Objective: Primary hyperparathyroidism can be cured by the successful removal of the culpable parathyroid adenoma. Successful localisation allows the specialist surgeon to perform safer and more cost-effective focused excisions rather than exploratory surgery. This study aims to identify possible factors that predict successful adenoma localisation using technetium99m-sestamibi. Design, Patients and Measurements: Retrospective analysis of 159 patients undergoing parathyroid localisation with technetium99m-sestamibi SPECT/CT. Patients were classified as successful or unsuccessful localisation when compared to the surgical site of a proven adenoma following successful parathyroidectomy. Preoperative and postoperative serum parathyroid hormone (PTH), calcium and 25-hydroxyvitamin D levels and pathological size of the parathyroid adenoma were recorded. Results: Larger specimen volume, weight and higher preoperative PTHs were strongly associated with successful localisation. The percentage change in serum calcium (calculated as the difference between pre- and post-op calcium) was also strongly associated with successful localisation. Higher preoperative serum calcium (> 2.85 mmol/L) was also associated with successful localisation although with a reduced statistical significance. Seventy percent of patients in our cohort underwent parathyroidectomy with a serum calcium < 2.85 mmol/L, of which 92% had pathologically confirmed adenomas and 67% had successful localisation with sestamibi. Conclusion: The serum PTH and change in serum calcium were most strongly associated with successful localisation. The degree of hypercalcaemia was also associated with successful localisation but without as strong an association when compared to the change in calcium. Several factors influence the degree of hypercalcaemia in patients with primary hyperparathyroidism including parathyroid adenoma size, 25-hydroxyvitamin D status and the individual's baseline calcium set point. Historic information (if available) on the patient's individual baseline set point prior to developing primary hyperparathyroidism, and subsequent elevation when primary hyperparathyroidism has developed, could aid decision-making for clinicians when deciding on parathyroidectomy.

Association of perioperative changes in biological markers with RFS (A) by categories of Ki67 relative change, (B) by categories of Ki67 absolute change, (C) by baseline TILs, (D) by surgery TILs. RFS is represented in the time interval 0 to 6 years, as no RFS events occurred beyond 6 years from randomization. All treatment groups are combined; log-rank tests are stratified by treatment group (P = P value). For A and B, a value of −100% Ki67 change (ΔKi67) has been imputed for patients with a pCR in breast. For B, we have categorized both baseline and surgery Ki67 into high if ≥10% or low if <10%. No patient increased Ki67 from low to high after 11 days of perioperative treatment. Because of small number of patients in the “low–low” group, we have compared patients with “high” value at surgery with patients with “low” value at surgery.

Percentage change in Ki67 between pretreatment (baseline) and surgery for part 1 (A) and part 2 (B); Kaplan–Meier estimates by treatment group for relapse free survival (C) and overall survival (D). A, Waterfall plots for part 1 and part 2: for each patient, bar height represents percentage change at surgery from baseline. Percentage change was calculated as [(surgery score + 0.1) − (pretreatment score + 0.1)]/[(pretreatment score + 0.1)]*100. The constant of 0.1 was added to accommodate cases with a value of 0%. Negative values represent decrease from baseline, positive values represent increase from baseline. pCR in breast: patients with pCR (no disease in ether breast or nodes) plus two additional patients with 0% breast cellularity but nodal involvement are represented as bars of height −120% at the left of the figures and noted “pCR in breast;” any existing Ki67 values for these patients have been excluded of the main analysis; in a sensitivity analysis, we imputed a value of −100% change for these patients (Appendix 2). Small triangles indicate patients with RCB1. Disease recurrences are also indicated at the top of each figure with circles and crosses. B, RFS is represented in the time interval of up to 6 years after randomization, as no RFS event occurred later. Overall survival is represented in the fully observed range of values. Log-rank test comparing concurrently randomized treatment groups are reported in the figures. In the figure, trastuzumab and control part 1 and part 2 groups are combined to improve readability. C, control; L, lapatinib; T, trastuzumab; T+L, combination; P1, part 1; P2, part 2; all, P1&P2; P, P value.

