Molecular subtyping of head and neck cancer - Clinical applicability and correlations with morphological characteristics.

AIM: We aimed to evaluate the applicability of a customized NanoString panel for molecular subtyping of recurrent or metastatic head and neck squamous cell carcinoma (R/M-HNSCC). Additionally, histological analyses were conducted, correlated with the molecular subtypes and tested for their prognostic value. MATERIAL AND METHODS: We conducted molecular subtyping of R/M-HNSCC according to the molecular subtypes defined by Keck et al. For molecular analyses a 231 gene customized NanoString panel (the most accurately subtype defining genes, based on previous analyses) was applied to tumor samples from R/M-HNSCC patients that were treated in the CeFCiD trial (AIO/IAG-KHT trial 1108). A total of 130 samples from 95 patients were available for sequencing, of which 80 samples from 67 patients passed quality controls and were included in histological analyses. H&E stained slides were evaluated regarding distinct morphological patterns (e.g. tumor budding, nuclear size, stroma content). RESULTS: Determination of molecular subtypes led to classification of tumor samples as basal (n = 46, 45 %), inflamed/mesenchymal (n = 31, 30 %) and classical (n = 26, 25 %). Expression levels of Amphiregulin (AREG) were significantly higher for the basal and classical subtypes compared to the mesenchymal subtype. While molecular subtypes did not have an impact on survival, high levels of tumor budding were associated with poor outcomes. No correlation was found between molecular subtypes and histological characteristics. CONCLUSIONS: Utilizing the 231-gene NanoString panel we were able to determine the molecular subtype of R/M-HNSCC samples by the use of FFPE material. The value to stratify for different treatment options remains to be explored in the future. The prognostic value of tumor budding was underscored in this clinically well annotated cohort.

Built to Last? Reproducibility and Reusability of Deep Learning Algorithms in Computational Pathology.

Recent progress in computational pathology has been driven by deep learning. While code and data availability are essential to reproduce findings from preceding publications, ensuring a deep learning model's reusability is more challenging. For that, the codebase should be well-documented and easy to integrate into existing workflows and models should be robust toward noise and generalizable toward data from different sources. Strikingly, only a few computational pathology algorithms have been reused by other researchers so far, let alone employed in a clinical setting. To assess the current state of reproducibility and reusability of computational pathology algorithms, we evaluated peer-reviewed articles available in PubMed, published between January 2019 and March 2021, in 5 use cases: stain normalization; tissue type segmentation; evaluation of cell-level features; genetic alteration prediction; and inference of grading, staging, and prognostic information. We compiled criteria for data and code availability and statistical result analysis and assessed them in 160 publications. We found that only one-quarter (41 of 160 publications) made code publicly available. Among these 41 studies, three-quarters (30 of 41) analyzed their results statistically, half of them (20 of 41) released their trained model weights, and approximately a third (16 of 41) used an independent cohort for evaluation. Our review is intended for both pathologists interested in deep learning and researchers applying algorithms to computational pathology challenges. We provide a detailed overview of publications with published code in the field, list reusable data handling tools, and provide criteria for reproducibility and reusability.

Carcinoid syndrome caused by a pulmonary carcinoid mimics intestinal manifestations of ulcerative colitis: A case report.

BACKGROUND: Pulmonary carcinoids are rare, low-grade malignant tumors characterized by neuroendocrine differentiation and relatively indolent clinical behavior. Most cases present as a slow-growing polypoidal mass in the major bronchi leading to hemoptysis and pulmonary infection due to blockage of the distal bronchi. Carcinoid syndrome is a paraneoplastic syndrome caused by the systemic release of vasoactive substances that presents in 5% of patients with neuroendocrine tumors. Due to such nonspecific presentation, most patients are misdiagnosed or diagnosed late and may receive several courses of antibiotics to treat recurrent pneumonia before the tumor is diagnosed. CASE SUMMARY: We report the case of a 48-year-old male who presented with cough, dyspnea, a history of recurrent pneumonitis, and therapy-refractory ulcerative colitis that completely subsided after the resection of a pulmonary carcinoid. CONCLUSION: We report and emphasize pulmonary carcinoid as a differential diagnosis in patients with nonresponding inflammatory bowel diseases and recurrent pneumonia.

Transformer-based biomarker prediction from colorectal cancer histology: A large-scale multicentric study.

