Digital quantification of PRAME for distinguishing melanoma from nevi compared to manual assessment.

AIMS: In this proof-of-concept study, we propose a new method for automated digital quantification of PRAME (PReferentially expressed Antigen of MElanoma) as a diagnostic aid to distinguish between benign and malignant melanocytic lesions. The proposed method utilizes immunohistochemical virtual double nuclear staining for PRAME and SOX10 to precisely identify the melanocytic cells of interest, which is combined with digital image analyse to quantify a PRAME-index. METHODS: Our study included 10 compound nevi, 3 halo nevi, and 10 melanomas. Tissue slides were stained with PRAME, scanned, the cover glass removed, stained with SOX10, scanned again, and finally analysed digitally. The digitally quantified PRAME-index was compared with a manual qualitative assessment by a dermatopathologist using the standard PRAME-scoring system. RESULTS: The digitally quantified PRAME-index showed a sensitivity of 70 % and a specificity of 100 % for separating melanomas from benign lesions. The manual qualitative PRAME-score showed a sensitivity of 60 % and a specificity of 100 %. Comparing the two methods using ROC-analyses, our digital quantitative method (AUC: 0.931, 95 % CI: 0.834;1.00, SD: 0.050) remains on par with the manual qualitative method (AUC: 0.877, 95 % CI: 0.725;1.00, SD: 0.078). CONCLUSION: We found our novel digital quantitative method was at least as precise at classifying lesions as benign or malignant as the current manual qualitative assessment. Our method has the advantages of being operator-independent, objective, and replicable. Furthermore, our method can easily be implemented in an already digitalized pathology department. Given the small cohort size, more studies are to be done to validate our findings.

A multidimensional analysis reveals distinct immune phenotypes and the composition of immune aggregates in pediatric acute myeloid leukemia.

Because of the low mutational burden and consequently, fewer potential neoantigens, children with acute myeloid leukemia (AML) are thought to have a T cell-depleted or 'cold' tumor microenvironment and may have a low likelihood of response to T cell-directed immunotherapies. Understanding the composition, phenotype, and spatial organization of T cells and other microenvironmental populations in the pediatric AML bone marrow (BM) is essential for informing future immunotherapeutic trials about targetable immune-evasion mechanisms specific to pediatric AML. Here, we conducted a multidimensional analysis of the tumor immune microenvironment in pediatric AML and non-leukemic controls. We demonstrated that nearly one-third of pediatric AML cases has an immune-infiltrated BM, which is characterized by a decreased ratio of M2- to M1-like macrophages. Furthermore, we detected the presence of large T cell networks, both with and without colocalizing B cells, in the BM and dissected the cellular composition of T- and B cell-rich aggregates using spatial transcriptomics. These analyses revealed that these aggregates are hotspots of CD8+ T cells, memory B cells, plasma cells and/or plasmablasts, and M1-like macrophages. Collectively, our study provides a multidimensional characterization of the BM immune microenvironment in pediatric AML and indicates starting points for further investigations into immunomodulatory mechanisms in this devastating disease.

Immunohistochemical double nuclear staining for cell-specific automated quantification of the proliferation index - A promising diagnostic aid for melanocytic lesions.

AIMS: Pathologists often use immunohistochemical staining of the proliferation marker Ki67 in their diagnostic assessment of melanocytic lesions. However, the interpretation of Ki67 can be challenging. We propose a new workflow to improve the diagnostic utility of the Ki67-index. In this workflow, Ki67 is combined with the melanocytic tumour-cell marker SOX10 in a Ki67/SOX10 double nuclear stain. The Ki67-index is then quantified automatically using digital image analysis (DIA). The aim of this study was to optimise and test three different multiplexing methods for Ki67/SOX10 double nuclear staining. METHODS: Multiplex immunofluorescence (mIF), multiplex immunohistochemistry (mIHC), and multiplexed immunohistochemical consecutive staining on single slide (MICSSS) were optimised for Ki67/SOX10 double nuclear staining. DIA applications were designed for automated quantification of the Ki67-index. The methods were tested on a pilot case-control cohort of benign and malignant melanocytic lesions (n = 23). RESULTS: Using the Ki67/SOX10 double nuclear stain, malignant melanocytic lesions could be completely distinguished from benign lesions by the Ki67-index. The Ki67-index cut-offs were 1.8% (mIF) and 1.5% (mIHC and MICSSS). The AUC of the automatically quantified Ki67-index based on double nuclear staining was 1.0 (95% CI: 1.0;1.0), whereas the AUC of conventional Ki67 single-stains was 0.87 (95% CI: 0.71;1.00). CONCLUSIONS: The novel Ki67/SOX10 double nuclear stain highly improved the diagnostic precision of Ki67 interpretation. Both mIHC and mIF were useful methods for Ki67/SOX10 double nuclear staining, whereas the MICSSS method had challenges in the current setting. The Ki67/SOX10 double nuclear stain shows potential as a valuable diagnostic aid for melanocytic lesions.

