A tumour-bed boost delivered after whole-breast radiotherapy increases local cancer-control rates but requires more patient visits and can increase breast hardness. IMPORT HIGH tested simultaneous ...integrated boost against sequential boost with the aim of reducing treatment duration while maintaining excellent local control and similar or reduced toxicity.
IMPORT HIGH is a phase 3, non-inferiority, open-label, randomised controlled trial that recruited women after breast-conserving surgery for pT1–3pN0–3aM0 invasive carcinoma from radiotherapy and referral centres in the UK. Patients were randomly allocated to receive one of three treatments in a 1:1:1 ratio, with computer-generated random permuted blocks used to stratify patients by centre. The control group received 40 Gy in 15 fractions to the whole breast and 16 Gy in 8 fractions sequential photon tumour-bed boost. Test group 1 received 36 Gy in 15 fractions to the whole breast, 40 Gy in 15 fractions to the partial breast, and 48 Gy in 15 fractions concomitant photon boost to the tumour-bed volume. Test group 2 received 36 Gy in 15 fractions to the whole breast, 40 Gy in 15 fractions to the partial breast, and 53 Gy in 15 fractions concomitant photon boost to the tumour-bed volume. The boost clinical target volume was the clip-defined tumour bed. Patients and clinicians were not masked to treatment allocation. The primary endpoint was ipsilateral breast tumour relapse (IBTR) analysed by intention to treat; assuming 5% 5-year incidence with the control group, non-inferiority was predefined as 3% or less absolute excess in the test groups (upper limit of two-sided 95% CI). Adverse events were assessed by clinicians, patients, and photographs. This trial is registered with the ISRCTN registry, ISRCTN47437448, and is closed to new participants.
Between March 4, 2009, and Sept 16, 2015, 2617 patients were recruited. 871 individuals were assigned to the control group, 874 to test group 1, and 872 to test group 2. Median boost clinical target volume was 13 cm3 (IQR 7 to 22). At a median follow-up of 74 months there were 76 IBTR events (20 for the control group, 21 for test group 1, and 35 for test group 2). 5-year IBTR incidence was 1·9% (95% CI 1·2 to 3·1) for the control group, 2·0% (1·2 to 3·2) for test group 1, and 3·2% (2·2 to 4·7) for test group 2. The estimated absolute differences versus the control group were 0·1% (–0·8 to 1·7) for test group 1 and 1·4% (0·03 to 3·8) for test group 2. The upper confidence limit for test group 1 versus the control group indicated non-inferiority for 48 Gy. Cumulative 5-year incidence of clinician-reported moderate or marked breast induration was 11·5% for the control group, 10·6% for test group 1 (p=0·40 vs control group), and 15·5% for test group 2 (p=0·015 vs control group).
In all groups 5-year IBTR incidence was lower than the 5% originally expected regardless of boost sequencing. Dose-escalation is not advantageous. 5-year moderate or marked adverse event rates were low using small boost volumes. Simultaneous integrated boost in IMPORT HIGH was safe and reduced patient visits.
Cancer Research UK.
Leiomyosarcomas of soft tissues are an aggressive group of tumors with a high incidence of recurrence. Little is known about the molecular genetic changes associated with clinical outcome. Therefore, ...we studied 28 leiomyosarcoma samples of similar grade using comparative genomic hybridization and DNA flow cytometry and identified a difference in survival time associated with ploidy status and the number of chromosomal aberrations. The average survival time was shown to decrease with increase in chromosomal aberrations identified using comparative genomic hybridization. The average survival time was shorter in the near-tetraploid group than in the diploid and triploid group. Gain of 5p14-pter was significantly more common in near-tetraploid tumors. The survival time of patients with near-tetraploidy together with gain of 5p14-pter was reduced, and 50% died within the 1st year. Furthermore, loss of 13q14-q21 was significantly more frequent in the <5-year than in the >5-year survival group (P = .01). These results suggest that 13q14-q21 loss and 5p14-pter gain at diagnosis could be used to identify patients with leiomyosarcoma who are likely to have a shorter survival time and who might benefit from early treatment intensification.