Deep learning (DL) can accelerate the prediction of prognostic biomarkers from routine pathology slides in colorectal cancer (CRC). However, current approaches rely on convolutional neural networks (CNNs) and have mostly been validated on small patient cohorts. Here, we develop a new transformer-based pipeline for end-to-end biomarker prediction from pathology slides by combining a pre-trained transformer encoder with a transformer network for patch aggregation. Our transformer-based approach substantially improves the performance, generalizability, data efficiency, and interpretability as compared with current state-of-the-art algorithms. After training and evaluating on a large multicenter cohort of over 13,000 patients from 16 colorectal cancer cohorts, we achieve a sensitivity of 0.99 with a negative predictive value of over 0.99 for prediction of microsatellite instability (MSI) on surgical resection specimens. We demonstrate that resection specimen-only training reaches clinical-grade performance on endoscopic biopsy tissue, solving a long-standing diagnostic problem.

Proteogenomic analysis reveals RNA as a source for tumor-agnostic neoantigen identification.

Systemic pan-tumor analyses may reveal the significance of common features implicated in cancer immunogenicity and patient survival. Here, we provide a comprehensive multi-omics data set for 32 patients across 25 tumor types for proteogenomic-based discovery of neoantigens. By using an optimized computational approach, we discover a large number of tumor-specific and tumor-associated antigens. To create a pipeline for the identification of neoantigens in our cohort, we combine DNA and RNA sequencing with MS-based immunopeptidomics of tumor specimens, followed by the assessment of their immunogenicity and an in-depth validation process. We detect a broad variety of non-canonical HLA-binding peptides in the majority of patients demonstrating partially immunogenicity. Our validation process allows for the selection of 32 potential neoantigen candidates. The majority of neoantigen candidates originates from variants identified in the RNA data set, illustrating the relevance of RNA as a still understudied source of cancer antigens. This study underlines the importance of RNA-centered variant detection for the identification of shared biomarkers and potentially relevant neoantigen candidates.

Independent Tissue-Based Biomarkers in Endometrioid Endometrial Cancer: Tumor Budding in Microsatellite Instability and WHO Grading in Copy-Number-Low Patients.

The molecular characterization of endometrial endometrioid adenocarcinomas has provided major advances in its prognostic stratification. However, risk assessment of microsatellite instability (MSI) and copy-number (CN)-low cases remains a challenge. Thus, we aimed to identify tissue-based morphologic biomarkers that might help in the prognostic stratification of these cases. Histomorphologic parameters (WHO grading, tumor budding (TB), tumor-stroma ratio (as a quantitative description of stromal desmoplasia), tumor-infiltrating lymphocytes (TIL), "microcystic, elongated, fragmented" (MELF) pattern) were analyzed in resection specimens of the TCGA-UCEC cohort (n = 228). For each quantitative parameter, a two-tiered system was developed utilizing systematically determined cutoffs. Associations with survival outcomes were calculated in univariate and multivariate analysis and validated in two independent cohorts. In MSI tumors, only TB remained an independent prognostic factor. TB (≥3 buds/high-power field) was associated with inferior outcomes and with lymph node metastases. The prognostic significance of TB was confirmed in two validation cohorts. For CN-low tumors, established grading defined by the WHO was independently prognostic with inferior outcomes for high-grade tumors. The evaluation of TB might help in identifying MSI-patients with unfavorable prognosis who, e.g., could benefit from lymphadenectomy. WHO-based grading facilitates independent prognostic stratification of CN-low endometrioid adenocarcinomas. Therefore, we propose the utilization of TB and WHO-based grading, two tissue-based and easy-to-assess biomarkers, in MSI/CN-low endometrial carcinomas for improved clinical management.

Tumour budding-based grading as independent prognostic biomarker in HPV-positive and HPV-negative head and neck cancer.

BACKGROUND: The prognostic significance of tumour budding (TB) and minimal cell nest size (MCNS) was shown in human papillomavirus (HPV)-negative head and neck squamous cell carcinomas (HNSCC). However, the optimisation of cutpoints, the prognostic impact in HPV-positive HNSCC, and the comparison with other histopathological grading systems are insufficiently investigated. METHODS: TB and MCNS were analysed digitally in 1 and 10 high-power fields (HPF) of 331 HPV-positive and HPV-negative cases from TCGA. Optimising the cutpoints a new cellular dissociation grading (CDG) system was defined and compared to the WHO grading and the Brandwein-Gensler (BG) risk model. RESULTS: The two-tiered CDG system based solely on TB yielded optimal prognostic stratification with shortened overall survival for CDG-high cases. Optimal cut-offs were two buds (1 HPF) and six buds (10 HPF), respectively. Analysing MCNS did not add prognostic significance to quantifying TB. CDG was a significant prognostic marker in HPV-negative and HPV-positive tumours and prognostically superior to the WHO and BG systems. High CDG was associated with clinically occult lymph-node metastases. CONCLUSIONS: The most comprehensive study of TB in HNSCC so far confirmed its prognostic impact in HPV-negative tumours and for the first time in HPV-positive tumours. Further studies are warranted to evaluate its applicability for therapy guidance in HNSCC.

