Single-cell transcriptomic analyses reveal distinct immune cell contributions to epithelial barrier dysfunction in checkpoint inhibitor colitis.

Immune checkpoint inhibitor (ICI) therapy has revolutionized oncology, but treatments are limited by immune-related adverse events, including checkpoint inhibitor colitis (irColitis). Little is understood about the pathogenic mechanisms driving irColitis, which does not readily occur in model organisms, such as mice. To define molecular drivers of irColitis, we used single-cell multi-omics to profile approximately 300,000 cells from the colon mucosa and blood of 13 patients with cancer who developed irColitis (nine on anti-PD-1 or anti-CTLA-4 monotherapy and four on dual ICI therapy; most patients had skin or lung cancer), eight controls on ICI therapy and eight healthy controls. Patients with irColitis showed expanded mucosal Tregs, ITGAEHi CD8 tissue-resident memory T cells expressing CXCL13 and Th17 gene programs and recirculating ITGB2Hi CD8 T cells. Cytotoxic GNLYHi CD4 T cells, recirculating ITGB2Hi CD8 T cells and endothelial cells expressing hypoxia gene programs were further expanded in colitis associated with anti-PD-1/CTLA-4 therapy compared to anti-PD-1 therapy. Luminal epithelial cells in patients with irColitis expressed PCSK9, PD-L1 and interferon-induced signatures associated with apoptosis, increased cell turnover and malabsorption. Together, these data suggest roles for circulating T cells and epithelial-immune crosstalk critical to PD-1/CTLA-4-dependent tolerance and barrier function and identify potential therapeutic targets for irColitis.

Immune Responses in Checkpoint Myocarditis Across Heart, Blood, and Tumor.

Immune checkpoint inhibitors (ICIs) are widely used anti-cancer therapies that can cause morbid and potentially fatal immune-related adverse events (irAEs). ICI-related myocarditis (irMyocarditis) is uncommon but has the highest mortality of any irAE. The pathogenesis of irMyocarditis and its relationship to anti-tumor immunity remain poorly understood. We sought to define immune responses in heart, tumor, and blood during irMyocarditis and identify biomarkers of clinical severity by leveraging single-cell (sc)RNA-seq coupled with T cell receptor (TCR) sequencing, microscopy, and proteomics analysis of 28 irMyocarditis patients and 23 controls. Our analysis of 284,360 cells from heart and blood specimens identified cytotoxic T cells, inflammatory macrophages, conventional dendritic cells (cDCs), and fibroblasts enriched in irMyocarditis heart tissue. Additionally, potentially targetable, pro-inflammatory transcriptional programs were upregulated across multiple cell types. TCR clones enriched in heart and paired tumor tissue were largely non-overlapping, suggesting distinct T cell responses within these tissues. We also identify the presence of cardiac-expanded TCRs in a circulating, cycling CD8 T cell population as a novel peripheral biomarker of fatality. Collectively, these findings highlight critical biology driving irMyocarditis and putative biomarkers for therapeutic intervention.

Classical Paroxysmal Nocturnal Hemoglobinuria Presenting With Severe Anemia and Pigmented Acute Kidney Injury.

Paroxysmal nocturnal hemoglobinuria is a rare form of intravascular hemolysis caused by an acquired deficiency of complement regulatory glycoproteins. In our case, a 53-year-old male presented with fatigue, discoloration of urine, and reduced urine output. Preliminary investigations showed severe anemia (3.7 g/dl) and hyperkalemia (7.6 mmol/L) in the setting of acute kidney injury, requiring urgent dialysis. Four units of packed cell volumes were transfused for the correction of anaemia. Following initial stabilization, flow cytometry and a fluorescein-labeled proaerolysin (FLAER) study showed a total deficiency of CD59 in 95.92% of granulocytes and a 97.14% deficiency in monocytes. A bone marrow biopsy showed erythroblast hyperplasia confirming the diagnosis of classical paroxysmal nocturnal hemoglobinuria. The patient was treated with steroids, androgens, and iron supplementation and made a complete recovery with a near-total resolution of his acute kidney injury. This paper aims to review the clinical features and investigations in order to focus on acute kidney injury as an outcome of paroxysmal nocturnal hemoglobinuria.

Clustered Incidence of Leukocytoclastic Vasculitis and Purpura Fulminans: A Case Series of a Rare Dermatological Manifestation of Rickettsial Disease.