The CONSORT diagram summarizes patients recruited into each part of the trial, patients randomized, patients eligible to start treatment, patients who started treatment, and those who completed perioperative treatment as per protocol. In part 1, 22 patients were allocated to control, 57 to trastuzumab, and 51 to lapatinib; in part 2, 29 were allocated to control, 32 to trastuzumab, and 66 to the combination. Overall, 255 (99%) patients were considered eligible to start treatment and included in the analysis of perioperative endpoints. Of the 204 patients in the treatment groups, 201 patients (99%) received some perioperative treatment, with 190/201 (95%) completing the 11 days of perioperative treatment. The figure also describes how many patients available for analysis of coprimary endpoints Ki67 and apoptosis. Only patients with both paired samples and enough tumor tissue for biomarker analysis were included in the analysis: 223 patients (88%) had paired Ki67 data and 193 (76%) had paired apoptosis data available for analysis. Patients with pCR or 0% breast cellularity were excluded from main analysis of Ki67 and apoptosis.

Patient demographics and tumor characteristics at baseline and at surgery, by randomized treatment group.

Analysis of Ki67 and apoptosis, by randomized treatment group.

Detection of molecular residual disease (MRD) allows for the identification of breast cancer patients at high-risk of recurrence, with the potential that early initiation of treatment at early stages of relapse could improve patient outcomes. The Invitae Personalized Cancer Monitoring™ assay (PCM) is a newly developed next-generation sequencing approach that utilizes up to 50 patient-specific, tumor-informed DNA variants, to detect circulating tumor DNA (ctDNA). The ability of the PCM assay to detect MRD before clinical relapse was evaluated. The cohort included 61 female patients with high-risk breast cancer who underwent neoadjuvant chemotherapy. Plasma samples were collected before and during neoadjuvant therapy, after surgery and during monitoring. PCM was used to detect ctDNA at each time point. The sensitivity to detect ctDNA in plasma from patients who relapsed during the monitoring phase was 76.9

Supplementary Figure 1. Correlations between expression levels of ER, PgR, HER2 or EGFR and Ki67 in baseline tumor samples

Introduction: Identification of Molecular Residual Disease (MRD) in patients with breast cancer with circulating tumor DNA (ctDNA) presents a strategy to identify patients at high risk of relapse. Approaches that detect ctDNA at lower concentrations are required to increase sensitivity and improve on the lead time between ctDNA detection and clinical relapse. Here we present results using novel highly sensitive tumor-informed sequencing assays for ctDNA detection of MRD based on detection of multiple patient specific mutations in ctDNA. Methods: 62 stage II-III breast cancer patients (23 hormone receptor positive HER2 negative (HR+HER2-), 20 HER2+, 15 triple negative breast cancer (TNBC) and 4 unknown receptor status) enrolled in the ChemoNEAR sample collection study were included. All patients received neoadjuvant chemotherapy, followed up by surgery, with samples taken at diagnosis, and post-surgery every 3 months for the first two years, followed by every 6 months for up to five years. Tumor DNA from FFPE samples and germline was Whole Exome Sequenced to identify patient specific mutations and design anchored-multiplex PCR (AMP™) Personalized Cancer Monitoring (PCMTM) assays to track mutations in plasma. Cell free DNA was extracted from 613 plasma samples (median volume 4ml, range 0.5-4.5ml) and sequenced with PCMTM assays, with 37-177 variants (median 52) per panel, to a depth of 100,000x per locus. A proprietary algorithm was used to identify ctDNA. Results: At a median follow-up of 52.7 months post-surgery (range 15.3-96.4 months), ctDNA was detected in 25.8% (16/62) of patients, with detected ctDNA levels ranging from allele frequency (AF) of 0.01%, to 32.5%) (median 0.24% AF). Detection of ctDNA was associated with a high risk of future relapse (HR 65.4, 95% CI 14.5-293.7), with a median lead-time from ctDNA detection to clinical relapse of 13.7 months (range 3.9-58.9). MRD was identified in 76.9% (10/13) of patients who relapsed. ctDNA was detected prior to relapse in both patients with brain only relapse, but with a reduced lead time over clinical relapse (5.73 and 3.90 months), which was previously not achievable with digital PCR MRD-detection assays. Of patients with assessable baseline samples, 81% (39/48) had ctDNA detected. No patients with undetected ctDNA, or detectable ctDNA with AF< 0.1%, relapsed during follow-up, whereas ctDNA was detected at baseline in all 10 patients who relapsed during follow-up (p=0.1). Conclusions: PCMTM detected breast cancer relapse with a long lead-time over clinical relapse, and strong association with relapse free survival, an advancement over previously published data with digital PCR MRD detection. Prospective, interventional trials are now required to assess whether treatment on the basis of MRD detection improves outcome, including the TRAK ER Trial (NCT04985266). Citation Format: Isaac Garcia-Murillas, Giselle Walsh-Crestani, Edward Phillips, Rosalind Cutts, Sarah Hrebien, Kathryn Dunne, Kally Sidhu, Robert Daber, Amber C. Carter, Lorena De La Peña, Stephen Johnston, Alistair Ring, Simon Russell, Abigail Evans, Anthony Skene, Duncan Wheatley, Ian Smith, Nicholas Turner. Personalized Cancer Monitoring (PCM): a novel ctDNA tool to detect molecular residual disease in patients with early-stage breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-05-01.