Comparison of PD-L1 expression in squamous cell cancer of unknown primary and oropharyngeal squamous cell carcinoma.

PURPOSE: The tumorigenesis of squamous cell cancer of unknown primary (SCCUP) in the head and neck area has not been decoded so far, while poor survival rates and limited therapeutic options pose a serious challenge. The aim of this project was to investigate immunological characteristics of SCCUPs and compare them to oropharyngeal squamous cell carcinoma (OPSCC). METHODS: PD-L1 expression (TC) was examined by immunohistochemistry in 50 lymph node metastases of SCCUP and 47 primaries of OPSCC. CD3 + and CD8 + lymphocytic infiltration was measured in 5 high power fields. Expression of p16 and HPV ISH were assessed. RESULTS: SCCUP demonstrated a significantly higher expression of PD-L1 than OPSCC. In p16-negative SCCUPs PD-L1 proved to be an independent prognostic factor to prioritize high-risk patients. CONCLUSIONS: Immunologic differences between SCCUP and OPSCC were detected. A higher PD-L1 expression in SCCUP could potentially facilitate further evaluation of checkpoint inhibitor therapy.

Cellular dissociation grading on biopsies of pulmonary squamous cell carcinoma provides prognostic information across all stages and is congruent with resection specimen grading.

Grading of squamous cell carcinomas (SCCs) based on tumour budding and cell nest size has been termed cellular dissociation grading (CDG) and was suggested as a robust outcome predictor when assessed in biopsies and resections of various extrapulmonary SCCs. In pulmonary SCC (pSCC), this has so far been shown only for resected cancers. As most lung cancers are inoperable, it is of utmost importance to clarify whether the prognostic impact of CDG is retained in the biopsy setting. Two independent pSCC biopsy cohorts from Munich (n = 134, non-resected) and Heidelberg (n = 135, resected) were assessed. Tumour budding and cell nest size measures were assembled into the three-tiered CDG system (G1-G3). Data were correlated with clinicopathological parameters and overall- (OS), disease-specific- (DSS), and disease-free survival (DFS). Interobserver variability and concordance between biopsy and resection specimen were also investigated. CDG was highly congruent between biopsy and resection specimens (κ = 0.77, p < 0.001). In both pSCC cohorts, biopsy-derived CDG strongly impacted on OS, DSS, and DFS (e.g. DFS: p < 0.001). In multivariate survival analyses, CDG remained a stage independent predictor of survival in both cohorts (DFS: p < 0.001 respectively; hazard ratio Munich cohort: CDG-G2: 4.31, CDG-G3; 5.14; Heidelberg cohort: CDG-G2: 5.87, CDG-G3: 9.07). Interobserver agreement for CDG was almost perfect (κ = 0.84, p < 0.001). We conclude that assessment of CDG based on tumour budding and cell nest size is feasible on pSCC biopsies and harbours stage independent prognostic information in resectable as well as non-resectable pSCC. Integration of this grading approach into clinicopathological routine should be considered.

Neoadjuvant Radiation in High-Grade Soft-Tissue Sarcomas: Histopathologic Features and Response Evaluation.

