Neoadjuvant radiotherapy in ER+, HER2+, and triple-negative -specific breast cancer based humanized tumor mice enhances anti-PD-L1 treatment efficacy.

Pre-operative radiation therapy is not currently integrated into the treatment protocols for breast cancer. However, transforming immunological "cold" breast cancers by neoadjuvant irradiation into their "hot" variants is supposed to elicit an endogenous tumor immune defense and, thus, enhance immunotherapy efficiency. We investigated cellular and immunological effects of sub-lethal, neoadjuvant irradiation of ER pos., HER2 pos., and triple-negative breast cancer subtypes in-vitro and in-vivo in humanized tumor mice (HTM). This mouse model is characterized by a human-like immune system and therefore facilitates detailed analysis of the mechanisms and efficiency of neoadjuvant, irradiation-induced "in-situ vaccination", especially in the context of concurrently applied checkpoint therapy. Similar to clinical appearances, we observed a gradually increased immunogenicity from the luminal over the HER2-pos. to the triple negative subtype in HTM indicated by an increasing immune cell infiltration into the tumor tissue. Anti-PD-L1 therapy divided the HER2-pos. and triple negative HTM groups into responder and non-responder, while the luminal HTMs were basically irresponsive. Irradiation alone was effective in the HER2-pos. and luminal subtype-specific HTM and was supportive for overcoming irresponsiveness to single anti-PD-L1 treatment. The treatment success correlated with a significantly increased T cell proportion and PD-1 expression in the spleen. In all subtype-specific HTM combination therapy proved most effective in diminishing tumor growth, enhancing the immune response, and converted non-responder into responder during anti-PD-L1 therapy. In HTM, neoadjuvant irradiation reinforced anti-PD-L1 checkpoint treatment of breast cancer in a subtype -specific manner. According to the "bench to bedside" principle, this study offers a vital foundation for clinical translating the use of neoadjuvant irradiation in the context of checkpoint therapy.

App-based support for breast cancer patients to reduce psychological distress during therapy and survivorship - a multicentric randomized controlled trial.

Introduction: The negative impact of unmanaged psychological distress on quality of life and outcome in breast cancer survivors has been demonstrated. Fortunately, studies indicate that distress can effectively be addressed and even prevented using evidence-based interventions. In Germany prescription-based mobile health apps, known as DiGAs (digital health applications), that are fully reimbursed by health insurances, were introduced in 2020. In this study, the effectiveness of an approved breast cancer DiGA was investigated: The personalized coaching app PINK! Coach supports and accompanies breast cancer patients during therapy and follow-up. Methods: PINK! Coach was specifically designed for breast cancer (BC) patients from the day of diagnosis to the time of Follow-up (aftercare). The app offers individualized, evidence-based therapy and side-effect management, mindfulness-based stress reduction, nutritional and psychological education, physical activity tracking, and motivational exercises to implement lifestyle changes sustainably in daily routine. A prospective, intraindividual RCT (DRKS00028699) was performed with n = 434 patients recruited in 7 German breast cancer centers from September 2022 until January 2023. Patients with BC were included independent of their stage of diseases, type of therapy and molecular characteristics of the tumor. Patients were randomized into one of two groups: The intervention group got access to PINK! over 12 weeks; the control group served as a waiting-list comparison to "standard of care." The primary endpoint was psychological distress objectified by means of Patient Health Questionnaire-9 (PHQ-9). Subgroups were defined to investigate the app's effect on several patient groups such as MBC vs. EBC patients, patients on therapy vs. in aftercare, patients who received a chemotherapy vs. patients who did not. Results: Efficacy analysis of the primary endpoint revealed a significant reduction in psychological distress (least squares estimate -1.62, 95% confidence interval [1.03; 2.21]; p<0.001) among intervention group patients from baseline to T3 vs, control group. Subgroup analysis also suggested improvements across all clinical situations. Conclusion: Patients with breast cancer suffer from psychological problems including anxiety and depression during and after therapy. Personalized, supportive care with the app PINK! Coach turned out as a promising opportunity to significantly improve psychological distress in a convenient, accessible, and low-threshold manner for breast cancer patients independent of their stage of disease (EBC/MBC), therapy phase (aftercare or therapy) or therapy itself (chemotherapy/other therapy options). The app is routinely available in Germany as a DiGA. Clinical Trial Registration: DRKS Trial Registry (DRKS00028699).

Clinical impact of delaying initiation of adjuvant chemotherapy in patients with early triple negative breast cancer.