Rickettsiae are a group of eukaryotic obligatory intracellular parasites with ticks and mites as vectors. Rickettsia conorii is the Indian counterpart of Rocky Mountain spotted fever causing the endemic variant - Indian tick typhus. This disease can cause severe illness in adults and children and can be missed despite the availability of serological tests. Initial screening for rickettsial diseases (RD) may include blood workup and a non-specific agglutination test, Weil-Felix (WF). In WF, agglutination against Proteus antigens is analyzed and can show false-negative results within the first week of presentation. Delayed immune reaction in patients with RD in the first week could also be responsible for negative specific IgM serology. The challenge for physicians is to differentiate between the two common diagnoses for fever with rash - viral exanthematous fever and rickettsial fever. By its endothelial cell tropism, RD rarely can lead to purpura fulminans, which is characterized by widespread progressive dermal vascular necrosis and hemorrhage. This case series demonstrates dermatologic presentations of rickettsial fever in three individuals from the same neighborhood within the same week. Based on serologic IgM levels, the patients were treated with doxycycline and made a full recovery. This case series aimed to highlight the need for awareness regarding the variable presentations of rickettsial fever including leukocytoclastic vasculitis and purpura fulminans.

Spontaneous Transvaginal Small Bowel Evisceration With Uterine Procidentia: A Report of a Rare Case.

Spontaneous transvaginal small bowel evisceration is an extremely rare condition with a few more than 100 documented cases to date. The complications of such a rare entity revolve around its preoperative presentation as well as its operative and postoperative complications. The complications seen are intestinal incarcerations and perforations with an increased risk of post-surgical ileus and peritonitis in a time-dependent fashion. In our case, a postmenopausal female presented with sudden onset bowel evisceration through a defect in the posterior vaginal apex during straining for defecation. Past medical history was significant for intermittent abdominal pain, bloating, and chronic constipation. The patient had no signs of trauma, signs of sexual assault, preceding events, or prior urinary disturbances. The patient was treated surgically with laparotomy and bowel-packing followed by Ward-Mayo's repair with added on anterior and posterior colporrhaphy, and site-specific repair. Postoperatively, following a period of prolonged ileus, the patient made a full recovery. This case report aims to provide a better understanding of the mechanism and occurrence of such an event and also intends to raise awareness of this rare presentation as an emergency condition requiring prompt surgical management.

Late Presentation of Xeroderma Pigmentosa With Squamous Cell Carcinoma in Septic Shock: Report of a Rare Case.

Xeroderma pigmentosum (XP) is a rare autosomal recessive pathology affecting nucleotide excision repair against ultraviolet radiation. This leads to an increased predisposition to developing ophthalmological, neurological, and cutaneous conditions with an increased cell turnover. This case reports a late presentation of XP presenting with metastatic squamous cell carcinoma (SCC) and septic shock in an eight-year-old Indian male. Emergency management with IV fluid boluses and broad-spectrum antibiotics showed no improvement in vitals. Urgent surgical debridement and tumor debulking failed to improve laboratory values. Postoperative leukocytosis with fever spikes warranted the need to transfer the patient to a super-specialty oncology unit. Such an adverse presentation is commonly seen in XP-related invasive squamous cell carcinoma. Preventive management requires early identification and a multidisciplinary approach involving dermatologists, ophthalmologists, and surgeons. Late presentations revolve around control of the disease process by sharp debridement and chemotherapy with regular surveillance as the lesions tend to reoccur even after excision and chemotherapy.

An Initial Evaluation of Human Plasma cMLC-1: A Potential Protein Biomarker for Trastuzumab-Induced Cardiotoxicity, Breast Cancer Screening and Progression.

