Comparative Molecular Analysis of Primary Central Nervous System Lymphomas and Matched Vitreoretinal Lymphomas by Vitreous Liquid Biopsy

Primary Central Nervous System Lymphoma (PCNSL) is a lymphoid malignancy of the brain that occurs in ~1500 patients per year in the US. PCNSL can spread to the vitreous and retina, where it is known as vitreoretinal lymphoma (VRL). While confirmatory testing for diagnosis is dependent on invasive brain tissue or cerebrospinal fluid sampling, the ability to access the vitreous as a proximal biofluid for liquid biopsy to diagnose PCNSL is an attractive prospect given ease of access and minimization of risks and complications from other biopsy strategies. However, the extent to which VRL, previously considered genetically identical to PCNSL, resembles PCNSL in the same individual with respect to genetic alterations, diagnostic strategies, and precision-medicine based approaches has hitherto not been explored. Furthermore, the degree of intra-patient tumor genomic heterogeneity between the brain and vitreous sites has not been studied. In this work, we report on targeted DNA next-generation sequencing (NGS) of matched brain and vitreous samples in two patients who each harbored VRL and PCSNL. Our strategy showed enhanced sensitivity for molecular diagnosis confirmation over current clinically used vitreous liquid biopsy methods. We observed a clonal relationship between the eye and brain samples in both patients, which carried clonal CDKN2A deep deletions, a highly recurrent alteration in VRL patients, as well as MYD88 p.L265P activating mutation in one patient. Several subclonal alterations, however, in the genes SETD2, BRCA2, TERT, and broad chromosomal regions showed heterogeneity between the brain and the eyes, between the two eyes, and among different regions of the PCNSL brain lesion. Taken together, our data show that NGS of vitreous liquid biopsies in PCNSL patients with VRL highlights shared and distinct genetic alterations that suggest a common origin for these lymphomas, but with additional site-specific alterations. Liquid biopsy of VRL accurately replicates the findings for PCNSL truncal (tumor-initiating) genomic alterations; it can also nominate precision medicine interventions and shows intra-patient heterogeneity in subclonal alterations. To the best of our knowledge, this study represents the first interrogation of genetic underpinnings of PCNSL with matched VRL samples. Our findings support continued investigation into the utility of vitreous liquid biopsy in precision diagnosis and treatment of PCNSL/VRL.     

Improving the Management of Endometrial Cancer Patients through the Use of Liquid Biopsy Analyses: A Case Report

Endometrial cancer (EC) is the 4th most common neoplasm of the female genital tract, with 15–20% of patients being of high risk of recurrence which leads to a significant decrease in patient survival. Current therapeutic options for patients with EC are poor, being the combined therapy of carboplatin and paclitaxel the standard of care, with limited efficacy. Therefore, new therapeutic options and better monitoring tools are needed to improve the management of the disease. In the current case report, we showcase the value of liquid biopsy analyses in a microsatellite instability EC patient with initially good prognosis that however underwent rapid progression disease within 6 months post-surgery; through the study of plasma cfDNA/ctDNA dynamics to assess the tumour evolution during treatment, as well as the study of the uterine aspirate as a valuable sample that captures the intra-tumour heterogeneity that allows a comprehensive genomic profiling of the disease to identify potential therapeutic options. Furthermore, preclinical models were generated at the time of tumour progression to assess the efficacy of the identified targeted therapies.     

Extracellular Vesicles in Diffuse Large B Cell Lymphoma: Characterization and Diagnostic Potential

Diffuse large B cell lymphoma (DLBCL) is an aggressive B cell lymphoma characterized by a heterogeneous behavior and in need of more accurate biological characterization monitoring and prognostic tools. Extracellular vesicles are secreted by all cell types and are currently established to some extent as representatives of the cell of origin. The present study characterized and evaluated the diagnostic and prognostic potential of plasma extracellular vesicles (EVs) proteome in DLBCL by using state-of-the-art mass spectrometry. The EV proteome is strongly affected by DLBCL status, with multiple proteins uniquely identified in the plasma of DLBCL. A proof-of-concept classifier resulted in highly accurate classification with a sensitivity and specificity of 1 when tested on the holdout test data set. On the other hand, no proteins were identified to correlate with non-germinal center B-cell like (non-GCB) or GCB subtypes to a significant degree after correction for multiple testing. However, functional analysis suggested that antigen binding is regulated when comparing non-GCB and GCB. Survival analysis based on protein quantitative values and clinical parameters identified multiple EV proteins as significantly correlated to survival. In conclusion, the plasma extracellular vesicle proteome identifies DLBCL cancer patients from healthy donors and contains potential EV protein markers for prediction of survival.     

