ABSTRACT. Marine microbial diversity is important yet poorly-known, due to low culturability and undersampling. However, 16s rRNA gene sequences cloned directly from biomass allow us to know what microbial types are present, irrespective of culturing, and to create probes suitable for biodiversity studies. Many sequences are needed for good probe design. Here we report on sequences from 57 deep sea clones, obtained by the polymerase chain reaction with 'universal' primers, from 500 m and 3000 m depths in the northeast Pacific and 1000 m depth in the subtropical Atlantic. The most common group, with 19 of the new sequences (10 Atlantic), was a recently reported crenarchaeal cluster, Group I. We also found 6 sequences in 2 other archaeal groups in the broad methanogen-halophile lineage; 2 of these were in a distinct lineage not previously reported. The bacterial sequences included 22 dispersed among the a and y Proteobacteria (8 related to SAR 1 l), 5 related to a previously reported broad group (Group A) of marine clones poorly affiliated with known (cultured and sequenced) major bacterial divisions, 6 in a second group with little affiliation to any previously reported division (we call this Group B), 1 In a third possible major novel group, 2 deeply branched with~n the 'Green Nonsulfur' lineage, and 1 branching with a soil clone. In contrast to the vast majority of the sequences, a cluster of 5 sequences was very close to a cultured marine proteobacterium, Alterornonas macleod~j. It appeared that 5 of the clones were chimeric, although this label is difficult to apply when sequences are only distantly related to those in the database, as was common. We conclude that the deep sea contains numerous novel and widespread major prokaryotic lineages. Given the huge volume of this habitat and typical bacteria.1 abundances, it appears that the previously unknown archaeal and bacterial groups may be the most abundant organisms on Earth.

Introduction: HER2 overexpression is observed on CTCs in advanced BC (ABC), but their significance is not known. We aimed to describe clinical, pathologic, and molecular associations with HER2 overexpression on CTCs in ABC patients (pts). Methods: We conducted a retrospective analysis of data from ABC pts treated at Thomas Jefferson University and Northwestern University who had evaluation of CTCs and circulating tumor DNA (ctDNA). CTCs were enumerated with the CellSearch immunomagnetic kit (Menarini Silicon Biosystems), HER2 expression on CTCs was evaluated using the CellSearch CXC Kit, and ctDNA was analyzed using the Guardant360 NGS assay (Guardant Health). Associations with the presence of HER2+ CTCs were explored through univariate and multivariate logistic regression. Kruskal-Wallis testing evaluating HER2+ CTCs as a continuous variable was also conducted to confirm consistency of findings. Time to development of HER2+ CTCs was evaluated using Cox proportional hazards regression analysis. Results: Baseline CTCs were evaluated in 209 pts (10% stage III, 90% stage IV) of whom 41% had no detectable CTCs, 23% had 1-4 CTCs, and 36% had >5 CTCs (stage IV aggressive). Twelve percent had CTC clusters. At least 1 HER2+ CTC was seen in 33% of pts at baseline draw. Of 39 patients with HER2+ BC, only 18% had HER2+ CTCs. Of patients with HER2+ CTCs, 55% had hormone receptor positive BC, 28% had triple negative BC, and 18% had HER2+ BC. On univariate logistic analysis, BC subtype or HER2 status was not associated with the presence of HER2+ CTCs. IBC pts represented 52% of pts and were less likely to have HER2+ CTCs (OR 0.40 95% CI 0.19-0.84). Bone metastases were associated with an increased likelihood of HER2+ CTCs (OR 2.46, 95% CI 1.12-5.38); however, other sites of metastases and number of metastatic sites were not correlated with HER2+ CTCs. Aggressive disease features including >5 CTCs and presence of CTC clusters were strongly associated with HER2+ CTCs (OR 15.72, 95% CI 6.89-35.8 and 8.97, 95% CI 3.23-24.89, respectively). Of 168 pts with ctDNA analysis, ERRB2 aberrations were seen in 22% of pts and were significantly associated with HER2+ CTCs (OR of 3.74, 95% CI 1.45-9.63). On multivariate analysis, the associations with >5 CTCs and ERBB2 alterations in ctDNA remained statistically significant. The associations of HER2+ CTCs with bone disease, >5 CTCs, CTC clusters, and ERBB2 alterations in ctDNA, and the inverse relationship with IBC were consistent when HER2+ CTCs were evaluated as a continuous variable with Kruskal-Wallis testing. Among pts without HER2+ CTCs at baseline, the time to detection of HER2+ CTCs correlated with the presence of bone metastases (HR 3.40, 95% CI 1.14-10.19), >5 CTCs (3.77, 95% CI 1.33-10.70), and visceral disease (HR 3.00, 95% CI 1.07-8.44). Conclusions: HER2+ CTCs are common in ABC, independent of HER2 status of the tumor, and, in fact, common in the luminal BC. HER2+ CTCs were also strongly associated with CTC characteristics of aggressive disease with poor survival (CTCs clusters and >5 CTCs) and ERBB2 aberrations in ctDNA. Further studies will be investigating the role of HER2+ CTCs in endocrine resistance and the potential of anti-HER2 therapy in this unique CTC-defined setting. Citation Format: Shah AN, Gerratana L, Davis AA, Zhang Q, Zhang Y, Rossi G, Wang C, Strickland K, Yang H, Flaum L, Abu-Khalaf M, Behdad A, Ye Z, Platanias L, Gradishar WJ, Cristofanilli M. HER2-positive circulating tumor cells (CTCs) in advanced breast cancer (BC): A feature independent of BC subtype [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-01-19.

FILM, a combination of 5-fluorouracil (5-FU) 750 mg/m(2), ifosfamide 1 g/m(2), leucovorin 200 mg/m(2) and mitomycin C 6 mg/m(2) (alternate cycles), was administered to 24 chemo-naive patients with inoperable disease, locally advanced or metastatic. Up to 6 x 3-weekly cycles of FILM were administered on an out-patient basis. Responses included 8 patients in complete remission (CR) and 12 showing a partial response (PR) (83%). Following analysis of these results, the FILM regimen was introduced as a standard out-patient protocol at the North Middlesex Hospital, United Kingdom. A further 66 patients have been treated in this setting. Retrospective analysis of these data confirm the trial results and allow conclusions regarding tolerability, toxicity, duration of response and survival to be drawn from a total cohort of 90 patients. A total of 524 cycles have been administered. Nineteen cycles (4%) were delayed owing to slow recovery of white blood cells (WBC), but no dose reductions were necessary. Five blood transfusions were required for anaemia. The most frequent non-haematological toxicities included nausea, vomiting and fatigue. Of 80 patients treated for inoperable or locally advanced disease, 56 (70%) remain in remission, and 69 (86%) remain alive after 5 years.