PURPOSE: The optimal time to initiation of adjuvant chemotherapy (TTAC) for triple negative breast cancer (TNBC) patients is unclear. This study evaluates the association between TTAC and survival in TNBC patients. METHODS: We conducted a retrospective study using data from a cohort of TNBC patients diagnosed between January 1, 2010 to December 31, 2018, registered in the Tumor Centre Regensburg was conducted. Data included demographics, pathology, treatment, recurrence and survival. TTAC was defined as days from primary surgery to first dose of adjuvant chemotherapy. The Kaplan-Meier method was used to evaluate impact of TTAC on overall survival (OS) and 5-year OS. RESULTS: A total of 245 TNBC patients treated with adjuvant chemotherapy and valid TTAC data were included. Median TTAC was 29 days. The group receiving systemic therapy within 22 to 28 days after surgery had the most favorable outcome, with median OS of 10.2 years. Groups receiving systemic therapy between 29-35 days, 36-42 days, and more than 6 weeks after surgery had significantly decreased median survival, with median OS of 8.3 years, 7.8 years, and 6.9 years, respectively. Patients receiving therapy between 22-28 days had significantly better survival compared to those receiving therapy between 29-35 days (p = 0.043), and patients receiving therapy after 22-28 days also demonstrated significantly better survival compared to those receiving therapy after more than 43 days (p = 0.033). CONCLUSION: Timing of adjuvant systemic therapy can influence OS in TNBC patients. Efforts should be made to avoid unnecessary delays in administering chemotherapy to ensure timely initiation of systemic therapy and optimize patient outcomes.

Applicability of magnetic seeds for target lymph node biopsy after neoadjuvant chemotherapy in initially node-positive breast cancer patients: data from the AXSANA study.

PURPOSE: Currently, various techniques are available to mark and selectively remove initially suspicious axillary lymph nodes (target lymph nodes, TLNs) in breast cancer patients receiving neoadjuvant chemotherapy (NACT). To date, limited data are available on whether the use of magnetic seeds (MS) is suitable for localizing TLNs. This study aimed to investigate the feasibility of MS in patients undergoing target lymph node biopsy (TLNB) or targeted axillary dissection (TAD) after NACT. METHODS: Prospective data from the ongoing multicentric AXSANA study were extracted from selected patients in whom the TLN had been marked with an MS before NACT and who were enrolled from June 2020 to June 2023. The endpoints of the analysis were the detection rate, the rate of lost markers, and the potential impairment on magnetic resonance imaging (MRI) assessment. RESULTS: In 187 patients from 27 study sites in seven countries, MS were placed into the TLN before NACT. In 151 of these, post-NACT surgery had been completed at the time of analysis. In 146 patients (96.0%), a TLN could successfully be detected. In three patients, the seed was removed but no lymphoid tissue was detected on histopathology. The rate of lost markers was 1.2% (2 out of 164 MS). In 15 out of 151 patients (9.9%), MRI assessment was reported to be compromised by MS placement. CONCLUSION: MS show excellent applicability for TLNB/TAD when inserted before NACT with a high DR and a low rate of lost markers. Axillary MS can impair MRI assessment of the breast. TRIAL REGISTRATION NUMBER: NCT04373655 (date of registration May 4, 2020).

Does timing of neoadjuvant chemotherapy influence the prognosis in patients with early triple negative breast cancer?

PURPOSE: For patients with triple negative breast cancer (TNBC), the optimal time to initiate neoadjuvant chemotherapy (TTNC) is unknown. This study evaluates the association between TTNC and survival in patients with early TNBC. METHODS: A retrospective study using data from of a cohort of TNBC patients diagnosed between January 1, 2010 to December 31, 2018 registered in the Tumor Centre Regensburg was performed. Data included demographics, pathology, treatment, recurrence, and survival. Interval to treatment was defined as days from pathology diagnosis of TNBC to first dose of neoadjuvant chemotherapy (NACT). The Kaplan-Meier and Cox regression methods were used to evaluate the impact of TTNC on overall survival (OS) and 5 year OS. RESULTS: A total of 270 patients were included. Median follow up was 3.5 years. The 5-year OS estimates according to TTNC were 77.4%, 66.9%, 82.3%, 80.6%, 88.3%, 58.3%, 71.1% and 66.7% in patients who received NACT within 0-14, 15-21, 22-28, 29-35, 36-42, 43-49, 50-56 and > 56 days after diagnosis. Patients who received systemic therapy early had the highest estimated mean OS of 8.4 years, while patients who received systemic therapy after more than 56 days survived an estimated 3.3 years. CONCLUSION: The optimal time interval between diagnosis and NACT remains to be determined. However, starting NACT more than 42 days after diagnosis of TNBC seems to reduce survival. Therefore, it is strongly recommended to carry out the treatment in a certified breast center with appropriate structures, in order to enable an adequate and timely care.