Potential of Aqueous Humor as a Liquid Biopsy for Uveal Melanoma

Tumor biopsy can identify prognostic biomarkers for metastatic uveal melanoma (UM), however aqueous humor (AH) liquid biopsy may serve as an adjunct. This study investigated whether the AH of UM eyes has sufficient circulating tumor DNA (ctDNA) to perform genetic analysis. This is a case series of 37 AH samples, taken before or after radiation, and one tumor wash sample, from 12 choroidal and 8 ciliary body (CB) melanoma eyes. AH was analyzed for nucleic acid concentrations. AH DNA and one tumor wash sample underwent shallow whole-genome sequencing followed by Illumina sequencing to detect somatic copy number alterations (SCNAs). Four post-radiation AH underwent targeted sequencing of BAP1 and GNAQ genes. Post-radiation AH had significantly higher DNA and miRNA concentrations than paired pre-radiation samples. Highly recurrent UM SCNAs were identified in 0/11 post-radiation choroidal and 6/8 post-radiation CB AH. SCNAs were highly concordant in a CB post-radiation AH with its matched tumor (r = 0.978). BAP1 or GNAQ variants were detected in 3/4 post-radiation AH samples. AH is a source of ctDNA in UM eyes, particularly in post-radiation CB eyes. For the first time, UM SCNAs and mutations were identified in AH-derived ctDNA. Suggesting that AH can serve as a liquid biopsy for UM.     

A Liquid Biopsy in Bladder Cancer—The Current Landscape in Urinary Biomarkers

The non-muscle invasive bladder cancer tends to recur and progress. Therefore, it requires frequent follow-ups, generating costs and making it one of the most expensive neoplasms. Considering the expensive and invasive character of the current gold-standard diagnostic procedure, white-light cystoscopy, efforts to find an alternative method are ongoing. Although the last decade has seen significant advancements in urinary biomarker tests (UBTs) for bladder cancer, international guidelines have not recommended them. Currently, the paramount urgency is to find and validate the test with the best specificity and sensitivity, which would allow for the optimizing of diagnosis, prognosis, and a treatment plan. This review aims to summarise the up-to-date state of knowledge relating to UBTs and new developments in the detection, prognosis, and surveillance of bladder cancer and their potential applications in clinical practice.     

Characterization of BCMA Expression in Circulating Rare Single Cells of Patients with Plasma Cell Neoplasms

B-cell maturation antigen (BCMA), a key regulator of B-cell proliferation and survival, is highly expressed in almost all cases of plasma cell neoplasms and B-lymphoproliferative malignancies. BCMA is a robust biomarker of plasma cells and a therapeutic target with substantial clinical significance. However, the expression of BCMA in circulating tumor cells of patients with hematological malignancies has not been validated for the detection of circulating plasma and B cells. The application of BCMA as a biomarker in single-cell detection and profiling of circulating tumor cells in patients’ blood could enable early disease profiling and therapy response monitoring. Here, we report the development and validation of a slide-based immunofluorescence assay (i.e., CD138, BCMA, CD45, DAPI) for enrichment-free detection, quantification, and morphogenomic characterization of BCMA-expressing cells in patients (N = 9) with plasma cell neoplasms. Varying morphological subtypes of circulating BCMA-expressing cells were detected across the CD138(+/−) and CD45(+/−) compartments, representing candidate clonotypic post-germinal center B cells, plasmablasts, and both normal and malignant plasma cells. Genomic analysis by single-cell sequencing and correlation to clinical FISH cytogenetics provides validation, with data showing that patients across the different neoplastic states carry both normal and altered BCMA-expressing cells. Furthermore, altered cells harbor cytogenetic events detected by clinical FISH. The reported enrichment-free liquid biopsy approach has potential applications as a single-cell methodology for the early detection of BCMA+ B-lymphoid malignancies and in monitoring therapy response for patients undergoing anti-BCMA treatments.     