Supplementary Table 2. Biomarker changes in placebo treated arm.

Health education can offer a valuable window onto conceptual and behavioral change. In Study 1, we mapped out 3rd-grade Chinese children’s beliefs about causes of colds and flu and ways they can be prevented. We also explored older adults’ beliefs as a possible source of the children’s ideas. In Study 2, we gave 3rd- and 4th-grade Chinese children either a conventional cold/flu education program or an experimental “Think Biology” program that focused on a biological causal mechanism for cold/flu transmission. The “Think Biology” program led children to reason about cold/flu causation and prevention more scientifically than the conventional program, and their reasoning abilities dovetailed with their mastery of the causal mechanism. Study 3, a modified replication of Study 2, found useful behavioral change as well as conceptual change among children who received the “Think Biology” program and documented coherence among knowledge enrichment, conceptual change, and behavioral change.

Supplementary Figure from Combined Perioperative Lapatinib and Trastuzumab in Early HER2-Positive Breast Cancer Identifies Early Responders: Randomized UK EPHOS-B Trial Long-Term Results

Supplementary Table 1. Toxicities: incidence and National Cancer Institute (NCI) CTC grading

Supplementary Table 4. Correlation of change in protein and mRNA biomarker values with change in Ki67 for both lapatinib-treated and placebo-treated control patients.

Supplementary Table 3. Correlation between baseline protein and mRNA expression biomarker values with Ki67 response (defined as >50% fall in Ki67).

Supplementary Data from Proliferation and AKT Activity Biomarker Analyses after Capivasertib (AZD5363) Treatment of Patients with ER+ Invasive Breast Cancer (STAKT)

Purpose Ki67 assessed at diagnosis (Ki67 baseline ) is an important prognostic factor in primary oestrogen receptor-positive (ER +) breast cancer. Proportional change in Ki67 after 2 weeks (∆Ki67 2week ) is associated with clinical benefit from endocrine therapies and residual Ki67 (Ki67 2week ) with recurrence-free survival. The aim was to define the association between Ki67 baseline and after aromatase inhibitor (AI) exposure ∆Ki67 2week and Ki67 2week with key prognostic and biologic factors utilising data from the POETIC study. Patients and methods In POETIC 4480 postmenopausal patients with primary ER and/or PgR + breast cancer were randomised 2:1 to 2 weeks’ presurgical AI (anastrozole or letrozole) or no presurgical treatment (control). Ki67 was measured centrally in core-cut biopsies taken prior to AI and in core-cuts or the excision biopsy at surgery. Relationships between the Ki67 and biologic factors were explored using linear regression. Results Established associations of Ki67 baseline with biologic factors including PgR status, tumour grade, tumour size, histological subtype, nodal status, and vascular invasion were confirmed in the HER2- subpopulation. In the HER2 + subpopulation only grade and tumour size were significantly associated with Ki67 baseline . In control group Ki67 2week was 18% lower than Ki67 baseline (p < 0.001) when Ki67 2week was measured in excision biopsies but not when measured in core-cuts. Median suppression by AIs (∆Ki67 2week ) was 79.3% (IQR: −89.9 to −54.6) and 53.7% (IQR: −78.9 to −21.1) for HER2-negative and HER2-positive cases, respectively. Significantly less suppression occurred in PgR- vs PgR + and HER2 + vs HER2- tumours which remained apparent after adjustment for 2-week sample type. Conclusions The magnitude of this study allowed characterisation of relationships between Ki67 baseline , ∆Ki67 2week and Ki67 2week with high degrees of confidence providing a reference source for other studies. Lower values of Ki67 occur when measured on excision biopsies and could lead to apparent but artefactual decreases in Ki67: this should be considered when either ∆Ki67 2week or Ki67 2week is used in routine clinical practice to aid treatment decisions or in clinical trials assessing new drug therapies .