In this study, we sought to determine the prognostic value of both the European Organization for Research and Treatment of Cancer-Soft Tissue and Bone Sarcoma Group (EORTC-STBSG) score and the histologic parameters viable tumor, coagulative necrosis, hyalinization/fibrosis, and infarction in patients (n=64) with localized, nonmetastatic high-grade soft-tissue sarcomas after preoperative radiomonotherapy. A standardized macroscopic workup for pretreated surgical specimen including evaluation of a whole section of high-grade soft tissue sarcomas in the largest diameter, was used. Association with overall survival and disease-free survival was assessed. Limb salvage could be accomplished in 98.4% of patients. Overall, 90.6% tumors had negative resection margins. The median postoperative tumor diameter was 9 cm. Undifferentiated pleomorphic sarcoma (42.2%) and myxofibrosarcoma (17.2%) were the most common diagnoses. In all, 9.4% of patients had local recurrence despite clear resection margins, and 50% had distant metastases. Morphologic mapping suggests an overall heterogenous intratumoral response to radiotherapy, with significant differences among histologic subtypes. Complete regression (0% vital tumor cells) was not seen. Categorizing the results according to the proposed EORTC-STBSG 5-tier response score, <1% viable tumor cells were seen in 3.1%, ≥1% to <10% viable tumor cells in 20.4%, ≥10% to <50% viable tumor cells in 35.9% and ≥50% viable tumor cells in 40.6% of cases. Mean values for viable tumor cells were 40% (range: 1% to 100%), coagulative necrosis 5% (0% to 60%), hyalinization/fibrosis 25% (0% to 90%) and infarction 15% (0% to 79%). Hyalinization/fibrosis was a significant independent prognostic factor for overall survival (hazard ratio=4.4; P =0.047), while the other histologic parameters including the EORTC-STBSG score were not prognostic.

Tumour cell budding and spread through air spaces in squamous cell carcinoma of the lung - Determination and validation of optimal prognostic cut-offs.

PURPOSE: Prognostic stratification of patients with squamous cell carcinomas of the lung (SCC-L) is challenging. Therefore, we investigated several histomorphological parameters (tumour cell budding (TCB), spread through air spaces (STAS), tumour-stroma-ratio, immune cell infiltration) which could potentially serve as prognostic parameters in SCC-L. We aimed to systematically determine optimal cut-off-values and assess the prognostic capability of these patterns. We furthermore assessed interobserver variability (IOV) for prognostically significant patterns TCB and STAS. EXPERIMENTAL DESIGN: The Cancer Genome Atlas (TCGA) study cohort consisted of 335 patients with SCC-L. Histomorphological parameters analysed comprised TCB, minimal cell nest size (MCNS), STAS, stroma content and immune cell infiltration. The most significant cut-off-values were determined and univariate and multivariate survival outcomes were estimated. The identified cut-off-points were validated in an independent SCC-L cohort (n = 346 patients). Two experienced pathologists probed IOV in the validation cohort. RESULTS: In the TCGA study cohort, TCB, STAS and immune cell infiltration were identified as significant prognostic parameters. TCB-high tumours, a high number of STAS foci, extensive STAS for distance of STAS in alveoli and a low immune cell infiltration remained as independent prognostic factors in multivariate Cox proportional hazard analyses for overall survival (OS). The significance of TCB, number of STAS foci and distance of STAS in alveoli for OS could be validated in the validation cohort. IOV reached a Kappa ≥ 0.89 for prognostic parameters. CONCLUSIONS: We determined optimal cut-offs and identified TCB and STAS (number of STAS foci, distance of STAS in alveoli) as independent and uncorrelated prognostic factors for patients with SCC-L. The significance was validated in a large independent cohort. IOV was almost perfect for prognostic parameters. We propose the application of TCB- and STAS-based grading in SCC-L as prognostic morphological classifiers.

NOTCH1 Intracellular Domain and the Tumor Microenvironment as Prognostic Markers in HNSCC.

(1) Background: NOTCH1 is the second most common mutated gene in whole-exome sequencing of HNSCC. The aim of this project was to gain further insight into the relevance of NOTCH1 in HNSCC, potentially establishing NOTCH1 as a prognostic marker or therapeutic target; (2) Methods: NOTCH1 was silenced via RNA interference in six HNSCC cell lines and the impact was evaluated in migration and proliferation assays. Subsequently, the protein expression of NOTCH1 intracellular domain (NICD) and NOTCH1 mRNA expression were examined in 70 oropharyngeal squamous cell cancer tissue samples. Lastly, the NICD expression was compared with the local infiltration of lymphocytes, measured with the immunoscore; (3) Results: Knockdown of NOTCH1 decreased migration and proliferation. A high NICD expression was associated with lower OS. A high immunoscore resulted in significantly better OS. NICD expression was independent of the immunoscore and as a whole differentiated three distinct prognostic groups; (4) Conclusions: These data suggest that NOTCH1 is involved in migration and proliferation of HNSCC cell lines. In vivo, NICD expression was associated with overall survival and could, therefore, be used as a prognostic marker. NICD expression differs from NOTCH1 mRNA levels, potentially explaining the previously suggested bimodal role as an oncogene and tumor suppressor in HNSCC.

