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Background: Translational research studies in the context of international clinical trials may incur prolonged transportation time of tumor specimens. The effect of prolonged transportation time and sample preservation method on RNA quality and on reliability of derived gene expression profiling (GEP) is unknown. Methods: Tumor material from surgical specimens of patients with primary breast cancer (BC) was obtained with cold ischemia time (CIT) recorded for each patient. Each sample was divided into 8 aliquots 4 that were placed in RNAlater and 4 that were frozen. To simulate prolonged transportation duration, samples were kept 1 week in a bucket of dry ice replenished daily, with temperature monitoring. The bucket was stored at room temperature. RNA extraction and GEP using Affymetrix HG-U133 Plus2 were performed at baseline and at day 2, 4 and 7. Linear mixed effect models were used to explore effects of transportation time and preservation method on RNA quality (RNA Integrity Number-RIN). Pre-defined single genes (n = 12), gene modules (n = 25) and molecular subtypes (PAM50) were evaluated. P < 0.01 was considered for significance. Results: Samples were collected from 13 patients with an average CIT of 37 minutes (range: 20-85). During the simulation period, the mean temperature at which samples were stored was -77.8°C (SD+/-1.6°C). RIN values decreased significantly over time (1-day change -0.08, CI = -0.12 to -0.04, p < 0.001) and by RNAlater (-0.29, CI =-0.07 to -0.51, p < .01), but remained always above 6.5. RNA degradation evaluated with actin 3/5 and gapdh 3/5 ratios was significantly higher over time for samples preserved in RNAlater compared with frozen (difference in changes 0.68, CI=0.49 to 0.87, p < 0.001 and 0.33,0.25 to 0.4, p < 0.001, respectively), and for 5 RNAlater (38%) samples these ratios exceeded the Affymetrix thresholds (3 and 1.25, respectively) at least at 1 time-point. Comparing tissue preservation methods, only the expression of PTEN gene and CASP3 module changed significantly more over time in RNAlater compared with frozen samples (interaction p < 0.01). Genes and gene modules that were significantly differently expressed over time, independent of preservation method, were AURKA, PTEN, CASP3 and WOUND (p < 0.01, range of 1-day changes in log2 expression:-0.05 to 0.10), while the expression of ESR1 and ERBB2 was unaffected. Those that were significantly influenced by sample preservation method, adjusted for time, were genes ESR1, PLAU, VEGF, PIK3CA, PTEN and gene modules GENE21 and GENE70 was (P < 0.01, range: -0.61 to 0.16). Using PAM50, 8 (61%) samples were classified at least once as a different subtype over time compared to baseline, and 4 (30%) samples fell in a different Genomic Grade Index (GGI) class risk (high/low) at least once over time. Conclusions: In this study, frozen samples were significantly less degraded over time compared with samples stored in RNAlater. Preservation method had a stronger influence on genes and gene modules values compared with prolonged transport duration. In international clinical trials we recommend freezing samples to assure more stable gene expression analysis and limit transport duration to less than 3 days. [ABSTRACT FROM AUTHOR]

Background: A significant proportion of breast cancer (BC) patients (pts) develop multiple synchronous unilateral breast tumors, also referred to as multifocal BC (MBC), which represent a diagnostic and therapeutic challenge. Here, we aimed first to better define the incidence of MBCs and then to compare different foci from ductal MBCs in a global analysis using genomic, transcriptomic and epigenomic data. Methods: The incidence of MBCs was sought via a systematic query of all pathology reports between 2000 and 2010 within the Institut Bordet. The biological characterization of MBCs focused on 5 ductal MBCs for which the foci did not differ in terms of grade, hormonal receptors and HER2 status, since this is the case for the majority of MBCs. It involved the identification of somatic rearrangements (Rs), transcriptomic and epigenomic profiling in 2 foci from each pt, via low-coverage whole genome sequencing, HG133 Plus 2.0 Chips and Infinium Methylation 450K arrays respectively. Genomic Rs were validated using an orthogonal sequencing platform. Results: MBC is a frequent finding since it concerns 24% (1410/5811) of primary BCs. When investigating the genomics of the 5 MBCs, we observed that the number of Rs varied in each pt, with 3 pts having few (<10) Rs whilst the other 2 pts showed > 100Rs with a particular tandem-duplication phenotype. All pts had Rs common to both foci, suggesting a shared genetic background. Strikingly, in 4 pts, we observed a branched evolutionary pattern since private Rs were present in each focus. In contrast, we observed a linear evolutionary pattern in the remaining pt, since private Rs were only found in 1 of the 2 foci. Two pts were genetically very similar with 75% common Rs. Although the transcriptomic profiles looked very similar between the 2 foci, significant epigenomic differences were observed in 1 of the 2 pts investigated. Two other pts had 50% common Rs. In one pt there were only minor differences in transcriptomic and methylation patterns between the 2 foci. Contrastingly, in the second pt, DNA methylation patterns were dramatically discordant between the foci, suggesting that genomic, transcriptomic and epigenomic patterns are not necessarily correlated. Only in 1 pt of the 5 investigated, the 2 foci were extremely different, with only 14% of common Rs. These genetic differences were associated with dramatic differences in methylation and transcriptomic profiles. Interestingly, for 4 pts some of the identified rearrangements were found in parts of tumor-adjacent histologically normal breast tissue. Conclusions: Today, the College of American Pathologists recommends analysing more than 1 focus from MBC if they differ in histology and grading (Lester 2009). Here, we demonstrated for the first time that different lesions from MBC, which concerns of the ductal BC population, can differ at the (epi)genetic and transcriptomic level even when the foci present similar histology , grading, hormonal and HER2 status. Since the number of genetic alterations with potential clinical utility is rapidly growing due to increasing numbers of targeted therapies, these findings suggest that interrogating only the largest lesion might not be sufficient for adequate management of MBCs. [ABSTRACT FROM AUTHOR]