Background: Trastuzumab is a targeted therapy for human epidermal growth factor receptor 2 (HER2)-positive breast cancer. However, trastuzumab-induced cardiotoxicity (TIC) has been reported when trastuzumab is administered to patients as a single agent or combined with anthracycline. Currently no means for detecting the early onset of TIC such as a protein biomarker is available. In this regard and based on promising results from a preliminary animal study, the potential of cardiac myosin light chain 1(cMLC-1) as a biomarker to predict TIC, screen patients for breast cancer and monitor tumor progression in breast cancer patients was evaluated. Methods: Archived plasma samples collected before and after trastuzumab treatment at various fixed time points from 15 HER2+ patients with or without cardiotoxicity, recently collected plasma samples from 79 breast cancer patients (40 HER2+, 39 HER2-), and 46 healthy donors were analyzed for cMLC-1 levels using an enzyme-linked immunosorbent assay (ELISA). Results: An elevated plasma cMLC-1 level was found to be associated with TIC in 3 out of 7 (43%) trastuzumab-treated HER2+ breast cancer patients. However, this study provided an opportunity for us to study plasma cMCL-1 levels in breast cancer patients. It was demonstrated that elevated plasma cMCL-1 is associated with breast cancer. The cutoff cMLC-1 concentration is estimated to be 44.99 ng/mL with a sensitivity of 59.49% (95%CI: 48.47%-69.63%) and specificity of 71.74% (95%CI: 57.45% -82.68%). We also found a noticeable but not significantly more elevated plasma cMCL-1 level in HER2- than in HER2+ breast cancer patients with the given sample sizes. As a result, improved sensitivity of 79.49% (95%CI: 64.47%-89.22%) with the specificity of 63.04% (95%CI:48.60%-75.48%) were obtained for cMLC-1 to predict HER2- breast cancer with the cutoff at 37.17 ng/mL. Moreover, this study determined that cMLC-1 level was significantly higher in patients with metastatic breast cancer than in patients with non-metastatic breast cancer. Conclusions: While the analysis of cMLC-1 levels in the plasma of a limited number of trastuzumab-treated HER2+ breast cancer patients failed to fully support its identification as a blood protein biomarker for predicting TIC, additional analyses of plasma cMLC-1 levels did significantly establish its correlations with breast cancer and disease progression. Our findings shed light on and filled, to some extent, the gap of knowledge of the potential of cMLC-1 as a blood protein biomarker for screening breast cancer and monitoring disease progression of breast cancer.

EGFR Inhibition Potentiates FGFR Inhibitor Therapy and Overcomes Resistance in FGFR2 Fusion-Positive Cholangiocarcinoma.

FGFR inhibitors are approved for the treatment of advanced cholangiocarcinoma harboring FGFR2 fusions. However, the response rate is moderate, and resistance emerges rapidly due to acquired secondary FGFR2 mutations or due to other less-defined mechanisms. Here, we conducted high-throughput combination drug screens, biochemical analysis, and therapeutic studies using patient-derived models of FGFR2 fusion-positive cholangiocarcinoma to gain insight into these clinical profiles and uncover improved treatment strategies. We found that feedback activation of EGFR signaling limits FGFR inhibitor efficacy, restricting cell death induction in sensitive models and causing resistance in insensitive models lacking secondary FGFR2 mutations. Inhibition of wild-type EGFR potentiated responses to FGFR inhibitors in both contexts, durably suppressing MEK/ERK and mTOR signaling, increasing apoptosis, and causing marked tumor regressions in vivo. Our findings reveal EGFR-dependent adaptive signaling as an important mechanism limiting FGFR inhibitor efficacy and driving resistance and support clinical testing of FGFR/EGFR inhibitor therapy for FGFR2 fusion-positive cholangiocarcinoma. SIGNIFICANCE: We demonstrate that feedback activation of EGFR signaling limits the effectiveness of FGFR inhibitor therapy and drives adaptive resistance in patient-derived models of FGFR2 fusion-positive cholangiocarcinoma. These studies support the potential of combination treatment with FGFR and EGFR inhibitors as an improved treatment for patients with FGFR2-driven cholangiocarcinoma. This article is highlighted in the In This Issue feature, p. 1171.

Cell-free DNA captures tumor heterogeneity and driver alterations in rapid autopsies with pre-treated metastatic cancer.

In patients with metastatic cancer, spatial heterogeneity of somatic alterations may lead to incomplete assessment of a cancer's mutational profile when analyzing a single tumor biopsy. In this study, we perform sequencing of cell-free DNA (cfDNA) and distinct metastatic tissue samples from ten rapid autopsy cases with pre-treated metastatic cancer. We show that levels of heterogeneity in genetic biomarkers vary between patients but that gene expression signatures representative of the tumor microenvironment are more consistent. Across nine patients with plasma samples available, we are able to detect 62/62 truncal and 47/121 non-truncal point mutations in cfDNA. We observe that mutation clonality in cfDNA is correlated with the number of metastatic lesions in which the mutation is detected and use this result to derive a clonality threshold to classify truncal and non-truncal driver alterations with reasonable specificity. In contrast, mutation truncality is more often incorrectly assigned when studying single tissue samples. Our results demonstrate the utility of a single cfDNA sample relative to that of single tissue samples when treating patients with metastatic cancer.

Temporal and spatial heterogeneity of host response to SARS-CoV-2 pulmonary infection.

