorescence in situ hybridization. RESULTS: pCR was achieved in 5.7% of luminal tumors (n = 8730), 24.6% of HER2-positive tumors (n = 4403), and 18.9% of triple-negative tumors (n = 3660). Among HER2-positive tumors, pCR was achieved in 31.6% of ER-negative tumors (n = 2252), 17.0% of ER-positive ones (n = 2132), 31.4% of patients who received trastuzumab as neoadjuvant chemotherapy (n = 2437), and 16.2% of patients who did not receive trastuzumab (n = 1966). Of the 2811 patients who were HER2-positive before treatment, 601 (21.4%) had HER2-negative tumors after neoadjuvant chemotherapy, whereas 340 (3.4%) of the 9947 patients with HER2-negative tumors before treatment had HER2-positive tumors afterward. Of the 10,973 patients with ER-positive tumors before treatment, 499 (4.6%) had ER-negative tumors after neoadjuvant chemotherapy, whereas 519 (9.3%) of the 5607 patients who were ER-negative before treatment had ER-positive tumors afterward. CONCLUSION: We confirmed that loss of HER2-positive status can occur after neoadjuvant treatment in patients with primary HER2-positive breast cancer. We also confirmed that in practice, differences in pCR rates between breast cancer subtypes are the same as in clinical trials. Our data strongly support the need for retest ER, PgR, HER2 of surgical sample after neoadjuvant therapy in order to accurately determine appropriate use of targeted therapy.

Y chromosome haplogroup D2a1 is significantly associated with high levels of luteinizing hormone in Japanese men.

The association between the Y chromosome haplogroup D2 and risk of azoospermia and low sperm motility has been previously studied, and it was indicated that haplogroups DE (YAP lineage) are associated with prostate cancer risk in Japanese males. Our assumption had been that Y chromosome haplogroups may be associated with sex hormone levels, because sex hormones have been deemed responsible for spermatogenesis and carcinogenesis. In this study, we assessed the association between Y chromosome haplogroups and sex hormone levels, including those of testosterone, sex hormone-binding globulin (SHBG), follicle-stimulating hormone (FSH), luteinizing hormone (LH), inhibin-B, and calculated free testosterone (cFT), in 901 young men from the general Japanese population (cohort 1) and 786 Japanese men of proven fertility (cohort 2). We found that the haplogroup D2a1 was significantly associated with high LH levels in a combined analysis involving two cohorts (ß = 0.068, SE = 0.025, p = 0.0075), following correction for multiple testing. To date, this result is the first evidence that implicates Y chromosome haplogroups in an association with sex hormone levels.

Arthroscopy of the distal radioulnar joint.

Wrist arthroscopy is now widely indicated for diagnosis and treatment of acute or chronic wrist pain, especially for triangular fibrocartilage complex (TFCC) lesions, as a gold standard. In most cases radiocarpal and midcarpal arthroscopy was performed, while DRUJ arthroscopy has been rarely performed because of its difficulties. Recent anatomic and biomechanical studies demonstrated that the radioulnar ligament (RUL), which is the proximal component of the TFCC facing to the DRUJ, is the primary stabilizer of the distal radioulnar joint (DRUJ). Rupture of the radioulnar ligament (RUL) at the fovea area, where it is the main attaching portion to the ulna and is the isometric point during forearm rotation as the rotation axis passes, is responsible for DRUJ instability. Although physical examination and imaging diagnosis may indicate a foveal detachment of the TFCC, DRUJ arthroscopy is potential for visualizing the RUL at the fovea. Role of DRUJ arthroscopy should be more important not only for diagnosis of rupture of the RUL but also for decision making of treatment option for RUL tear. DRUJ arthroscopy also demonstrates the joint surface of both the sigmoid notch and ulnar head, and the proximal surface of the TFCC.

Gene expression profiles of inflammatory breast cancer: correlation with response to neoadjuvant chemotherapy and metastasis-free survival.