Immune Checkpoint Profiling in Humanized Breast Cancer Mice Revealed Cell-Specific LAG-3/PD-1/TIM-3 Co-Expression and Elevated PD-1/TIM-3 Secretion.

Checkpoint blockade is particularly based on PD-1/PD-L1-inhibiting antibodies. However, an efficient immunological tumor defense can be blocked not only by PD-(L)1 but also by the presence of additional immune checkpoint molecules. Here, we investigated the co-expression of several immune checkpoint proteins and the soluble forms thereof (e.g., PD-1, TIM-3, LAG-3, PD-L1, PD-L2 and others) in humanized tumor mice (HTM) simultaneously harboring cell line-derived (JIMT-1, MDA-MB-231, MCF-7) or patient-derived breast cancer and a functional human immune system. We identified tumor-infiltrating T cells with a triple-positive PD-1, LAG-3 and TIM-3 phenotype. While PD-1 expression was increased in both the CD4 and CD8 T cells, TIM-3 was found to be upregulated particularly in the cytotoxic T cells in the MDA-MB-231-based HTM model. High levels of soluble TIM-3 and galectin-9 (a TIM-3 ligand) were detected in the serum. Surprisingly, soluble PD-L2, but only low levels of sPD-L1, were found in mice harboring PD-L1-positive tumors. Analysis of a dataset containing 3039 primary breast cancer samples on the R2 Genomics Analysis Platform revealed increased TIM-3, galectin-9 and LAG-3 expression, not only in triple-negative breast cancer but also in the HER2+ and hormone receptor-positive breast cancer subtypes. These data indicate that LAG-3 and TIM-3 represent additional key molecules within the breast cancer anti-immunity landscape.

Lightweight implants in breast reconstruction.

OBJECTIVE: Since the first use of silicone implants by Cronin in 1962, there have been several attempts to introduce alternative filling materials for breast implants on the market. A promising new development are lightweight implants, whose filler material is one third lighter than conventional silicone gel. While these implants have been used primarily for aesthetic augmentation, a benefit could be expected particularly in post-mastectomy reconstruction. MATERIALS AND METHODS: Since 2019, 92 operations using lightweight implants have been performed at our clinic, 61 of them for breast reconstruction after mastectomy. These have been compared to 92 breast reconstructions using conventional silicone implants. RESULTS: The average volume of the lightweight implants was 30% higher than of the conventional implants (452 ml resp. 347 ml), whereas the implant weight was comparable in both groups (317 g resp. 347 g). Grade 3-4 capsular fibrosis was seen in 6 cases in both groups; revision was required 9 times (lightweight implants) and 7 times (conventional silicone implants) during the follow-up period. DISCUSSION: To our knowledge, this is the first study to investigate the use of lightweight implants in breast reconstruction. With exception of the filler material, the implants used in the two groups were comparable in shape and surface. The inserted lightweight implants had a greater volume but nearly the same weight as the conventional implants and were used in patients with a higher body mass index. Thus, lightweight implants were preferred in patients whose reconstruction required a larger implant volume. CONCLUSION: Lightweight implants are a new alternative for breast reconstruction especially in case that larger implant volume is demanded. The increased complication rate has to be verified in further studies.

Automated breast ultrasound (ABUS) for intraoperative margin control on surgical specimens in breast conserving surgery.