A multidimensional analysis reveals distinct immune phenotypes and tertiary lymphoid structure-like aggregates in the bone marrow of pediatric acute myeloid leukemia.

Because of the low mutational burden, children with acute myeloid leukemia (AML) are thought to have a 'cold' tumor microenvironment and consequently, a low likelihood of response to T cell-directed immunotherapies. Here, we provide a multidimensional overview of the tumor immune microenvironment in newly diagnosed pediatric AML. On a cohort level, we demonstrate wide variation in T cell infiltration with nearly one-third of cases harboring an immune-infiltrated bone marrow. These immune-infiltrated cases are characterized by a decreased abundance of M2-like macrophages, which we find to be associated with response to T cell-directed immunotherapy in adult AML. On an organizational level, we reveal the composition of spatially organized immune aggregates in pediatric AML, and show that in the adult setting such aggregates in post-treatment bone marrow and extramedullary sites associate with response to ipilimumab-based therapy. Altogether, our study provides immune correlates of response to T cell-directed immunotherapies and indicates starting points for further investigations into immunomodulatory mechanisms in AML.

Fueling the flames of colon cancer - does CRP play a direct pro-inflammatory role?

Background: Systemic inflammation, diagnostically ascribed by measuring serum levels of the acute phase reactant C-reactive protein (CRP), has consistently been correlated with poor outcomes across cancer types. CRP exists in two structurally and functionally distinct isoforms, circulating pentameric CRP (pCRP) and the highly pro-inflammatory monomeric isoform (mCRP). The aim of this pilot study was to map the pattern of mCRP distribution in a previously immunologically well-defined colon cancer (CC) cohort and explore possible functional roles of mCRP within the tumor microenvironment (TME). Methods: Formalin-fixed, paraffin-embedded (FFPE) tissue samples from 43 stage II and III CC patients, including 20 patients with serum CRP 0-1 mg/L and 23 patients with serum CRP >30 mg/L were immunohistochemically (IHC) stained with a conformation-specific mCRP antibody and selected immune and stromal markers. A digital analysis algorithm was developed for evaluating mCRP distribution within the primary tumors and adjacent normal colon mucosa. Results: mCRP was abundantly present within tumors from patients with high serum CRP (>30 mg/L) diagnostically interpreted as being systemically inflamed, whereas patients with CRP 0-1 mg/L exhibited only modest mCRP positivity (median mCRP per area 5.07‰ (95%CI:1.32-6.85) vs. 0.02‰ (95%CI:0.01-0.04), p<0.001). Similarly, tissue-expressed mCRP correlated strongly with circulating pCRP (Spearman correlation 0.81, p<0.001). Importantly, mCRP was detected exclusively within tumors, whereas adjacent normal colon mucosa showed no mCRP expression. Double IHC staining revealed colocalization of mCRP with endothelial cells and neutrophils. Intriguingly, some tumor cells also colocalized with mCRP, suggesting a direct interaction or mCRP expression by the tumor itself. Conclusion: Our data show that the pro-inflammatory mCRP isoform is expressed in the TME of CC, primarily in patients with high systemic pCRP values. This strengthens the hypothesis that CRP might not only be an inflammatory marker but also an active mediator within tumors.

SARS-CoV-2 detection using reverse transcription strand invasion based amplification and a portable compact size instrument.

Rapid nucleic-acid based tests that can be performed by non-professionals outside laboratory settings could help the containment of the pandemic SARS-CoV-2 virus and may potentially prevent further widespread lockdowns. Here, we present a novel compact portable detection instrument (the Egoo Health System) for extraction-free detection of SARS-CoV-2 using isothermal reverse transcription strand invasion based amplification (RT-SIBA). The SARS-CoV-2 RT-SIBA assay can be performed directly on crude oropharyngeal swabs without nucleic acid extraction with a reaction time of 30 min. The Egoo Health system uses a capsule system, which is automatically sealed tight in the Egoo instrument after applying the sample, resulting in a closed system optimal for molecular isothermal amplification. The performance of the Egoo Health System is comparable to the PCR instrument with an analytical sensitivity of 25 viral RNA copies per SARS-CoV-2 RT-SIBA reaction and a clinical sensitivity and specificity between 87.0-98.4% and 96.6-98.2% respectively.

Hypoxia-inducible factor-1α expression and breast cancer recurrence in a Danish population-based case control study.