BACKGROUND: Inflammatory breast cancer (IBC) is an aggressive disease. To date, no molecular feature reliably predicts either the response to chemotherapy (CT) or the survival. Using DNA microarrays, we searched for multigene predictors. PATIENTS AND METHODS: The World IBC Consortium generated whole-genome expression profiles of 137 IBC and 252 non-IBC (nIBC) samples. We searched for transcriptional profiles associated with pathological complete response (pCR) to neoadjuvant anthracycline-based CT and distant metastasis-free survival (DMFS) in respective subsets of 87 and 106 informative IBC samples. Correlations were investigated with predictive and prognostic gene expression signatures published in nIBC (nIBC-GES). Supervised analyses tested genes and activation signatures of 19 biological pathways and 234 transcription factors. RESULTS: Three of five tested prognostic nIBC-GES and the two tested predictive nIBC-GES discriminated between IBC with and without pCR, as well as two interferon activation signatures. We identified a 107-gene signature enriched for immunity-related genes that distinguished between responders and nonresponders in IBC. Its robustness was demonstrated by external validation in three independent sets including two IBC sets and one nIBC set, with independent significant predictive value in IBC and nIBC validation sets in multivariate analysis. We found no robust signature associated with DMFS in patients with IBC, and neither of the tested prognostic GES, nor the molecular subtypes were informative, whereas they were in our nIBC series (220 stage I-III informative samples). CONCLUSION: Despite the relatively small sample size, we show that response to neoadjuvant CT in IBC is, as in nIBC, associated with immunity-related processes, suggesting that similar mechanisms responsible for pCR exist. Analysis of a larger IBC series is warranted regarding the correlation of gene expression profiles and DMFS.

Ink4a/Arf(-/-) and HRAS(G12V) transform mouse mammary cells into triple-negative breast cancer containing tumorigenic CD49f(-) quiescent cells.

Intratumoral heterogeneity within individual breast tumors is a well-known phenomenon that may contribute to drug resistance. This heterogeneity is dependent on several factors, such as types of oncogenic drivers and tumor precursor cells. The purpose of our study was to engineer a mouse mammary tumor model with intratumoral heterogeneity by using defined genetic perturbations. To achieve this, we used mice with knockout (-/-) of Ink4a/Arf, a tumor suppressor locus; these mice are known to be susceptible to non-mammary tumors such as fibrosarcoma. To induce mammary tumors, we retrovirally introduced an oncogene, HRAS(G12V), into Ink4a/Arf(-/-) mammary cells in vitro, and those cells were inoculated into syngeneic mice mammary fat pads. We observed 100% tumorigenesis. The tumors formed were negative for estrogen receptor, progesterone receptor and HER2. Further, they had pathological features similar to those of human triple-negative breast cancer (TNBC) (for example, pushing borders, central necrosis). The tumors were found to be heterogeneous and included two subpopulations: CD49f(-) quiescent cells and CD49f(+)cells. Contrary to our expectation, CD49f(-) quiescent cells had high tumor-initiating potential and CD49f(+)cells had relatively low tumor-initiating potential. Gene expression analysis revealed that CD49f(-) quiescent cells overexpressed epithelial-to-mesenchymal transition-driving genes, reminiscent of tumor-initiating cells and claudin-low breast cancer. Our animal model with intratumoral heterogeneity, derived from defined genetic perturbations, allows us to test novel molecular targeted drugs in a setting that mimics the intratumoral heterogeneity of human TNBC.

Long-term treatment efficacy in primary inflammatory breast cancer by hormonal receptor- and HER2-defined subtypes.