T315 Decreases Acute Myeloid Leukemia Cell Viability through a Combination of Apoptosis Induction and Autophagic Cell Death

T315, an integrin-linked kinase (ILK) inhibitor, has been shown to suppress the proliferation of breast cancer, stomach cancer and chronic lymphocytic leukemia cells. Here we demonstrate that T315 decreases cell viability of acute myeloid leukemia (AML) cell lines (HL-60 and THP-1) and primary leukemia cells from AML patients in a dose-responsive manner. Normal human bone marrow cells are less sensitive than leukemia cells to T315. T315 down regulates protein kinase B (Akt) and p-Akt and induces caspase activation, poly-ADP-ribose polymerase (PARP) cleavage, apoptosis and autophagy through an ILK-independent manner. Interestingly, pretreatment with autophagy inhibitors rescues cells from apoptosis and concomitant PARP cleavage, which implicates a key role of autophagic cell death in T315-mediated cytotoxicity. T315 also demonstrates efficacy in vivo, suppressing the growth of THP-1 xenograft tumors in athymic nude mice when administered intraperitoneally. This study shows that autophagic cell death and apoptosis cooperatively contribute to the anticancer activity of T315 in AML cells. In conclusion, the complementary roles of apoptotic and autophagic cell death should be considered in the future assessment of the translational value of T315 in AML therapy.     

Berberine Delays Onset of Collagen-Induced Arthritis through T Cell Suppression

There is evidence that berberine (BBR), a clinically relevant plant compound, ameliorates clinically apparent collagen-induced arthritis (CIA) in vivo. However, to date, there are no studies involving the use of BBR which explore its prophylactic potential in this model of rheumatoid arthritis (RA). The aim of this study was to determine if prophylactic BBR use during the preclinical phase of collagen-induced arthritis would delay arthritic symptom onset, and to characterize the cellular mechanism underlying such an effect. DBA/1J mice were injected with an emulsion of bovine type II collagen (CII) and complete Freund’s adjuvant (day 0) and a booster injection of CII in incomplete Freund’s adjuvant (day 18) to induce arthritis. Mice were then given i.p. injections of 1 mg/kg/day of BBR or PBS (vehicle with 0.01% DMSO) from days 0 to 28, were left untreated (CIA control), or were in a non-arthritic control group (n = 15 per group). Incidence of arthritis in BBR-treated mice was 50%, compared to 90% in both the CIA and PBS controls. Populations of B and T cells from the spleens and draining lymph nodes of mice were examined on day 14 (n = 5 per group) and day 28 (n = 10 per group). BBR-treated mice had significantly reduced populations of CD4⁺T_h and CD4⁺CXCR5⁺ T_fh cells, and an increased proportion of Foxp3⁺ T_reg at days 14 and 28, as well as reduced expression of co-stimulatory molecules CD28 and CD154 at both endpoints. The effect seen on T cell populations and co-stimulatory molecule expression in BBR-treated mice was not mirrored in CD19⁺ B cells. Additionally, BBR-treated mice experienced reduced anti-CII IgG2a and anti-CII total IgG serum concentrations. These results indicate a potential role for BBR as a prophylactic supplement for RA, and that its effect may be mediated specifically through T cell suppression. However, the cellular effector involved raises concern for BBR prophylactic use in the context of vaccine efficacy and other primary adaptive immune responses.     