BACKGROUND: Hypoxia-inducible factor-1α (HIF-1α) is a transcription factor that facilitates the adaptation of cancer cells to hypoxic conditions and may be prognostic of breast cancer recurrence. We evaluated the association of HIF-1α expression with breast cancer recurrence, and its association with timing of breast cancer recurrence. METHODS: In this population-based case-control study, we included women diagnosed with stage I-III breast cancer between 1985 and 2001, aged 35-69 years, registered in the Danish Breast Cancer Group. We identified 541 cases of breast cancer recurrence among women with estrogen receptor (ER)-positive disease who were treated with tamoxifen for at least 1 year (ER+ TAM+). We also enrolled 300 breast cancer recurrence cases among women with ER-negative disease, not treated with tamoxifen, who survived at least 1 year (ER-/TAM-). Controls were recurrence-free breast cancer patients at the time of case diagnosis, matched to recurrence cases on ER/TAM status, date of surgery, menopausal status, cancer stage, and county of residence. Expression of HIF-1α was measured by immunohistochemistry on tissue microarrays. We fitted logistic regression models to compute odds ratios (ORs) and 95% confidence intervals (CIs) associating HIF-1α expression with recurrence, and with timing of recurrence. RESULTS: HIF-1α expression was observed in 23% of cases and 20% of controls in the ER+/TAM+ stratum, and in 47% of cases and 48% of controls in the ER-/TAM- stratum. We observed a near-null association between HIF-1α expression in both ER/TAM groups (ER+/TAM+ OR = 1.21, 95%CI 0.88, 1.67 and ER-/TAM- OR = 0.97, 95%CI 0.68, 1.39). HIF-1α expression was not associated with time to recurrence among women in the ER+/TAM+ stratum, but was associated with early recurrence among women in the ER-/TAM- stratum. CONCLUSION: In this study, HIF-1α expression was not associated with breast cancer recurrence overall but may be associated with early recurrence among women diagnosed with ER- breast cancer.

Metabolic Pathway Analysis and Effectiveness of Tamoxifen in Danish Breast Cancer Patients.

BACKGROUND: Tamoxifen and its metabolites compete with estrogen to occupy the estrogen receptor. The conventional dose of adjuvant tamoxifen overwhelms estrogen in this competition, reducing breast cancer recurrence risk by nearly half. Phase I metabolism generates active tamoxifen metabolites, and phase II metabolism deactivates them. No earlier pharmacogenetic study has comprehensively evaluated the metabolism and transport pathways, and no earlier study has included a large population of premenopausal women. METHODS: We completed a cohort study of 5,959 Danish nonmetastatic premenopausal breast cancer patients, in whom 938 recurrences occurred, and a case-control study of 541 recurrent cases in a cohort of Danish predominantly postmenopausal breast cancer patients, all followed for 10 years. We collected formalin-fixed paraffin-embedded tumor blocks and genotyped 32 variants in 15 genes involved in tamoxifen metabolism or transport. We estimated conventional associations for each variant and used prior information about the tamoxifen metabolic path to evaluate the importance of metabolic and transporter pathways. RESULTS: No individual variant was notably associated with risk of recurrence in either study population. Both studies showed weak evidence of the importance of phase I metabolism in the clinical response to adjuvant tamoxifen therapy. CONCLUSIONS: Consistent with prior knowledge, our results support the role of phase I metabolic capacity in clinical response to tamoxifen. Nonetheless, no individual variant substantially explained the modest phase I effect on tamoxifen response. IMPACT: These results are consistent with guidelines recommending against genotype-guided prescribing of tamoxifen, and for the first time provide evidence supporting these guidelines in premenopausal women.

Expression of survivin does not appear to influence breast cancer recurrence risk.

BACKGROUND: Survivin is an inhibitor of apoptosis, and its expression associates with poor outcomes in multiple cancers. It may be a therapeutic target due to its unique expression in cancer cells. METHODS: We estimated the association between nuclear and cytoplasmic survivin expression in primary tumors and breast cancer recurrence. In this case-control study, we included women age 35-69, diagnosed with stage I-III breast cancer between 1985 and 2001, and registered with the Danish Breast Cancer Group. We identified 541 patients with breast cancer recurrence with estrogen receptor-positive disease who were treated with tamoxifen for at least 1 year (ER+/TAM+) and 300 with estrogen receptor-negative carcinomas, not treated with tamoxifen, and who survived at least 1 year (ER-/TAM-). Controls were matched to cases on ER/TAM status, date of surgery, menopausal status, stage and county. Survivin expression was estimated by immunohistochemistry on tissue microarrays. We fit logistic regression models to estimate odds ratios (ORs) and 95% confidence intervals (CIs) associating nuclear and cytoplasmic survivin expression with recurrence. RESULTS: Associations between nuclear and cytoplasmic survivin expression and breast cancer recurrence were near-null in both ER+/TAM + and ER-/TAM - strata. For the cytoplasmic to nuclear ratio (CNR) of survivin expression, we found a null association in the ER+/TAM + group comparing CNR ≥5 with CNR <5, but an association (OR =2.48, 95% CI: 1.15, 5.31) in the ER-/TAM - group. CONCLUSIONS: Survivin expression was not associated with breast cancer recurrence in this study. The CNR ratio may warrant further investigation especially among ER - tumors.