Thymidylate
synthase (TS) is an important target for cancer chemotherapy. However,
several mechanisms of resistance to TS inhibitors have been described.
One mechanism that may be relevant to ...short-term exposure to TS
inhibitors occurs as a result of disruption of the autoregulatory loop,
which allows TS to control its own translation. This disruption leads
to up-regulation of TS protein and is generally thought to decrease
efficacy. This study has investigated TS protein up-regulation using a
range of TS inhibitors in both tumor and nonmalignant cell lines
in vitro and in vivo.
Up-regulation of TS protein showed a time-, dose-, and
cell-type-specific response to treatment with ZD9331. This response was
observed in W1L2 cells treated for 24 h at equitoxic doses of
raltitrexed (6-fold), ZD9331 (10-fold), fluorouracil (5-fold), LY231514
(7-fold), AG337 (7-fold), and BW1843U89 (3-fold). Up-regulation was
observed over a range of doses. Elevation of TS protein only persisted
up to 12 h after removal of drug. The extent of induction does not
depend on basal TS levels. Nontransformed human fibroblasts showed
significantly greater up-regulation of TS protein than tumor cells
exposed to an equitoxic dose of ZD9331. In vivo
experiments using the L5178Y thymidine kinase −/− mouse lymphoma
implanted into DBA2 mice also showed greater up-regulation of TS
protein in normal intestinal epithelial cells compared with tumor
cells.
These results confirm that TS up-regulation is a common feature
of TS inhibition in tumor cells and that it may occur to a greater
extent in normal tissues, although the clinical implications of these
findings remain to be determined.
Wild-type p53 is frequently mutated in late-stage ovarian cancer and has been proposed as a determinant of cisplatin chemosensitivity.
We have therefore established a human ovarian cancer cell line ...differing only in p53 status and characterized its response
after treatment with different platinum complexes. The wild-type p53-expressing cell line A2780 was stably transfected with
HPV-16 E6 (E6) or an empty vector (VC) as control. Parental A2780 and VC had similar cisplatin sensitivities, whereas E6 was 3- to
4-fold more sensitive as measured by sulforhodamine B and clonogenic assay. E6 was 2- to 3-fold more sensitive to transplatin
and the novel cisplatin analog ZD0473 than VC, whereas the trans -platinum analog JM335 was approximately equitoxic. Platinum uptake was similar for all of the cell lines after cisplatin.
The removal of platinum-DNA adducts, as measured by atomic absorption spectroscopy, was reduced in E6 compared with VC after
cisplatin but similar after JM335. After 10 μM cisplatin, the G 1 population (0â96 h) was reduced in E6 cells compared with VC, whereas the S phase (8â48 h) and G 2 phase (48â96 h) were increased. Similar proportions of VC and E6 cells died by apoptosis, as detected by annexin V binding,
but more E6 cells died by necrosis relative to VC. Our results suggest that the loss of functional p53 can increase cisplatin
cytotoxicity in A2780, with loss of G 1 /S checkpoint control and decreased cisplatin-DNA adduct repair, but these effects can be circumvented by the use of JM335,
which forms different DNA-platinum adducts.
Background
Whole-breast radiotherapy (WBRT) is the standard treatment for breast cancer following breast-conserving surgery. Evidence shows that tumour recurrences occur near the original cancer: the ...tumour bed. New treatment developments include increasing dose to the tumour bed during WBRT (synchronous integrated boost) and irradiating only the region around the tumour bed, for patients at high and low risk of tumour recurrence, respectively. Currently, standard imaging uses bony anatomy to ensure accurate delivery of WBRT. It is debatable whether or not more targeted treatments such as synchronous integrated boost and partial-breast radiotherapy require image-guided radiotherapy (IGRT) focusing on implanted tumour bed clips (clip-based IGRT).