PURPOSE: As breast-conserving surgery (BCS) has become the standard for treatment of early breast cancer, the need for new technologies to improve intraoperative margin assessment has become clear. Close or positive margins during BCS lead to additional surgeries, treatment delay, additional stress for patients and increasing healthcare cost. Automated three-dimensional breast ultrasound (ABUS) systems are meant to overcome the shortcomings of hand-held ultrasound (HHUS). In this study, we investigate the feasibility of ABUS to conduct ultrasound on surgical specimens in breast conserving therapy. METHODS: In this monocentric, non-interventional study, specimens of 40 women were examined via ABUS. A construction with isotonic saline solution, gel pads and ABUS membranes was invented by our team to produce images of breast cancer specimens using ABUS. Evaluation of the ABUS images was carried out by two independent physicians trained on ABUS evaluation. RESULTS: ABUS was conducted on 40 specimens. 90% of the generated images were of high quality. Measured tumor sizes with ABUS were bigger than measured tumor size with HHUS (mean tumor size 22.9 vs. 18.1 mm, CI 2.38-7.35, p < 0.05). The mean difference between the ABUS tumor size and the pathological tumor size was 1.8 mm (CI - 0.84-4.53, p = 0.17). The mean difference between the HHUS tumor size and the pathological tumor size was 3.2 mm (CI - 5.35 to - 1.03, p = 0.005). CONCLUSION: ABUS seems to be a suitable method to conduct specimen ultrasound. Further studies are required to evaluate the accuracy of ABUS for intraoperative margin assessment and possible implementation in clinical work routine.

Critical evaluation of molecular tumour board outcomes following 2 years of clinical practice in a Comprehensive Cancer Centre.

BACKGROUND: Recently, molecular tumour boards (MTBs) have been integrated into the clinical routine. Since their benefit remains debated, we assessed MTB outcomes in the Comprehensive Cancer Center Ostbayern (CCCO) from 2019 to 2021. METHODS AND RESULTS: In total, 251 patients were included. Targeted sequencing was performed with PCR MSI-evaluation and immunohistochemistry for PD-L1, Her2, and mismatch repair enzymes. 125 treatment recommendations were given (49.8%). High-recommendation rates were achieved for intrahepatic cholangiocarcinoma (20/30, 66.7%) and gastric adenocarcinoma (10/16, 62.5%) as opposed to colorectal cancer (9/36, 25.0%) and pancreatic cancer (3/18, 16.7%). MTB therapies were administered in 47 (18.7%) patients, while 53 (21.1%) received alternative treatment regimens. Thus 37.6% of recommended MTB therapies were implemented (47/125 recommendations). The clinical benefit rate (complete + partial + mixed response + stable disease) was 50.0% for MTB and 63.8% for alternative treatments. PFS2/1 ratios were 34.6% and 16.1%, respectively. Significantly improved PFS could be achieved for m1A-tier-evidence-based MTB therapies (median 6.30 months) compared to alternative treatments (median 2.83 months; P = 0.0278). CONCLUSION: The CCCO MTB yielded a considerable recommendation rate, particularly in cholangiocarcinoma patients. The discrepancy between the low-recommendation rates in colorectal and pancreatic cancer suggests the necessity of a weighted prioritisation of entities. High-tier recommendations should be implemented predominantly.

The effect of radiotherapy on fat engraftment for complete breast reconstruction using lipofilling only.

PURPOSE: Lipofilling has been established as a standard technique for contour enhancement following breast reconstruction. However, there is a paucity in current literature regarding the use of this technique for complete reconstruction of the female breast as an alternative to conventional techniques, such as expander or flap-based procedures. In particular, the influence of pre-operative irradiation for successful reconstruction has rarely been examined in published studies. Here, the authors describe their experience with successful fat injection in pre-radiated breasts in comparison with non-pre-radiated patients. METHODS: In this retrospective study, we examined a total of 95 lipofilling treatments on 26 patients (28 breasts). All of them experienced mastectomy following breast cancer; local breast defects after partial resection of the gland were not included in this study. In total, 47 lipofilling procedures in 12 non-irradiated patients (14 breasts) and 48 procedures in 14 irradiated women (also 14 breasts) were performed. Per session, approximately 297 ± 112 cc of adipose tissue was grafted in group A (no radiotherapy) and approximately 259 ± 93 cc was grafted in group B (radiotherapy). RESULTS: Among the group of women without pre-operative radiation, 71% of breast reconstructions limited to lipofilling only showed constant engraftment of fat tissue with a successful reconstructive result, whereas only 21% of the patients with pre-radiated breasts showed complete reconstruction of the breast with a permanent fat in-growth. CONCLUSION: Preoperative radiotherapy significantly impedes successful completion of breast reconstructions planned only by autologous fat transfer. Patients should be selected individually and carefully for complete breast reconstruction using lipofilling only.

Advanced Immune Cell Profiling by Multiparameter Flow Cytometry in Humanized Patient-Derived Tumor Mice.

