Influence of HER2 expression on prognosis in metastatic triple-negative breast cancer-results from an international, multicenter analysis coordinated by the AGMT Study Group.

BACKGROUND: Triple-negative breast cancer (TNBC) is associated with poor prognosis, and new treatment options are urgently needed. About 34%-39% of primary TNBCs show a low expression of human epidermal growth factor receptor 2 (HER2-low), which is a target for new anti-HER2 drugs. However, little is known about the frequency and the prognostic value of HER2-low in metastatic TNBC. PATIENTS AND METHODS: We retrospectively included patients with TNBC from five European countries for this international, multicenter analysis. Triple-negativity had to be shown in a metastatic site or in the primary breast tumor diagnosed simultaneously or within 3 years before metastatic disease. HER2-low was defined as immunohistochemically (IHC) 1+ or 2+ without ERBB2 gene amplification. Survival probabilities were calculated by the Kaplan-Meier method, and multivariable hazard ratios (HRs) were estimated by Cox regression models. RESULTS: In total, 691 patients, diagnosed between January 2006 and February 2021, were assessable. The incidence of HER2-low was 32.0% [95% confidence interval (CI) 28.5% to 35.5%], with similar proportions in metastases (n = 265; 29.8%) and primary tumors (n = 425; 33.4%; P = 0.324). The median overall survival (OS) in HER2-low and HER2-0 TNBC was 18.6 and 16.1 months, respectively (HR 1.00; 95% CI 0.83-1.19; P = 0.969). Similarly, in multivariable analysis, HER2-low had no significant impact on OS (HR 0.95; 95% CI 0.79-1.13; P = 0.545). No difference in prognosis was observed between HER2 IHC 0/1+ and IHC 2+ tumors (HR 0.89; 95% CI 0.69-1.17; P = 0.414). CONCLUSIONS: In this large international dataset of metastatic TNBC, the frequency of HER2-low was 32.0%. Neither in univariable nor in multivariable analysis HER2-low showed any influence on OS.

Sustained response to brigatinib in a patient with refractory metastatic pheochromocytoma harboring R1192P anaplastic lymphoma kinase mutation: a case report from the Austrian Group Medical Tumor Therapy next-generation sequencing registry and discussion of the literature.

Metastatic pheochromocytoma and paraganglioma (PPGL) are rare diseases with dismal prognosis and standard therapies are lacking. We herein report the first case of a germline anaplastic lymphoma kinase (ALK) mutation in a patient with chemorefractory metastatic pheochromocytoma in the absence of mutations of known PPGL-associated predisposing genes. Therapy with the ALK inhibitor (ALKi) brigatinib led to dramatic and durable disease remission, despite previous disease progression on the ALKi alectinib. This case underscores the potential clinical use of molecular profiling in rare diseases with limited treatment options and suggests that the ALK-R1192P point mutation might predict sensitivity to brigatinib.

A clinical validation study of MammaPrint in hormone receptor-positive breast cancer from the Austrian Breast and Colorectal Cancer Study Group 8 (ABCSG-8) biomarker cohort.

BACKGROUND: MammaPrint is a prognostic assay based on gene expression in tumors from patients with early breast cancer. MammaPrint has been extensively validated and Food and Drug Administration cleared in fresh and formalin-fixed and paraffin-embedded (FFPE) tissue. We aimed to assess its prognostic performance in the biomarker cohort of the Austrian Breast and Colorectal Cancer Study Group 8 (ABCSG-8) patient population, and to obtain a higher level of evidence with regard to its clinical validity after RNA extraction from FFPE biobank tissue. PATIENTS AND METHODS: A prespecified retrospective analysis to test the prognostic performance of the MammaPrint test to predict distant recurrence-free survival at 5 and 10 years as primary end point was carried out. MammaPrint risk, clinicopathological factors (after central pathological review), and clinical risk (using a modified version of Adjuvant! Online) were evaluated by Cox regression analyses. RESULTS: From 1347 available samples, 607 (45%) failed quality control after RNA extraction. In total, 658 (49%) patients were included in survival analyses: MammaPrint low risk versus high risk is a significant prognostic factor for distant recurrence-free survival at 5 years (94.0% versus 91.6%) with a significant risk reduction of 6.5% at 10 years (log-rank P value = 0.017, low risk 91.3% versus high risk 84.8%). The multivariable models suggest that hazard ratio (HR) is primarily driven by tumor stage (5-year HR 3.89; confidence interval 1.97-7.71) and nodal status (5-year HR 1.73; confidence interval 0.91-3.21). After adjustment for clinical risk groups, MammaPrint HRs remain stable with values just below 2.0 after the first 3 years. CONCLUSIONS: The MammaPrint test showed significant prognostic performance at 5 and 10 years of follow-up. In the particular cohort of ABCSG-8, the statistical independence from clinically assessed covariates remains unclear, and no conclusions concerning the clinical validity of the test can be drawn.