Liquid Biopsy as a Source of Nucleic Acid Biomarkers in the Diagnosis and Management of Lynch Syndrome

Lynch syndrome (LS) is an autosomal dominant inherited cancer predisposition disorder, which may manifest as colorectal cancer (CRC), endometrial cancer (EC) or other malignancies of the gastrointestinal and genitourinary tract as well as the skin and brain. Its genetic cause is a defect in one of the four key DNA mismatch repair (MMR) loci. Testing of patients at risk is currently based on the absence of MMR protein staining and detection of mutations in cancer tissue and the germline, microsatellite instability (MSI) and the hypermethylated state of the MLH1 promoter. If LS is shown to have caused CRC, lifetime follow-up with regular screening (most importantly, colonoscopy) is required. In recent years, DNA and RNA markers extracted from liquid biopsies have found some use in the clinical diagnosis of LS. They have the potential to greatly enhance the efficiency of the follow-up process by making it minimally invasive, reproducible, and time effective. Here, we review markers reported in the literature and their current clinical applications, and we comment on possible future directions.     

Tracking the Molecular Fingerprint of Head and Neck Cancer for Recurrence Detection in Liquid Biopsies

The 5-year relative survival for patients with head and neck cancer, the seventh most common form of cancer worldwide, was reported as 67% in developed countries in the second decade of the new millennium. Although surgery, radiotherapy, chemotherapy, or combined treatment often elicits an initial satisfactory response, relapses are frequently observed within two years. Current surveillance methods, including clinical exams and imaging evaluations, have not unambiguously demonstrated a survival benefit, most probably due to a lack of sensitivity in detecting very early recurrence. Recently, liquid biopsy monitoring of the molecular fingerprint of head and neck squamous cell carcinoma has been proposed and investigated as a strategy for longitudinal patient care. These innovative methods offer rapid, safe, and highly informative genetic analysis that can identify small tumors not yet visible by advanced imaging techniques, thus potentially shortening the time to treatment and improving survival outcomes. In this review, we provide insights into the available evidence that the molecular tumor fingerprint can be used in the surveillance of head and neck squamous cell carcinoma. Challenges to overcome, prior to clinical implementation, are also discussed.     

Size and Methylation Index of Cell-Free and Cell-Surface-Bound DNA in Blood of Breast Cancer Patients in the Contest of Liquid Biopsy

Aberrantly methylated circulating DNA (cirDNA) has proven to be a good cancer marker, but its detection is limited by low concentrations, fragmentation, and insufficiency. Since the methylated cirDNA was shown to be more stable in circulation than the unmethylated one and was shown to bind with the blood cell surface, we studied the concentration, representation, and fragmentation of tumor-derived methylated DNA in cell-free and cell-surface-associated DNA. We found that long DNA fragments (more than 10 kb) are mainly associated with the surface of blood cells. However, in plasma short DNA fragments (100–1000 bp) were also found along with long DNA fragments. Isolation of short fragments after separation of cirDNA in 6% PAGE followed by quantitative PCR (L1 element) has shown that short DNA fragments in healthy females represent 22% versus 0.5–4.4% in breast cancer patients. The methylated form of the RARβ2 gene was detected only in long DNA fragments by Real-time TaqMan PCR of bisulfite-converted DNA. The methylation index of cirDNA from healthy women was estimated at 0%, 9%, and 7% in plasma, PBS-EDTA, and trypsin eluates from the surface of blood cells, respectively. The methylation index of breast cancer patients’ DNA was found to be 33%, 15%, and 61% in the same fractions confirming the overrepresentation of methylated DNA in csbDNA.     

MCAM/MUC18/CD146 as a Multifaceted Warning Marker of Melanoma Progression in Liquid Biopsy

Human malignant melanoma shows a high rate of mortality after metastasization, and its incidence is continuously rising worldwide. Several studies have suggested that MCAM/MUC18/CD146 plays an important role in the progression of this malignant disease. MCAM/MUC18/CD146 is a typical single-spanning transmembrane glycoprotein, existing as two membrane isoforms, long and short, and an additional soluble form, sCD146. We previously documented that molecular MCAM/MUC18/CD146 expression is strongly associated with disease progression. Recently, we showed that MCAM/MUC18/CD146 and ABCB5 can serve as melanoma-specific-targets in the selection of highly primitive circulating melanoma cells, and constitute putative proteins associated with disease spreading progression. Here, we analyzed CD146 molecular expression at onset or at disease recurrence in an enlarged melanoma case series. For some patients, we also performed the time courses of molecular monitoring. Moreover, we explored the role of soluble CD146 in different cohorts of melanoma patients at onset or disease progression, rather than in clinical remission, undergoing immune therapy or free from any clinical treatment. We showed that MCAM/MUC18/CD146 can be considered as: (1) a membrane antigen suitable for identification and enrichment in melanoma liquid biopsy; (2) a highly effective molecular “warning” marker for minimal residual disease monitoring; and (3) a soluble protein index of inflammation and putative response to therapeutic treatments.     