"Humanized" mice have been widely used for the characterization of human cancer progression and as a powerful preclinical model. Standardization of multicolor phenotyping could help to identify immune cell patterns involved in checkpoint-related complications. Therefore, we applied established protocols for immune cell profiling to our humanized Patient-Derived Xenograft (hPDX) model. hPDX are characterized by the co-existence of a human immune system and a patient-derived tumor transplant. These mice possess a human-like immune system after CD34+ stem cell transplantation while the reconstitution level of the immune system was not related to the quantity of transplanted CD34+ cells. Contamination ≤ 1.2% by CD3+ cells in the hematopoietic stem cell (HSC) transplant did not trigger abnormal T cell maturation. Different B and T cell differentiation stages were identified, as well as regulatory T cells (Tregs) and exhausted T cells that expressed TIGIT, PD-1, or KLRG1. Overall, the application of standardized protocols for the characterization of immune cells using flow cytometry will contribute to a better understanding of immune-oncologic processes.

Implants Versus Lipograft: Analysis of Long-Term Results Following Congenital Breast Asymmetry Correction.

AIMS: Congenital breast asymmetry represents a particular challenge to the classic techniques of plastic surgery given the young age of patients at presentation. This study reviews and compares the long-term results of traditional breast augmentation using silicone implants and the more innovative technique of lipografting. METHODS: To achieve this, we not only captured subjective parameters such as satisfaction with outcome and symmetry, but also objective parameters including breast volume and anthropometric measurements. The objective examination was performed manually and by using the Vectra® H2 photogrammetry scanning system. RESULTS: Differences between patients undergoing either implant augmentation or lipograft were revealed not to be significant with respect to patient satisfaction with surgical outcome (p = 0.55) and symmetry (p = 0.69). Furthermore, a breast symmetry of 93 % was reported in both groups. Likewise, no statistically significant volume difference between the left and right breasts was observed in both groups (p < 0.41). However, lipograft patients needed on average 2.9 procedures to achieve the desired result, compared with 1.3 for implant augmentation. In contrast, patients treated with implant augmentation may require a number of implant changes during their lifetime. CONCLUSION: Both methods may be considered for patients presenting with congenital breast asymmetry. LEVEL OF EVIDENCE III: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Autologous fat grafting for breast reconstruction after breast cancer: a 12-year experience.

PURPOSE: The aim of our study was to examine the surgical outcome and complications (efficiency) as well as the incidence of locoregional recurrence and distant metastases (oncological safety) in patients who underwent autologous fat grafting (AFG) of the breast following breast cancer surgery. METHODS: In our monocentric cohort study, retrospective and prospective data were collected from all consecutive patients who underwent AFG after breast cancer between 2008 and 2020; a total of 93 patients met the inclusion criteria. RESULTS: Our long-term results showed no increase in tumor recurrence and distant metastases in the studied collective when compared to the available literature. We observed 1 local recurrence (1.1%), 2 distant metastases (2.2%), and 1 tumor-related death (1.1%). There was a high degree of patient satisfaction; 67.12% of patients reported adequate satisfaction with autologous fat grafting. CONCLUSION: Currently, to our knowledge, this is the study with the longest follow-up time (mean 6.7 years after AFG and 11.5 years after tumor resection). The results of our clinical study will contribute to improve evidence in the broad field of AFG, adipose stem cell and tumor research. Consistent with our study, the literature review shows a clear tendency of clinical trial results with a low incidence rate of tumor recurrence and metastasis following the use of AFG. AFG seems to be a safe procedure also after breast cancer treatment.

Feasibility of ABUS as an Alternative to Handheld Ultrasound for Response Control in Neoadjuvant Breast Cancer Treatment.

INTRODUCTION: The Invenia Automated Breast Ultrasound Screening (ABUS) is indicated as an adjunct to mammography for breast cancer screening in asymptomatic women with high-density breast tissue. ABUS provides time-efficient evaluation of the 3-dimensional recordings within 3 to 6 minutes. The role and advantages of ABUS in everyday clinical practice, especially in routine examination during neoadjuvant chemotherapy (NACT), is not clear. The aim of this monocentric, noninterventional retrospective study is to evaluate the use of ABUS in patients who are under NACT treatment for response control. METHODS: Regular sonographic response check with handheld ultrasound (HHUS) examination and with ABUS were conducted in 83 women who underwent NACT. The response controls were conducted every 3 to 6 weeks during NACT. The handheld sonography was performed with GE Voluson S8. Handheld sonographic measurements and ABUS measurements were compared with the final pathologic tumor size. RESULTS: There was no statistical difference between the measurements with HHUS examination or ABUS compared with final pathologic tumor size (P = .47). The average difference from ABUS measured tumor size to final pathologic tumor size was 9.8 mm. The average difference from handheld measured tumor size to final pathologic tumor size was 9/3 mm. Both the specificity of ABUS and HHUS examination in predicting pathologic complete remission was 100%. CONCLUSION: ABUS seems to be a suitable method to conduct response control in neoadjuvant breast cancer treatment. ABUS may facilitate preoperative planning and offers remarkable time saving for physicians compared with HHUS examination and thus should be considered for clinical practice.