Loss of CDX2 in colorectal cancer is associated with histopathologic subtypes and microsatellite instability but is prognostically inferior to hematoxylin-eosin-based morphologic parameters from the WHO classification.

BACKGROUND: Immunohistochemical loss of CDX2 has been proposed as a biomarker of dismal survival in colorectal carcinoma (CRC), especially in UICC Stage II/III. However, it remains unclear, how CDX2 expression is related to central hematoxylin-eosin (HE)-based morphologic parameters defined by 2019 WHO classification and how its prognostic relevance is compared to these parameters. METHODS: We evaluated CDX2 expression in 1003 CRCs and explored its prognostic relevance compared to CRC subtypes, tumour budding and WHO grade in the overall cohort and in specific subgroups. RESULTS: CDX2-low/absent CRCs were enriched in specific morphologic subtypes, right-sided and microsatellite-instable (MSI-H) CRCs (P < 0.001) and showed worse survival characteristics in the overall cohort/UICC Stage II/III (e.g. DFS: P = 0.005) and in microsatellite stable and left-sided CRCs, but not in MSI-H or right-sided CRCs. Compared with CDX2, all HE-based markers showed a significantly better prognostic discrimination in all scenarios. In multivariate analyses including all morphologic parameters, CDX2 was not an independent prognostic factor. CONCLUSION: CDX2 loss has some prognostic impact in univariate analyses, but its prognostic relevance is considerably lower compared to central HE-based morphologic parameters defined by the WHO classification and vanishes in multivariate analyses incorporating these factors.

Functional analysis of peripheral and intratumoral neoantigen-specific TCRs identified in a patient with melanoma.

BACKGROUND: Neoantigens derived from somatic mutations correlate with therapeutic responses mediated by treatment with immune checkpoint inhibitors. Neoantigens are therefore highly attractive targets for the development of therapeutic approaches in personalized medicine, although many aspects of their quality and associated immune responses are not yet well understood. In a case study of metastatic malignant melanoma, we aimed to perform an in-depth characterization of neoantigens and respective T-cell responses in the context of immune checkpoint modulation. METHODS: Three neoantigens, which we identified either by immunopeptidomics or in silico prediction, were investigated using binding affinity analyses and structural simulations. We isolated seven T-cell receptors (TCRs) from the patient's immune repertoire recognizing these antigens. TCRs were compared in vitro by multiparametric analyses including functional avidity, multicytokine secretion, and cross-reactivity screenings. A xenograft mouse model served to study in vivo functionality of selected TCRs. We investigated the patient's TCR repertoire in blood and different tumor-related tissues over 3 years using TCR beta deep sequencing. RESULTS: Selected mutated peptide ligands with proven immunogenicity showed similar binding affinities to the human leukocyte antigen complex and comparable disparity to their wild-type counterparts in molecular dynamic simulations. Nevertheless, isolated TCRs recognizing these antigens demonstrated distinct patterns in functionality and frequency. TCRs with lower functional avidity showed at least equal antitumor immune responses in vivo. Moreover, they occurred at high frequencies and particularly demonstrated long-term persistence within tumor tissues, lymph nodes and various blood samples associated with a reduced activation pattern on primary in vitro stimulation. CONCLUSIONS: We performed a so far unique fine characterization of neoantigen-specific T-cell responses revealing defined reactivity patterns of neoantigen-specific TCRs. Our data highlight qualitative differences of these TCRs associated with function and longevity of respective T cells. Such features need to be considered for further optimization of neoantigen targeting including adoptive T-cell therapies using TCR-transgenic T cells.

Kallikrein-Related Peptidase 6 Is Associated with the Tumour Microenvironment of Pancreatic Ductal Adenocarcinoma.