Altered Circulating Follicular T Helper Cell Subsets and Follicular T Regulatory Cells Are Indicators of a Derailed B Cell Response in Lupus,Which Could Be Modified by Targeting IL-21R

Systemic lupus erythematosus (SLE) is characterized by the breakdown of self-tolerance, the production of high-affinity pathogenic autoantibodies and derailed B cell responses, which indicates the importance of central players, such as follicular T helper (T_FH) subsets and follicular T regulatory (T_FR) cells, in the pathomechanism of the disease. In this study, we aimed to analyze the distribution of the circulating counterparts of these cells and their association with disease characteristics and B cell disproportions in SLE. We found that the increased percentage of activated circulating T_FH (cT_FH) and cT_FR cells was more pronounced in cutaneous lupus; however, among cT_FH subsets, the frequency of cT_FH17 cells was decreased in patients with lupus nephritis. Furthermore, the decreased proportion of cT_FH17 cells was associated with low complement C4 levels and high disease activity scores. We also investigated whether the blocking of the IL-21 receptor (IL-21R) with an anti-IL-21R monoclonal antibody inhibits the B cell response, since IL-21 primarily produced by T_FH cells potentially promotes humoral immunity. We observed that anti-IL-21R inhibited plasmablast generation and immunoglobulin production. Our study demonstrated that, besides cT_FR/cT_FH imbalance, cT_FH17 cells play a crucial role in SLE pathogenesis, and modulating cT_FH-B cell interaction through the IL-21/IL-21R pathway may be a promising therapeutic strategy to suppress the pathological B cell response.     

Identification of miR-20a-5p as Robust Normalizer for Urine microRNA Studies in Renal Cell Carcinoma and a Profile of Dysregulated microRNAs

Renal cell carcinoma (RCC) is the third most frequent urinary malignancy and one of the most lethal. Current diagnostic and follow-up techniques are harmful and unspecific in low-grade tumors. Novel minimally invasive markers such as urine microRNAs (miRNAs) are under study. However, discrepancies arise among studies in part due to lack of consent regarding normalization. We aimed to identify the best miRNA normalizer for RCC studies performed in urine samples together with a miRNA profile with diagnostic value and another for follow-up. We evaluated the performance of 120 candidate miRNAs in the urine of 16 RCC patients and 16 healthy controls by RT-qPCR followed by a stability analysis with RefFinder. In this screening stage, miR-20a-5p arose as the most stably expressed miRNA in RCC and controls, with a good expression level. Its stability was validated in an independent cohort of 51 RCC patients and 32 controls. Using miR-20a-5p as normalizer, we adjusted and validated a diagnostic model for RCC with three miRNAs (miR-200a-3p, miR-34a-5p and miR-365a-3p) (AUC = 0.65; Confidence Interval 95% [0.51, 0.79], p = 0.043). let-7d-5p and miR-205-5p were also upregulated in patients compared to controls. Comparing RCC samples before surgery and fourteen weeks after, we identified let-7d-5p, miR-152-3p, miR-30c-5p, miR-362-3p and miR-30e-3p as potential follow-up profile for RCC. We identified validated targets of most miRNAs in the renal cell carcinoma pathway. This is the first study that identifies a robust normalizer for urine RCC miRNA studies, miR-20a-5p, which may allow the comparison of future studies among laboratories. Once confirmed in a larger independent cohort, the miRNAs profiles identified may improve the non-invasive diagnosis and follow-up of RCC.     