BACKGROUND: Subtypes defined by hormonal receptor (HR) and HER2 status have not been well studied in inflammatory breast cancer (IBC). We characterized clinical parameters and long-term outcomes, and compared pathological complete response (pCR) rates by HR/HER2 subtype in a large IBC patient population. We also compared disease-free survival (DFS) and overall survival (OS) between IBC patients who received targeted therapies (anti-hormonal, anti-HER2) and those who did not. PATIENTS AND METHODS: We retrospectively reviewed the records of patients diagnosed with IBC and treated at MD Anderson Cancer Center from January 1989 to January 2011. Of those, 527 patients had received neoadjuvant chemotherapy and had available information on estrogen receptor (ER), progesterone receptor (PR), and HER2 status. HR status was considered positive if either ER or PR status was positive. Using the Kaplan-Meier method, we estimated median DFS and OS durations from the time of definitive surgery. Using the Cox proportional hazards regression model, we determined the effect of prognostic factors on DFS and OS. Results were compared by subtype. RESULTS: The overall pCR rate in stage III IBC was 15.2%, with the HR-positive/HER2-negative subtype showing the lowest rate (7.5%) and the HR-negative/HER2-positive subtype, the highest (30.6%). The HR-negative, HER2-negative subtype (triple-negative breast cancer, TNBC) had the worst survival rate. HR-positive disease, irrespective of HER2 status, had poor prognosis that did not differ from that of the HR-negative/HER2-positive subtype with regard to OS or DFS. Achieving pCR, no evidence of vascular invasion, non-TNBC, adjuvant hormonal therapy, and radiotherapy were associated with longer DFS and OS. CONCLUSIONS: Hormone receptor and HER2 molecular subtypes had limited predictive and prognostic power in our IBC population. All molecular subtypes of IBC had a poor prognosis. HR-positive status did not necessarily confer a good prognosis. For all IBC subtypes, novel, specific treatment strategies are needed in the neoadjuvant and adjuvant settings.

Statin use in primary inflammatory breast cancer: a cohort study.

BACKGROUND: Some studies have suggested that statins, which have cholesterol-lowering and anti-inflammatory properties, may have antitumor effects. Effects of statins on inflammatory breast cancer (IBC) have never been studied. METHODS: We reviewed 723 patients diagnosed with primary IBC in 1995-2011 and treated at The University of Texas MD Anderson Cancer Center. Statin users were defined as being on statins at the initial evaluation. Based on Ahern et al's statin classification (JNCI, 2011), clinical outcomes were compared by statin use and type (weakly lipophilic to hydrophilic (H-statin) vs lipophilic statins (L-statin)). We used the Kaplan-Meier method to estimate the median progression-free survival (PFS), overall survival (OS) and disease-specific survival (DSS), and a Cox proportional hazards regression model to test the statistical significance of potential prognostic factors. RESULTS: In the multivariable Cox model, H-statins were associated with significantly improved PFS compared with no statin (hazard ratio=0.49; 95% confidence interval=0.28-0.84; P<0.01); OS and DSS P-values were 0.80 and 0.85, respectively. For L-statins vs no statin, P-values for PFS, DSS, and OS were 0.81, 0.4, and 0.74, respectively. CONCLUSION: H-statins were associated with significantly improved PFS. A prospective randomised study evaluating the survival benefits of statins in primary IBC is warranted.

Clinical benefit from neoadjuvant chemotherapy in oestrogen receptor-positive invasive ductal and lobular carcinomas.

BACKGROUND: The aim of this study was to compare clinical and pathological outcomes after neoadjuvant chemotherapy between oestrogen receptor (ER)-positive invasive pure lobular carcinoma (ILC) and invasive ductal carcinoma (IDC). METHODS: This analysis included 1895 patients (n=177 ILC; n=1718 IDC), with stage I-III breast cancer, who received neoadjuvant chemotherapy. Clinical and pathological response rates, the frequency of positive surgical margins and rate of breast-conserving surgery were compared. RESULTS: There was a trend for fewer good clinical responses in ILC compared with IDC. Tumour downstaging was significantly less frequent in ILC. Positive or close surgical resection margins were more frequent in ILC, and breast-conserving surgery was less common (P<0.001). These outcome differences remained significant in multivariate analysis, including tumour size, nodal status, age, grade and type of chemotherapy. Invasive pure lobular carcinoma was also associated with a significantly lower pathological complete response (pCR) rate in univariate analysis, but this was no longer significant after adjusting for tumour size and grade. CONCLUSION: Neoadjuvant chemotherapy results in lower rates of clinical benefit, including less downstaging, more positive margins and fewer breast-conserving surgeries in ER-positive ILC compared with ER-positive IDC. Pathological complete responses are rare in both groups, but do not significantly differ after adjusting for other variables.