The immune landscape of hormone receptor positive, HER2-negative metastatic breast cancer (HR+/HER2- mBC), the most common subtype of BC, remains understudied. This is mainly due to reduced sample acquisition opportunities from metastases as compared to primary tumors. In this study, we explored stromal tumor-infiltrating lymphocytes (sTIL) in metastatic samples collected through our post-mortem tissue donation program UPTIDER (NCT04531696). sTIL were scored as a continuous parameter according to international guidelines on 427 metastases and 38 primary untreated tumors acquired from 20 patients with HR+/HER2- mBC. ER-status was evaluated on 362 metastases with a cut-off for positivity set at 1% according to ASCO/CAP guidelines. Our analyses show that 54% and 15% of metastases had sTIL levels >1% and >5% respectively. sTIL levels tended to be lower in metastases as compared to their respective primary tumor (Estimate: -2.83, 95%CI: -5.77-0.11, p:0.07). sTIL levels were lower in metastases from invasive lobular carcinoma than in metastases from invasive breast carcinoma of no special type (Estimate: -1.67, 95%CI: -2.35--0.98, p:<0.001). A loss of ER expression was observed in 14% of all metastases, yet a negative ER-status was not significantly associated with increased sTIL levels. Finally, sTIL levels were significantly higher in lung and axillary lymph node metastases compared to all metastases. While these analyses were conducted on multiple metastases obtained at the end of life after several lines of treatment, the data provides novel and valuable insights into the state of immune infiltration in patients with metastatic HR+/HER2- BC., (Copyright © 2024. Published by Elsevier Inc.)

Purpose: The ZEPHIR clinical trial evaluated the role of [89Zr]trastuzumab-PET/CT (HER2-PET/CT) and 2-[18F]fluoro-2-deoxy-D-glucose PET/CT ([18F]FDG-PET/CT) in predicting outcomes in patients with advanced HER2-positive breast cancer treated with trastuzumab emtansine (T-DM1). Here, we combined molecular/metabolic imaging and transcriptomic data to investigate the biological processes associated with [89Zr]trastuzumab and [18F]FDG uptake, and to dissect the mechanisms involved in T-DM1 resistance., Experimental Design: RNA was extracted from metastasis biopsies obtained in the ZEPHIR trial. HER2-PET/CT and [18F]FDG-PET/CT imaging data of biopsied lesions were integrated with transcriptomic data. Lesions were compared based on the level of [89Zr]trastuzumab uptake as well as on the presence/absence of metabolic response, defined comparing baseline and on-treatment [18F]FDG-PET/CT., Results: We analyzed matched transcriptomic and molecular/metabolic imaging data for 24 metastases. Genes and pathways involved in extracellular matrix (ECM) organization and glycosylphosphatidylinositol synthesis were enriched in lesions presenting low [89Zr]trastuzumab uptake. [18F]FDG uptake at baseline correlated with proliferation and immune-related processes. Hypoxia and ECM-related processes were enriched in lesions showing no metabolic response to T-DM1, while immune-related processes were associated with high [89Zr]trastuzumab uptake and metabolic response. Gene signatures including differentially expressed genes according to [89Zr]trastuzumab uptake and metabolic response showed predictive value in an external cohort., Conclusions: To our knowledge, this study represents the first correlative analysis between [89Zr]trastuzumab tumor uptake and gene expression profiling in humans. Our findings suggest a role of ECM in impairing [89Zr]trastuzumab tumor uptake and T-DM1 metabolic response in advanced HER2-positive breast cancer, highlighting the potential of molecular imaging to depict tumor microenvironment features.