Surgery of congenital breast asymmetry-which objective parameter influences the subjective satisfaction with long-term results.

PURPOSE: Congenital breast asymmetry is a serious gynecological malformation for affected patients. The condition hits young women in puberty and is associated with socio-esthetic handicap, depression, and psychosexual problems. Surgical treatment is usually early in the patient's lifetime, so a long-term sustainable solution is important. Although postoperative outcome has been evaluated in several studies before, this study is the first to analyze which objective parameters have the greatest influence on subjective satisfaction with long-term results. METHODS: Thirty-four patients diagnosed with congenital breast asymmetry that underwent either lipofilling or implant therapy between the years of 2008 to 2019 were examined. On average, our collective comprised patients seven years after surgery. Data were mainly gathered through manual measurements, patient-reported outcome measures (Breast Q™), and breast volumetry based on 3D scans (Vectra® H2, Canfield Scientific). RESULTS: Among all analyzed parameters, only areolar diameter correlated significantly negatively with the subjective outcome satisfaction of the patient. Regarding the subjective assessment of postoperative satisfaction with similarity of the breasts, again the mean areolar diameter, but also the difference in areolar diameter and breast volume between the right and left breasts correlated significantly negatively. CONCLUSION: Areolar diameter was revealed as being a significant factor influencing subjective long-term satisfaction in breast asymmetry patients. Moreover, 3D volumetry proves to be an effective tool to substantiate subjective patient assessments. Our findings may lead to further improvements to surgical planning and will be expanded in further studies.

New aspects in digital breast assessment: further refinement of a method for automated digital anthropometry.

PURPOSE: In this trial, we used a previously developed prototype software to assess aesthetic results after reconstructive surgery for congenital breast asymmetry using automated anthropometry. To prove the consensus between the manual and automatic digital measurements, we evaluated the software by comparing the manual and automatic measurements of 46 breasts. METHODS: Twenty-three patients who underwent reconstructive surgery for congenital breast asymmetry at our institution were examined and underwent 3D surface imaging. Per patient, 14 manual and 14 computer-based anthropometric measurements were obtained according to a standardized protocol. Manual and automatic measurements, as well as the previously proposed Symmetry Index (SI), were compared. RESULTS: The Wilcoxon signed-rank test revealed no significant differences in six of the seven measurements between the automatic and manual assessments. The SI showed robust agreement between the automatic and manual methods. CONCLUSION: The present trial validates our method for digital anthropometry. Despite the discrepancy in one measurement, all remaining measurements, including the SI, showed high agreement between the manual and automatic methods. The proposed data bring us one step closer to the long-term goal of establishing robust instruments to evaluate the results of breast surgery. LEVEL OF EVIDENCE: IV.

Compared DNA and RNA quality of breast cancer biobanking samples after long-term storage protocols in - 80 °C and liquid nitrogen.

Molecular investigations are crucial for further developments in precision medicine. RNA sequencing, alone or in combination with further omic-analyses, resulted in new therapeutic strategies. In this context, biobanks represent infrastructures to store tissue samples and body fluids in combination with clinical data to promote research for new predictive and prognostic biomarkers as well as therapeutic candidate molecules. Until today, the optimal storage conditions are a matter of debate especially with view to the storage temperature. In this unique approach we compared parallel samples from the same tumour, one half stored at - 80 °C and one half in the vapor phase of liquid nitrogen, with almost identical pre-analytical conditions. We demonstrated that RNA isolated from breast cancer samples revealed significantly higher RINe-values after 10 years of storage in the vapor phase of liquid nitrogen compared to storage at - 80 °C. In contrast, no significant difference was found regarding the DIN-values after DNA isolation. Morphological changes of the nucleus and cytoplasm, especially in the samples stored at - 80 °C, gave insights to degenerative effects, most possibly due to the storage protocol and its respective peculiarities. In addition, our results indicate that exact point-to point documentation beginning at the sample preparation is mandatory.