Cryopreservation of human cancers conserves tumour heterogeneity for single-cell multi-omics analysis

Background: High throughput single-cell RNA sequencing (scRNA-Seq) has emerged as a powerful tool for exploring cellular heterogeneity among complex human cancers. scRNA-Seq studies using fresh human surgical tissue are logistically difficult, preclude histopathological triage of samples, and limit the ability to perform batch processing. This hindrance can often introduce technical biases when integrating patient datasets and increase experimental costs. Although tissue preservation methods have been previously explored to address such issues, it is yet to be examined on complex human tissues, such as solid cancers and on high throughput scRNA-Seq platforms. Methods: Using the Chromium 10X platform, we sequenced a total of ~ 120,000 cells from fresh and cryopreserved replicates across three primary breast cancers, two primary prostate cancers and a cutaneous melanoma. We performed detailed analyses between cells from each condition to assess the effects of cryopreservation on cellular heterogeneity, cell quality, clustering and the identification of gene ontologies. In addition, we performed single-cell immunophenotyping using CITE-Seq on a single breast cancer sample cryopreserved as solid tissue fragments. Results: Tumour heterogeneity identified from fresh tissues was largely conserved in cryopreserved replicates. We show that sequencing of single cells prepared from cryopreserved tissue fragments or from cryopreserved cell suspensions is comparable to sequenced cells prepared from fresh tissue, with cryopreserved cell suspensions displaying higher correlations with fresh tissue in gene expression. We showed that cryopreservation had minimal impacts on the results of downstream analyses such as biological pathway enrichment. For some tumours, cryopreservation modestly increased cell stress signatures compared to freshly analysed tissue. Further, we demonstrate the advantage of cryopreserving whole-cells for detecting cell-surface proteins using CITE-Seq, which is impossible using other preservation methods such as single nuclei-sequencing. Conclusions: We show that the viable cryopreservation of human cancers provides high-quality single-cells for multiomics analysis. Our study guides new experimental designs for tissue biobanking for future clinical single-cell RNA sequencing studies

Atypical Ductal Hyperplasia: Factors predicting upstaging to carcinoma

Aim: Percutaneous core needle biopsy (CNB) is considered the gold standard technique for initial histological diagnosis of suspicious breast lesions seen on screening mammogram, but it is less reliable for diagnosing atypical ductal hyperplasia (ADH) due to significant rates of diagnosis upstaging to malignant disease after excision biopsy. The purpose of this study was to identify factors that predict diagnosis upstage to carcinoma in patients diagnosed with ADH on core biopsy. Methods: A retrospective database search identified 52 consecutive CNB of suspicious breast lesions revealing pure ADH. Inclusion criteria included asymptomatic women presenting for screening mammogram, who subsequently underwent surgical excision. Logistic regression analysis evaluated clinical, radiological, and histological factors. Results: A total of 52 patients with ADH on CNB were identified who met our criteria. Twenty-six of 52 patients (50%) were upstaged to ductal carcinoma in situ or invasive carcinoma, based on histological interpretation of the surgically excised specimen. Lesion size was showed to be a statistically significant predictor on univariable logistic regression analysis. Multivariate logistic regression analysis revealed Asian ethnicity and lesion size as independent predictors of malignancy (p = 0.050 and 0.011, respectively). Conversely, women of Middle Eastern and European origin and lesions < 15 mm on mammography were negative predictors of malignancy. Conclusion: Lesion size ≥ 15 mm on mammography and Asian ethnicity are independent risk factors for breast carcinoma in asymptomatic patients diagnosed with ADH on CNB

Adenomyoepithelioma with Ductal Carcinoma In Situ: A Case Report and Review of the Literature

Adenomyoepithelioma (AME) with microglandular adenosis-like growth pattern and superimposed ductal carcinoma in situ (DCIS) was identified in a 55-year-old female after biopsy of an atypical lesion identified through routine breast screening. A literature review reveals that this association has rarely been described

Phyllodes tumour with heterologous sarcomatous differentiation: Case series with literature review

Introduction: Phyllodes tumours are rare fibroepithelial malignancies of the breast, accounting for less than 1% of malignant breast tumours. Further malignant differentiation of phyllodes tumours can occur, resulting in cases of extremely rare heterologous sarcomatous differentiation. Presentation of case: Two females in their fifties were diagnosed with malignant phyllodes tumour associated with heterologous sarcomatous differentiation. The first patient, aged 50 had phyllodes tumour with chondrosarcoma, osteosarcoma and ductal carcinoma-in-situ. The second patient, aged 53 had phyllodes tumour with osteosarcoma and liposarcoma. Discussion: The association of phyllodes tumour and heterologous sarcomatous differentiation is rare, with only 4 previously reported cases in English literature. The paucity of evidence presents challenges in its management with uncertain prognosis and monitoring requirements for two aforementioned patients. Conclusion: Further case series and long-term follow up is required for accurate characterisation of phyllodes tumours with heterologous sarcomatous differentiation

ALK1-Negative Anaplastic Large Cell Lymphoma of the Breast from a Nonprosthesis Cyst

Summary: Anaplastic large cell lymphoma of the breast is a rare malignancy associated with prosthetic breast implants. We present a case of a woman with no prior history of breast implants who developed anaplastic lymphoma kinase-1 negative anaplastic large cell lymphoma on a background of a previous benign cyst aspiration

Colonization with antibiotic-resistant Streptococcus pneumoniae in children with sickle cell disease

Objective: Because of a susceptibility to severe pneumococcal infection, children with sickle cell disease (SCD) routinely receive penicillin prophylaxis. Increasing rates of penicillin resistance have been reported throughout the world. Our objective was to assess the prevalence of nasopharyngeal colonization with Streptococcus pneumoniae and to assess the antimicrobial susceptibility of the organisms in children with SCD

Positive Anterior Margins in Breast Conserving Surgery: Does It Matter? A Systematic Review of the Literature

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