As cancer-associated factors, kallikrein-related peptidases (KLKs) are components of the tumour microenvironment, which represents a rich substrate repertoire, and considered attractive targets for the development of novel treatments. Standard-of-care therapy of pancreatic cancer shows unsatisfactory results, indicating the need for alternative therapeutic approaches. We aimed to investigate the expression of KLKs in pancreatic cancer and to inhibit the function of KLK6 in pancreatic cancer cells. KLK6, KLK7, KLK8, KLK10 and KLK11 were coexpressed and upregulated in tissues from pancreatic cancer patients compared to normal pancreas. Their high expression levels correlated with each other and were linked to shorter survival compared to low KLK levels. We then validated KLK6 mRNA and protein expression in patient-derived tissues and pancreatic cancer cells. Coexpression of KLK6 with KRT19, αSMA or CD68 was independent of tumour stage, while KLK6 was coexpressed with KRT19 and CD68 in the invasive tumour area. High KLK6 levels in tumour and CD68+ cells were linked to shorter survival. KLK6 inhibition reduced KLK6 mRNA expression, cell metabolic activity and KLK6 secretion and increased the secretion of other serine and aspartic lysosomal proteases. The association of high KLK levels and poor prognosis suggests that inhibiting KLKs may be a therapeutic strategy for precision medicine.

Morphology Matters: A Critical Reappraisal of the Clinical Relevance of Morphologic Criteria From the 2019 WHO Classification in a Large Colorectal Cancer Cohort Comprising 1004 Cases.

The 2019 World Health Organization (WHO) classification of colorectal carcinoma (CRC) profoundly reclassified CRC subtypes and introduces tumor budding as a second major grading criterion, while condensing conventional grade into a 2-tiered system. So far it remains largely unexplored how these parameters interact with each other and whether they truly have an independent impact on patient prognosis. We reclassified a large single-center cohort of 1004 CRCs spanning 2 decades for adjusted WHO grade (low vs. high), tumor budding (Bd1/Bd2/Bd3), and CRC subtype (adenocarcinoma not otherwise specified, micropapillary, mucinous, serrated, medullary, adenoma-like, signet-ring cell, mixed adenoneuroendocrine carcinoma/neuroendocrine carcinoma, undifferentiated) according to the criteria of the 2019 WHO classification. We investigated the interaction of these parameters, their connection to stage/microsatellite status, and their significance for patient survival in the different subgroups. Specific subtypes other than adenocarcinoma not otherwise specified represented one third of all CRCs and were unevenly distributed throughout stage and microsatellite subgroups. Subtypes, WHO grade and tumor budding profoundly impacted all survival parameters (P<0.001 for all analyses), with CRC subtypes and tumor budding-but not WHO grade-being stage-independent prognosticators for all survival comparisons. WHO grade had very limited prognostic value in CRC subtypes, while tumor budding retained its strong prognostic impact in most scenarios. Accurate delineation of CRC subtypes introduced in the 2019 WHO classification provides strong stage-independent prognostic information, arguing that they should be considered in pathology reports and in clinical trials. Of the morphology-based grading schemes included in the 2019 WHO, tumor budding outperforms WHO grade.

TP53 germline mutations in the context of families with hereditary breast and ovarian cancer: a clinical challenge.

PURPOSE: TP53germline (g) mutations, associated with the Li-Fraumeni syndrome (LFS), have rarely been reported in the context of hereditary breast and ovarian cancer (HBOC). The prevalence and cancer risks in this target group are unknown and counseling remains challenging. Notably an extensive high-risk surveillance program is implemented, which evokes substantial psychological discomfort. Emphasizing the lack of consensus about clinical implications, we aim to further characterize TP53g mutations in HBOC families. METHODS: Next-generation sequencing was conducted on 1876 breast cancer (BC) patients who fulfilled the inclusion criteria for HBOC. RESULTS: (Likely) pathogenic variants in TP53 gene were present in 0.6% of the BC cohort with higher occurrence in early onset BC < 36 years. (1.1%) and bilateral vs. unilateral BC (1.1% vs. 0.3%). Two out of eleven patients with a (likely) pathogenic TP53g variant (c.542G > A; c.375G > A) did not comply with classic LFS/Chompret criteria. Albeit located in the DNA-binding domain of the p53-protein and therefore revealing no difference to LFS-related variants, they only displayed a medium transactivity reduction constituting a retainment of wildtype-like anti-proliferative functionality. CONCLUSION: Among our cohort of HBOC families, we were able to describe a clinical subgroup, which is distinct from the classic LFS-families. Strikingly, two families did not adhere to the LFS criteria, and functional analysis revealed a reduced impact on TP53 activity, which may suit to the attenuated phenotype. This is an approach that could be useful in developing individualized screening efforts for TP53g mutation carrier in HBOC families. Due to the low incidence, national/international cooperation is necessary to further explore clinical implications. This might allow providing directions for clinical recommendations in the future.