TP53 Targeted Deep Sequencing of Cell-Free DNA in Esophageal Squamous Cell Carcinoma Using Low-Quality Serum: Concordance with Tumor Mutation

Circulating cell-free DNA (cfDNA) is emerging as a potential tumor biomarker. CfDNA-based biomarkers may be applicable in tumors without an available non-invasive screening method among at-risk populations. Esophageal squamous cell carcinoma (ESCC) and residents of the Asian cancer belt are examples of those malignancies and populations. Previous epidemiological studies using cfDNA have pointed to the need for high volumes of good quality plasma (i.e., >1 mL plasma with 0 or 1 cycles of freeze-thaw) rather than archival serum, which is often the main available source of cfDNA in retrospective studies. Here, we have investigated the concordance of TP53 mutations in tumor tissue and cfDNA extracted from archival serum left-over from 42 cases and 39 matched controls (age, gender, residence) in a high-risk area of Northern Iran (Golestan). Deep sequencing of TP53 coding regions was complemented with a specialized variant caller (Needlestack). Overall, 23% to 31% of mutations were concordantly detected in tumor and serum cfDNA (based on two false discovery rate thresholds). Concordance was positively correlated with high cfDNA concentration, smoking history (p-value = 0.02) and mutations with a high potential of neoantigen formation (OR; 95%CI = 1.9 (1.11–3.29)), suggesting that tumor DNA release in the bloodstream might reflect the effects of immune and inflammatory context on tumor cell turnover. We identified TP53 mutations in five controls, one of whom was subsequently diagnosed with ESCC. Overall, the results showed that cfDNA mutations can be reliably identified by deep sequencing of archival serum, with a rate of success comparable to plasma. Nonetheless, 70% non-identifiable mutations among cancer patients and 12% mutation detection in controls are the main challenges in applying cfDNA to detect tumor-related variants when blindly targeting whole coding regions of the TP53 gene in ESCC.     

Molecular Characterization of a Rare Case of Monozygotic Dichorionic Diamniotic Twin Pregnancy after Single Blastocyst Transfer in Preimplantation Genetic Testing (PGT)

Preimplantation genetic testing (PGT) is widely used to select unaffected embryos, increasing the odds of having a healthy baby. During the last few decades, it was accepted that monozygotic dichorionic diamniotic twin pregnancies occurred from the embryo splitting before Day 3 postfertilization according to Corner’s dogma. Hence, the occurrence of a dichorionic diamniotic twin pregnancy after a single blastocyst transfer was considered a dizygotic pregnancy resulting from blastocyst transfer and concurrent natural fertilization. In our study, we have provided for the first time molecular proof that a single blastocyst transfer can result in a monozygotic dichorionic diamniotic twin pregnancy, invalidating Corner’s dogma. In this case, we recommend systematically assessing the genetic status of dichorionic twins after single blastocyst transfer using prenatal diagnosis to exclude the risk from a potential concurrent spontaneous pregnancy and to ensure that both fetuses are unaffected. To achieve this goal, we have developed here an innovative noninvasive prenatal diagnosis by exclusion of paternal variants with droplet digital PCR, maximizing the reliability of genetic diagnosis. Further multicentric prospective studies using genetic testing are now required to establish the rate of blastocyst splitting leading to dichorionic pregnancy in PGT and to identify the risk factors.     

Successful Treatment of Persistent SARS-CoV-2 Infection in a B-Cell Depleted Patient with Activated Cytotoxic T and NK Cells: A Case Report

We report a lymphoma patient with profound B-cell deficiency after chemotherapy combined with anti-CD20 antibody successfully treated with remdesivir and convalescent plasma for prolonged SARS-CoV-2 infection. Viral clearance was likely attributed to the robust expansion and activation of TCR Vβ2 CD8+ cytotoxic T cells and CD16 + CD56- NK cells. This is the first presentation of TCR-specific T cell oligoclonal response in COVID-19. Our study suggests that B-cell depleted patients may effectively respond to anti-SARS-CoV-2 treatment when NK and antigen-specific Tc cell response is induced.