Supplementary Table and Figure Legends. A list of all the supplementary table and figure legends.

AbstractPurpose: EPHOS-B aimed to determine whether perioperative anti-HER2 therapy inhibited proliferation and/or increased apoptosis in HER2-positive breast cancer. Patients and Methods: This randomized phase II, two-part, multicenter trial included newly diagnosed women with HER2-positive invasive breast cancer due to undergo surgery. Patients were randomized to: part 1 (1:2:2), no treatment (control), trastuzumab or lapatinib; part 2 (1:1:2) control, trastuzumab, or lapatinib and trastuzumab combination. Treatment was given for 11 days presurgery. Coprimary endpoints were change in Ki67 and apoptosis between baseline and surgery tumor samples (biologic response: ≥30% change). Central pathology review scored residual cancer burden (RCB). Relapse-free survival (RFS) explored long-term effects. Results: Between November 2010 and September 2015, 257 patients were randomized (part 1: control 22, trastuzumab 57, lapatinib 51; part 2: control 29, trastuzumab 32, combination 66). Ki67 response was evaluable for 223 patients: in part 1 Ki67 response occurred in 29/44 (66%) lapatinib versus 18/49 (37%) trastuzumab (P = 0.007) and 1/22 (5%) control (P < 0.0001); in part 2 in 36/49 (74%) combination versus 14/31 (45%) trastuzumab (P = 0.02) and 2/28 (7%) control (P < 0.0001). No significant increase in apoptosis after 11 days was seen in treatment groups. Six patients achieved complete pathologic response (pCR, RCB0) and 13 RCB1, all but two in the combination group. After 6 years median follow-up, 28 (11%) had recurrence and 19 (7%) died. No recurrences or deaths were observed among patients who achieved a pCR. Ki67% falls ≥50% associated with fewer recurrences (P = 0.002). Conclusions: Early response after short duration anti-HER2 dual therapy identifies cancers dependent on the HER2 pathway providing a strategy for exploring risk-adapted individualized treatment de-escalation.

Background In clinical practice, oestrogen receptor (ER) analysis is almost entirely by immunohistochemistry (IHC). ASCO/CAP recommends cut-offs of < 1% (negative) and 1–10% (low) cells positive. There is uncertainty whether patients with ER low tumours benefit from endocrine therapy. We aimed to assess IHC and mRNA cut-points for ER versus biological response of primary breast cancer to 2 weeks’ aromatase inhibitor treatment as measured by change in Ki67. Methods Cases were selected from the aromatase inhibitor treatment group of POETIC. We selected the 15% with the poorest Ki67 response (PR, < 40% Ki67 suppression, n = 230) and a random 30% of the remainder categorised as intermediate (IR, 40–79% Ki67 suppression, n = 150) and good-responders (GR, ≥ 80% Ki67 suppression, n = 230) from HER2 − group. All HER2 + cases available were selected irrespective of their response category ( n = 317). ER expression was measured by IHC and qPCR. Results ER IHC was available from 515 HER2 − and 186 HER2 + tumours and ER qPCR from 367 HER2 − and 171 HER2 + tumours. Ninety-one percentage of patients with ER IHC < 10% were PRs with similar rates in HER2 − and HER2 + cases. At or above ER IHC 10% substantial numbers of patients showed IR or GR. Similar proportions of patients were defined by cut-points of ER IHC < 10% and ER mRNA < 5 units. In addition, loss of PgR expression altered ER anti-proliferation response with 92% of PgR − cases with ER IHC < 40% being PRs. Conclusions There was little responsiveness at IHC < 10% and no distinction between < 1% and 1–10% cells positive. Similar separation of PRs from IR/GRs was achieved by IHC and mRNA.