Clonal evolution in diffuse large B-cell lymphoma with central nervous system recurrence.

BACKGROUND: The prognosis of patients with secondary central nervous system lymphoma (SCNSL) is poor and despite massive advances in understanding the mutational landscape of primary diffuse large B-cell lymphoma (DLBCL), the genetic comparison to SCNSL is still lacking. We therefore collected paired samples from six patients with DLBCL with available biopsies from a lymph node (LN) at primary diagnosis and the central nervous system (CNS) at recurrence. PATIENTS AND METHODS: A targeted, massively parallel sequencing approach was used to analyze 216 genes recurrently mutated in DLBCL. Healthy tissue from each patient was also sequenced in order to exclude germline mutations. The results of the primary biopsies were compared with those of the CNS recurrences to depict the genetic background of SCNSL and evaluate clonal evolution. RESULTS: Sequencing was successful in five patients, all of whom had at least one discordant mutation that was not detected in one of their samples. Four patients had mutations that were found in the CNS but not in the primary LN. Discordant mutations were found in genes known to be important in lymphoma biology such as MYC, CARD11, EP300 and CCND3. Two patients had a Jaccard similarity coefficient below 0.5 indicating substantial genetic differences between the primary LN and the CNS recurrence. CONCLUSIONS: This analysis gives an insight into the genetic landscape of SCNSL and confirms the results of our previous study on patients with systemic recurrence of DLBCL with evidence of substantial clonal diversification at relapse in some patients, which might be one of the mechanisms of treatment resistance.

Correction: The influence of FCGR2A and FCGR3A polymorphisms on the survival of patients with recurrent or metastatic squamous cell head and neck cancer treated with cetuximab.

In the abstract and in other parts of the manuscript the authors wrote that the mutation rs396991 causes a valine (V) to phenylalanine (F) substitution at position 157. However, the correct codon number is 158. These errors have not been fixed in the original Article.

The influence of FCGR2A and FCGR3A polymorphisms on the survival of patients with recurrent or metastatic squamous cell head and neck cancer treated with cetuximab.

FCGR2A-H131R and FCGR3A-V157F are single-nucleotide polymorphisms known to influence the outcome of patients treated with rituximab, cetuximab and trastuzumab. We investigated the impact of these polymorphisms on the clinical outcome of 103 patients with recurrent or metastatic squamous cell carcinoma of the head and neck treated with a platinum compound, fluorouracil and cetuximab as palliative first-line therapy. The survival of patients with FCGR2A-131H/H and/or FCGR3A-157V/V genotypes was significantly longer compared with patients carrying 131R and 157F alleles (median progression-free survival (PFS): 5.5 vs 4.1 months, P=0.02; median overall survival: 10.2 vs 7.2 months, P=0.04). In multivariate analysis, the FCGR2A and 3A genotypes as well as the time between initial diagnosis and relapse of disease not amenable to curative therapy remained the only independent prognostic factors for PFS. The results are in line with previous reports in colorectal cancer patients and confirm the possible value of genetic polymorphisms of immunocompetent cells for the success of cetuximab treatment.

Endothelin-1 genetic polymorphism as predictive marker for bevacizumab in metastatic breast cancer.

Biomarkers for bevacizumab efficacy in metastatic breast cancer (MBC) are of urgent need. The genetic variability of genes involved in angiogenesis could explain the interpatient variability of drug effects. For this biomarker study DNA was extracted from tumor blocks or blood samples of patients with human epidermal growth factor receptor 2 (HER2)-negative MBC treated with bevacizumab in combination with chemotherapy (bevacizumab cohort, 163 patients) or chemotherapy only (control cohort, 105 patients). We assessed the correlation of 10 single-nucleotide polymorphisms (SNPs) in genes modulating angiogenesis (vascular endothelial growth factor-A (VEGF-A), VEGF receptor 1 (VEGFR-1), serine threonine kinase 39 (STK39)) or hypertension (endothelin-1 and uromodulin) with outcome and toxicity. In the bevacizumab cohort, the SNP rs5370-TT in endothelin-1 (EDN1) showed a significantly shorter median overall survival (OS, 6.3 vs 21.3 months; hazard ratio (HR) 2.89, 95% confidence interval (CI) 1.34-6.26; log-rank P=0.0069) and a trend toward worse median progression-free survival (3.5 vs 7.9 months; HR 2.05, 95% CI 0.96-4.39; log-rank P=0.065) compared with the alternate genotypes combined. Similarly, patients harboring the VEGF-936 (rs3025039) TT alleles showed a significantly shorter median OS than patients with VEGF-936 CC or CT (14.9 vs 21.3 months; HR 2.37, 95% CI 1.09-5.13; P=0.0286). In multivariate analysis including important clinical parameters like disease-free survival (DFS), adjuvant chemotherapy, ECOG (Eastern Cooperative Oncology Group) performance score, histologic subtype, grade, hormone receptor status, visceral metastases and treatment line, only the association of rs5370 (EDN1) with OS was still statistically significant (P=0.012). In the control cohort, no association of the EDN1 genotype with outcome was seen, suggesting a predictive value for bevacizumab. In conclusion, the SNP rs5370 in endothelin-1 could help identifying patients who unlikely gain any benefit from bevacizumab.