Glycosphingolipids regulate ameloblastin expression in dental epithelial cells.

Neurotrophin 4 (NT-4) and its receptors regulate the differentiation of ameloblasts in tooth development. Gangliosides, sialic acids that contain glycosphingolipids (GSLs), are involved in a variety of membrane-associated cell physiological functions such as ligand-receptor signal transmission. However, the expression patterns and functions of GSLs during tooth development remain unclear. In this study, we identified strong expressions of GM3 and LacCer in dental epithelium, which give rise to differentiation into enamel-secreting ameloblasts. Exogenous GM3 and LacCer in dental epithelial cells induced the expression of ameloblastin (Ambn), while it was also interesting that GM3 synergistically exerted enhancement of NT-4-mediated Ambn expression. In addition, consistently exogenous GM3 and LacCer in dental epithelial cells induced distinct activation of extracellular signal-regulated kinase 1/2 (ERK1/2), an event upstream of the expression of Ambn. Furthermore, depletion of GSLs from dental epithelial cells by D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) inhibited Ambn expression as well as phosphorylation of ERK1/2. In contrast, exogenous addition of GM3 or LacCer rescued the phosphorylation of ERK1/2 repressed by pre-treatment with D-PDMP. Taken together, these results suggest that GM3 and LacCer are essential for NT-4-mediated Ambn expression, and contribute to dental epithelial cell differentiation into ameloblasts.

Lack of detection of a putative retrovirus associated with haemic neoplasia in the soft shell clam Mya arenaria.

Haemic neoplasia (HN) is a leukemia-like disease that affects at least 20 species of marine bivalves including soft shell clam, Mya arenaria. Since the disease was discovered in 1969, the etiology remains unknown. A retroviral etiology has been suggested based on the detection of reverse transcriptase activity and electron microscopic observation of retroviral-like particles using negative staining. To date, however no virus isolate and no retroviral sequence from HN has been obtained. Moreover, transmission of the disease by cell-free filtrate from affected clams has not been reproduced. In the current study, we reinvestigated the association of HN with a putative retrovirus. Sucrose gradient centrifugation followed by assessment of reverse transcriptase activity, electrophoretic analysis of protein and RNA, and electron microscopic examinations of fractions corresponding to retroviral density were employed. Detection of retroviral pol sequences using degenerate RT-PCR approaches was also attempted. Our results showed visible bands at the expected density of retrovirus in HN-positive and HN-negative clam tissues and both with reverse transcriptase activity. Electron microscopy, RNA analysis, protein analysis, and PCR systems targeting the pol gene of retroviruses did not however provide clear evidence supporting presence of a retrovirus. We point out that the retrovirus etiology of HN of Mya arenaria proposed some 25 years ago should be reconsidered in the absence of a virus isolate or virus sequences.

Structural and biophysical properties of a synthetic channel-forming peptide: designing a clinically relevant anion selective pore.

The design, synthesis, modeling and in vitro testing of channel-forming peptides derived from the cys-loop superfamily of ligand-gated ion channels are part of an ongoing research focus. Over 300 different sequences have been prepared based on the M2 transmembrane segment of the spinal cord glycine receptor α-subunit. A number of these sequences are water-soluble monomers that readily insert into biological membranes where they undergo supramolecular assembly, yielding channels with a range of selectivities and conductances. Selection of a sequence for further modifications to yield an optimal lead compound came down to a few key biophysical properties: low solution concentrations that yield channel activity, greater ensemble conductance, and enhanced ion selectivity. The sequence NK(4)-M2GlyR T19R, S22W (KKKKPARVGLGITTVLTMRTQW) addressed these criteria. The structure of this peptide has been analyzed by solution NMR as a monomer in detergent micelles, simulated as five-helix bundles in a membrane environment, modified by cysteine-scanning and studied for insertion efficiency in liposomes of selected lipid compositions. Taken together, these results define the structural and key biophysical properties of this sequence in a membrane. This model provides an initial scaffold from which rational substitutions can be proposed and tested to modulate anion selectivity. This article is part of a Special Issue entitled: Protein Folding in Membranes.