Objectives
Primary – to compare accuracy of patient set-up using standard imaging compared with clip-based IGRT. Secondary – comparison of imaging techniques using (1) tumour bed radiotherapy safety margins, (2) volume of breast tissue irradiated around tumour bed, (3) estimated breast toxicity following development of a normal tissue control probability model and (4) time taken.
Design
Multicentre observational study embedded within a national randomised trial: IMPORT-HIGH (Intensity Modulated and Partial Organ Radiotherapy – HIGHer-risk patient group) testing synchronous integrated boost and using clip-based IGRT.
Setting
Five radiotherapy departments, participating in IMPORT-HIGH.
Participants
Two-hundred and eighteen patients receiving breast radiotherapy within IMPORT-HIGH.
Interventions
There was no direct intervention in patients’ treatment. Experimental and control intervention were clip-based IGRT and standard imaging, respectively. IMPORT-HIGH patients received clip-based IGRT as routine; standard imaging data were obtained from clip-based IGRT images.
Main outcome measures
Difference in (1) set-up errors, (2) safety margins, (3) volume of breast tissue irradiated, (4) breast toxicity and (5) time, between clip-based IGRT and standard imaging.
Results
The primary outcome of overall mean difference in clip-based IGRT and standard imaging using daily set-up errors was 2–2.6 mm (
p
< 0.001). Heterogeneity testing between centres found a statistically significant difference in set-up errors at one centre. For four centres (179 patients), clip-based IGRT gave a mean decrease in the systematic set-up error of between 1 mm and 2 mm compared with standard imaging. Secondary outcomes were as follows: clip-based IGRT and standard imaging safety margins were less than 5 mm and 8 mm, respectively. Using clip-based IGRT, the median volume of tissue receiving 95% of prescribed boost dose decreased by 29 cm
3
(range 11–193 cm
3
) compared with standard imaging. Difference in median time required to perform clip-based IGRT compared with standard imaging was X-ray imaging technique dependent (range 8–76 seconds). It was not possible to estimate differences in breast toxicity, the normal tissue control probability model indicated that for breast fibrosis maximum radiotherapy dose is more important than volume of tissue irradiated.
Conclusions and implications for clinical practice
Margins of less than 8 mm cannot be used safely without clip-based IGRT for patients receiving concomitant tumour bed boost, as there is a risk of geographical miss of the tumour bed being treated. In principle, smaller but accurately placed margins may influence local control and toxicity rates, but this needs to be evaluated from mature clinical trial data in the future.
Funding
The National Institute for Health Research Efficacy and Mechanism Evaluation programme.
Abstract Background: Most ipsilateral breast tumor recurrences (IBTR) following breast conserving surgery occur near the tumor bed (Veronisi et al, Ann Oncol 2001) and are considered to represent ...‘true recurrence’ (derived from residual malignant cells of the index cancer). A smaller number occur elsewhere in the breast and are thought to be ‘new primaries’ (independently occurring cancer). The IMPORT LOW (Coles et al, The Lancet 2017) and IMPORT HIGH (Coles et al, The Lancet 2023) trials investigated adaptation of radiotherapy dose-volume to this spatially varying risk, testing partial breast irradiation and simultaneous integrated boost in patients with tumors at low and high risk of IBTR respectively. We analysed genomic relationships between index cancers and recurrences in these trials, to ascertain the frequency of true recurrences and unrelated new primaries, and their spatial distribution. Methodology: FFPE blocks were obtained from 137 patients who developed subsequent ipsi- or contralateral breast cancer: 66 from IMPORT HIGH and 71 from IMPORT LOW (4 with bilateral second cancers). DNA extracted from index and subsequent cancers underwent shallow whole genome sequencing (sWGS) using the Illumina NovaSeq 6000 system. Copy number profiles were derived from sWGS data using the R package QDNAseq(Scheinin et al, Genome Res 2014); those that failed QC criteria were removed. Relatedness of tumor pairs was determined using the R package Breakclone (Lips et al, Nat Genet 2022). This approach uses individual copy number aberration breakpoint position rather than events at chromosomal arm level. The p value cutoff for relatedness was 0.01; values between 0.01 and 0.05 were considered ambiguous. Researchers were blinded to clinical information prior to results. Results: In 80/137 tumor pairs both copy number profiles met QC criteria and clonal relationship could be ascertained. In total 26/80 were considered related, 20/80 ambiguous and 34/40 non-related (Table 1). 