Leading edge movement and ultrastructure in mouse macrophages.

The first event in the process of translocation of a cell over a substrate is the forward protrusion of a thin layer of cytoplasm, sometimes referred to as the leading edge. To gain more direct information on structural reorganizations associated with protrusion we have documented the ultrastructure of the actin cytoskeleton of mouse macrophages whose history of locomotion prior to fixation for electron microscopy had been recorded by video microscopy. It is shown that rapid protrusion is associated with the formation of a dense, diagonal network of actin filaments, lacking organized bundles. In cell edges that showed minor fluctuations back and forth over a period of 30 sec or more no dense meshworks were found: instead, a loose peripheral bundle of actin filaments was commonly observed. Cell edges that first protruded and then retracted showed a similar ultrastructure to those that exhibited only forward movement, but the width of the leading edge meshwork was, by comparison, reduced. Measurements showed that there was an approximate correlation between the leading edge mesh width and the net forward translocation observed during the terminal 30 sec, up to fixation. The results are discussed in relation to present concepts of the protrusion mechanism.

Contact formation during fibroblast locomotion: involvement of membrane ruffles and microtubules.

We have correlated the motility of the leading edge of fibroblasts, monitored by phase-contrast cinematography, with the relative distributions of several cytoskeletal elements (vinculin, tubulin, and actin) as well as with the contact patterns determined by interference reflection microscopy. This analysis has revealed the involvement of both ruffles and microspikes, as well as microtubules in the initiation of focal contact formation. Nascent vinculin sites within the leading edge or at its base, taken as primordial cell-substrate contacts, were invariably colocalized with sites that showed a history of transient, prolonged, or cyclic ruffling activity. Extended microspike structures, often preceded the formation of ruffles. Immunofluorescent labeling indicated that some of these primordial contacts were in close apposition to the ends of microtubules that penetrated into the leading edge. By fluorescence and electron microscopy short bundles of actin filaments found at the base of the leading edge were identified as presumptive, primordial contacts. It is concluded that ruffles and microspikes, either independently or in combination, initiate and mark the sites for future contact. Plaque proteins then accumulate (within 10-30 s) at the contract site and, beneath ruffles, induce localized bundling of actin filaments. We propose that all primordial contacts support traction for leading edge protrusion but that only some persist long enough to nucleate stress fiber assembly. Microtubules are postulated as the elements that select, stabilize, and potentiate the formation of these latter, long-lived contacts.

Coumarin-phalloidin: a new actin probe permitting triple immunofluorescence microscopy of the cytoskeleton.

7-Diethylamino-3-(4-isothiocyanotophenyl)-4-methylcoumarin (CPITC) was coupled to amino-methyldithiolanophalloidin to produce a new phalloidin derivative, coumarin-phalloidin, fluorescent in the blue region of the spectrum. Coumarin-phalloidin binds to actin with around 100-fold less affinity than unconjugated phalloidin, but with enough avidity to make it a useful stain for actin filaments. Appropriate filter combinations permit triple immunofluorescence microscopy of the cytoskeleton with fluorescein and rhodamine conjugates together with coumarin-phalloidin.

Microfilament-organizing centers in areas of cell contact: cytoskeletal interactions during cell attachment and locomotion.

In this article we discuss three aspects of cell contact formation: (a) the molecular architecture of the cytomatrix in cell-to-substrate focal contacts, (b) the dynamic properties of membrane- and microfilament-associated proteins in the contact areas, and (c) the involvement of microtubules in the coordinated and directed formation of new substrate contacts during cell locomotion. We show that different microfilament-associated proteins exhibit distinct patterns of association with focal contacts: some proteins are specifically associated with focal contacts (vinculin and talin); alpha-actinin is enriched in the contact areas but also is present along the stress fibers and in the lamellipodium; actin and filamin are detected throughout the contact areas but in apparently reduced amounts compared with the associated stress fibers; and tropomyosin, myosin, and spectrin are either absent from the endofacial surfaces of contact areas or are present in only very small amounts. Fluorescence photobleaching recovery analyses performed with living cells microinjected with fluorescently labeled actin, vinculin, and alpha-actinin indicate that each of these proteins maintains a dynamic equilibrium between a soluble cytoplasmic pool and a membrane-bound fraction. Correlation of the distribution of vinculin and tubulin in motile fibroblasts to local movements of the leading edge of the same cells indicates that free-end microtubules extend into actively ruffling areas along the lamellipodium and that new vinculin-containing contacts are preferentially formed in these protruding regions.