Metastatic breast cancer (mBC) remains incurable and liver metastases (LM) are observed in approximately 50% of all patients with mBC. In some cases, surgical resection of breast cancer liver metastases (BCLM) is associated with prolonged survival. However, there are currently no validated marker to identify these patients. The interactions between the metastatic cancer cells and the liver microenvironment result in two main histopathological growth patterns (HGP): replacement (r-HGP), characterized by a direct contact between the cancer cells and the hepatocytes, and desmoplastic (d-HGP), in which a fibrous rim surrounds the tumor cells. In patients who underwent resection of BCLM, the r-HGP is associated with a worse postoperative prognosis than the d-HGP. Here, we aim at unraveling the biological differences between these HGP within ten patients presenting both HGP within the same metastasis. The transcriptomic analyses reveal overexpression of genes involved in cell cycle, DNA repair, vessel co-option and cell motility in r-HGP while angiogenesis, wound healing, and several immune processes were found overexpressed in d-HGP LM. Understanding the biology of the LM could open avenues to refine treatment of BC patients with LM., (© 2024. The Author(s).)

Invasive lobular carcinoma (ILC) is the second most frequent type of breast cancer (BC) and its peculiar morphology is mainly driven by inactivation of CDH1, the gene coding for E-cadherin cell adhesion protein. ILC-specific therapeutic and disease-monitoring approaches are gaining momentum in the clinic, increasing the importance of accurate ILC diagnosis. Several essential and desirable morphologic diagnostic criteria are currently defined by the World Health Organization, the routine use of immunohistochemistry (IHC) for E-cadherin is not recommended. Disagreement in the diagnosis of ILC has been repeatedly reported, but interpathologist agreement increases with the use of E-cadherin IHC. In this study, we aimed to harmonize the pathological diagnosis of ILC by comparing 5 commonly used E-cadherin antibody clones (NCH-38, EP700Y, Clone 36, NCL-L-E-cad [Clone 36B5], and ECH-6). We determined their biochemical specificity for the E-cadherin protein and IHC staining performance according to type and location of mutation on the CDH1 gene. Western blot analysis on mouse cell lines with conditional E-cadherin expression revealed a reduced specificity of EP700Y and NCL-L-E-cad for E-cadherin, with cross-reactivity of Clone 36 to P-cadherin. The use of IHC improved interpathologist agreement for ILC, lobular carcinoma in situ, and atypical lobular hyperplasia. The E-cadherin IHC staining pattern was associated with variant allele frequency and likelihood of nonsense-mediated RNA decay but not with the type or position of CDH1 mutations. Based on these results, we recommend the indication for E-cadherin staining, choice of antibodies, and their interpretation to standardize ILC diagnosis in current pathology practice., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Primary tumors with a mixed invasive breast carcinoma of no-special type (IBC-NST) and invasive lobular cancer (ILC) histology are present in approximately five percent of all patients with breast cancer and are understudied at the metastatic level. Here, we characterized the histology of metastases from two patients with primary mixed IBC-NST/ILC from the postmortem tissue donation program UPTIDER (NCT04531696). The 14 and 43 metastatic lesions collected at autopsy had morphological features and E-cadherin staining patterns consistent with pure ILC. While our findings still require further validation, they may challenge current clinical practice and imaging modalities used in these patients., Competing Interests: Declaration of Competing interest All authors declare to have no conflict of interest, (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

While there is a great clinical need to understand the biology of metastatic cancer in order to treat it more effectively, research is hampered by limited sample availability. Research autopsy programmes can crucially advance the field through synchronous, extensive, and high-volume sample collection. However, it remains an underused strategy in translational research. Via an extensive questionnaire, we collected information on the study design, enrolment strategy, study conduct, sample and data management, and challenges and opportunities of research autopsy programmes in oncology worldwide. Fourteen programmes participated in this study. Eight programmes operated 24 h/7 days, resulting in a lower median postmortem interval (time between death and start of the autopsy, 4 h) compared with those operating during working hours (9 h). Most programmes (n = 10) succeeded in collecting all samples within a median of 12 h after death. A large number of tumour sites were sampled during each autopsy (median 15.5 per patient). The median number of samples collected per patient was 58, including different processing methods for tumour samples but also non-tumour tissues and liquid biopsies. Unique biological insights derived from these samples included metastatic progression, treatment resistance, disease heterogeneity, tumour dormancy, interactions with the tumour micro-environment, and tumour representation in liquid biopsies. Tumour patient-derived xenograft (PDX) or organoid (PDO) models were additionally established, allowing for drug discovery and treatment sensitivity assays. Apart from the opportunities and achievements, we also present the challenges related with postmortem sample collections and strategies to overcome them, based on the shared experience of these 14 programmes. Through this work, we hope to increase the transparency of postmortem tissue donation, to encourage and aid the creation of new programmes, and to foster collaborations on these unique sample collections. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland., (© 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.)