16/26 ipsilateral subsequent cancers in IMPORT HIGH were clonally related to the primary tumor and could be considered true recurrences. In 5/26 it was impossible to accurately call clonality as pairs shared only common copy number changes such as 1q or 8q gain and 16q loss. 5/26 were considered new primaries. In the lower risk IMPORT LOW cohort, subsequent cancers appear to have similar chances of being related or independent. As expected, most contralateral tumors in IMPORT LOW showed no evidence of a clonal relationship to the index tumors. In both trials an ambiguous subgroup of pairs shared some common copy number changes suggesting similar phenotype.5/14 contralateral tumors in IMPORT HIGH, in which some women were also at high risk of systemic relapse, shared very similar copy number profiles to the index tumor and may be metastases rather than new primaries. Discussion: A considerable number of tumors classed as IBTR appear genomically unrelated to the index cancer particularly in low risk cancer; this biology is relevant to clinical management both initially and at ‘recurrence’. The finding that 5/14 contralateral cancers in the high risk group appear to be clonally related suggests that some may be metastases. Results are being confirmed by targeted sequencing and will be correlated with recorded spatial and clinicopathological data and patient outcome. Future work will analyse spatial relationships more precisely using deformable image registration. *From the 77 patients with baseline circulating tumor DNA samples, 2 patients were excluded because their samples failed during assay quality control. Table. PIK3CA and ESR1 alterations detected in circulating tumor DNA at baseline and post abemaciclib treatment Citation Format: Sara Lightowlers, Maria Roman-Escorza, Elena Provenzano, Judith Bliss, Jason Carroll, H Y Charlie Chan, Clare Griffin, Joanne Haviland, Monica Jefford, Anna Kirby, Navita Somaiah, Mark Sydenham, Jenny Titley, John Yarnold, Charlotte Coles, Elinor Sawyer. Genomic analysis of local recurrences following risk adapted breast radiotherapy in the IMPORT trials defines 'true recurrences' and 'new primaries' abstract. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-22-04.
We reject the inference of a survival benefit for patients receiving partial-breast irradiation within the IMPORT LOW trial and caution against any such interpretation when the number of events ...reported is so small.1 There is no suggestion of a difference in disease-free and overall survival across IMPORT LOW treatment groups.1
Lineage analysis in vitro of heterogeneous tissues such as mammary epithelium requires the separation of constituent cell types and their growth as clones. The separation of virgin mouse mammary ...luminal epithelial and myoepithelial cells by fluorescence-activated cell-sorting, their growth at clonal density, and the phenotyping of the clones obtained with cell-type specific markers are described in this paper. Epithelial cells were isolated by collagenase digestion followed by trypsinization, and the luminal and myoepithelial cells were flow-sorted with the rat monoclonal antibodies 33A10 and JB6, respectively. Sorted cells were cloned under, using low oxygen conditions (<5% vol/vol), in medium containing cholera toxin and insulin, with an irradiated feeder layer of 3T3-L1 cells. Clones were characterized morphologically, and antigenically by multiple immunofluorescence with a panel of antibodies to cytoskeletal antigens specific to either luminal epithelial or myoepithelial cells in situ. Whereas sorted myoepithelial cells gave a single clone type, sorted luminal cells gave three morphological clone types, two of which grew rapidly. All myoepithelially derived clones showed a limited proliferative capacity in vitro, in contrast to their rat and human counterparts, as shown in previous studies. The present results with sorted mouse cells have also allowed the stability of the differentiated phenotype in mouse, rat, and human mammary luminal epithelial and myoepithelial cells in primary clonal culture to be compared. They show that the mouse mammary cells are the least stable in terms of expression of differentiation-specific cytoskeletal markers in vitro.