The relationship of SARS-CoV-2 pulmonary infection and severity of disease is not fully understood. Here we show analysis of autopsy specimens from 24 patients who succumbed to SARS-CoV-2 infection using a combination of different RNA and protein analytical platforms to characterize inter-patient and intra-patient heterogeneity of pulmonary virus infection. There is a spectrum of high and low virus cases associated with duration of disease. High viral cases have high activation of interferon pathway genes and a predominant M1-like macrophage infiltrate. Low viral cases are more heterogeneous likely reflecting inherent patient differences in the evolution of host response, but there is consistent indication of pulmonary epithelial cell recovery based on napsin A immunohistochemistry and RNA expression of surfactant and mucin genes. Using a digital spatial profiling platform, we find the virus corresponds to distinct spatial expression of interferon response genes demonstrating the intra-pulmonary heterogeneity of SARS-CoV-2 infection.

Temporal and Spatial Heterogeneity of Host Response to SARS-CoV-2 Pulmonary Infection.

The relationship of SARS-CoV-2 lung infection and severity of pulmonary disease is not fully understood. We analyzed autopsy specimens from 24 patients who succumbed to SARS-CoV-2 infection using a combination of different RNA and protein analytical platforms to characterize inter- and intra- patient heterogeneity of pulmonary virus infection. There was a spectrum of high and low virus cases that was associated with duration of disease and activation of interferon pathway genes. Using a digital spatial profiling platform, the virus corresponded to distinct spatial expression of interferon response genes and immune checkpoint genes demonstrating the intra-pulmonary heterogeneity of SARS-CoV-2 infection.

PTEN Loss Mediates Clinical Cross-Resistance to CDK4/6 and PI3Kα Inhibitors in Breast Cancer.

The combination of CDK4/6 inhibitors with antiestrogen therapies significantly improves clinical outcomes in ER-positive advanced breast cancer. To identify mechanisms of acquired resistance, we analyzed serial biopsies and rapid autopsies from patients treated with the combination of the CDK4/6 inhibitor ribociclib with letrozole. This study revealed that some resistant tumors acquired RB loss, whereas other tumors lost PTEN expression at the time of progression. In breast cancer cells, ablation of PTEN, through increased AKT activation, was sufficient to promote resistance to CDK4/6 inhibition in vitro and in vivo. Mechanistically, PTEN loss resulted in exclusion of p27 from the nucleus, leading to increased activation of both CDK4 and CDK2. Because PTEN loss also causes resistance to PI3Kα inhibitors, currently approved in the post-CDK4/6 setting, these findings provide critical insight into how this single genetic event may cause clinical cross-resistance to multiple targeted therapies in the same patient, with implications for optimal treatment-sequencing strategies. SIGNIFICANCE: Our analysis of serial biopsies uncovered RB and PTEN loss as mechanisms of acquired resistance to CDK4/6 inhibitors, utilized as first-line treatment for ER-positive advanced breast cancer. Importantly, these findings have near-term clinical relevance because PTEN loss also limits the efficacy of PI3Kα inhibitors currently approved in the post-CDK4/6 setting.This article is highlighted in the In This Issue feature, p. 1.

Author Correction: Liquid versus tissue biopsy for detecting acquired resistance and tumor heterogeneity in gastrointestinal cancers.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Liquid versus tissue biopsy for detecting acquired resistance and tumor heterogeneity in gastrointestinal cancers.

During cancer therapy, tumor heterogeneity can drive the evolution of multiple tumor subclones harboring unique resistance mechanisms in an individual patient1-3. Previous case reports and small case series have suggested that liquid biopsy (specifically, cell-free DNA (cfDNA)) may better capture the heterogeneity of acquired resistance4-8. However, the effectiveness of cfDNA versus standard single-lesion tumor biopsies has not been directly compared in larger-scale prospective cohorts of patients following progression on targeted therapy. Here, in a prospective cohort of 42 patients with molecularly defined gastrointestinal cancers and acquired resistance to targeted therapy, direct comparison of postprogression cfDNA versus tumor biopsy revealed that cfDNA more frequently identified clinically relevant resistance alterations and multiple resistance mechanisms, detecting resistance alterations not found in the matched tumor biopsy in 78% of cases. Whole-exome sequencing of serial cfDNA, tumor biopsies and rapid autopsy specimens elucidated substantial geographic and evolutionary differences across lesions. Our data suggest that acquired resistance is frequently characterized by profound tumor heterogeneity, and that the emergence of multiple resistance alterations in an individual patient may represent the 'rule' rather than the 'exception'. These findings have profound therapeutic implications and highlight the potential advantages of cfDNA over tissue biopsy in the setting of acquired resistance.