IMPORT HIGH is a multicentre randomized UK trial testing dose-escalated intensity-modulated radiotherapy (IMRT) after tumour excision in females with early breast cancer and higher than average local ...recurrence risk. A survey was carried out to investigate the impact of this trial on the adoption of advanced breast radiotherapy (RT) techniques in the UK.
A questionnaire was sent to all 26 IMPORT HIGH recruiting RT centres to determine whether the trial has influenced non-trial breast RT techniques in terms of volume delineation, dosimetry, treatment delivery and verification. In order to compare the clinical practice of breast RT between IMPORT HIGH and non-IMPORT HIGH centres, parts of the Royal College of Radiologists (RCR) breast RT audit result were used in this study.
26/26 participating centres completed the questionnaire. After joining the trial, the number of centres routinely using tumour bed clips to guide whole-breast RT rose from 5 (19%) to 21 (81%). 20/26 (77%) centres now contour target volumes and organs at risk (OARs) in some or all patients compared with 14 (54%) before the trial. 14/26 (54%) centres offer inverse-planned IMRT for selected non-trial patients with breast cancer, and 10/14 (71%) have adopted the IMPORT HIGH trial protocol for target volume and OARs dose constraints. Only 2/26 (8%) centres used clip information routinely for breast treatment verification prior to IMPORT HIGH, a minority that has since risen to 7/26 (27%). Data on 1386 patients was included from the RCR audit. This suggested that more cases from IMPORT HIGH centres had surgical clips implanted (83 vs 67%), were treated using CT guided planning with full three-dimensional dose compensation (100 vs 75%), and were treated with photon boost RT (30 vs 8%).
The study suggests that participation in the IMPORT HIGH trial has played an important part in providing the guidance and support networks needed for the safe integration of advanced RT techniques, where appropriate, as a standard of care for breast cancer patients treated at participating cancer centres.
We investigated the impact of the IMPORT HIGH trial on the adoption of advanced breast RT techniques in the UK and the trial has influenced non-trial breast RT techniques in terms of volume delineation, dosimetry, treatment delivery and verification.
Inter- and intralobular mammary fibroblasts have been separated from normal human breast tissue and cultured to study the differential expression of ectoenzymes present within the stroma of the ...normal gland and associated with breast cancers. Specific ectoenzymes were identified by indirect immunofluorescence and quantified by flow cytometry and semi-quantitative PCR. A consistent difference was noted between the two fibroblast sub-populations at early passage in respect of dipeptidyl peptidase IV (DPP IV) and aminopeptidase N (APN) expression. Early passage intralobular fibroblasts were positive for APN but negative for DPP IV, as seen in the intact tissue. However, with continued sub-culture they gradually began to express DPP IV, until at later passages they became indistinguishable from the interlobular fibroblasts, which were APN and DPP IV-positive at all stages in culture, as they are in intact tissue. Neutral endopeptidase (NEP/CALLA/CD10) is not expressed by normal adult breast fibroblasts but is found in the stroma associated with over 60% of breast cancers. It was up-regulated in vitro on both inter- and intralobular fibroblasts, with final levels that were significantly (< 14 times) higher on the former in all pairs of preparations from individual donors analysed. This difference persisted with continued passage, and levels of the ectoenzyme and its messenger RNA were further up-regulated by hydrocortisone in both populations. These results demonstrate that phenotypically distinct cultures of human mammary fibroblast sub-populations can be used to study the regulation of these stromal ectoenzymes.
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