What's new in molecular genetic pathology 2022: immune checkpoint inhibitor biomarkers and select solid tumors.

Predictive biomarker testing plays a critical role in targeted immuno-oncology, including the use of immune checkpoint inhibitors (ICI) for various solid tumors. Molecular advancements in cancers of the breast, kidney and brain have continued to propel tumor classification and precision therapy.

What's new in molecular genetic pathology 2021: solid tumors and NGS panel selection.

The linchpin of precision medicine is molecular genetic and genomic testing. Molecular biomarkers are important for establishing precise diagnoses and for predicting therapeutic responses that enable cancer patients to receive personalized and targeted treatment. Below are highlights of the current considerations in next generation sequencing (NGS) panel selection, and in molecular testing of solid tumors of the lung, digestive system, thyroid and soft tissue.

Identification of Antifungal Compounds against Multidrug-Resistant Candida auris Utilizing a High-Throughput Drug-Repurposing Screen.

Candida auris is an emerging fatal fungal infection that has resulted in several outbreaks in hospitals and care facilities. Current treatment options are limited by the development of drug resistance. Identification of new pharmaceuticals to combat these drug-resistant infections will thus be required to overcome this unmet medical need. We have established a bioluminescent ATP-based assay to identify new compounds and potential drug combinations showing effective growth inhibition against multiple strains of multidrug-resistant Candida auris The assay is robust and suitable for assessing large compound collections by high-throughput screening (HTS). Utilizing this assay, we conducted a screen of 4,314 approved drugs and pharmacologically active compounds that yielded 25 compounds, including 6 novel anti-Candida auris compounds and 13 sets of potential two-drug combinations. Among the drug combinations, the serine palmitoyltransferase inhibitor myriocin demonstrated a combinational effect with flucytosine against all tested isolates during screening. This combinational effect was confirmed in 13 clinical isolates of Candida auris.

MEPicides: potent antimalarial prodrugs targeting isoprenoid biosynthesis.

The emergence of Plasmodium falciparum resistant to frontline therapeutics has prompted efforts to identify and validate agents with novel mechanisms of action. MEPicides represent a new class of antimalarials that inhibit enzymes of the methylerythritol phosphate (MEP) pathway of isoprenoid biosynthesis, including the clinically validated target, deoxyxylulose phosphate reductoisomerase (Dxr). Here we describe RCB-185, a lipophilic prodrug with nanomolar activity against asexual parasites. Growth of P. falciparum treated with RCB-185 was rescued by isoprenoid precursor supplementation, and treatment substantially reduced metabolite levels downstream of the Dxr enzyme. In addition, parasites that produced higher levels of the Dxr substrate were resistant to RCB-185. Notably, environmental isolates resistant to current therapies remained sensitive to RCB-185, the compound effectively treated sexually-committed parasites, and was both safe and efficacious in malaria-infected mice. Collectively, our data demonstrate that RCB-185 potently and selectively inhibits Dxr in P. falciparum, and represents a promising lead compound for further drug development.

Preparation and Evaluation of Potent Pentafluorosulfanyl-Substituted Anti-Tuberculosis Compounds.

The global fight to stop tuberculosis (TB) remains a great challenge, particularly with the increase in drug-resistant strains and a lack of funding to support the development of new treatments. To bolster a precarious drug pipeline, we prepared a focused panel of eight pentafluorosulfanyl (SF5 ) compounds which were screened for their activity against Mycobacterium tuberculosis (Mtb) H37Rv in three different assay conditions and media. All eight compounds had sub-micromolar potency, and four displayed MICs <100 nm. Seven compounds were evaluated against non-replicating and mono-drug-resistant Mtb, and for their ability to inhibit Mtb within the macrophage. The greatest potency was observed against intracellular Mtb (MIC <10 nm for three compounds), which is often the most challenging to target. In general, the SF5 -bearing compounds were very similar to their CF3 counterparts, with the major differences observed being their in vitro ADME properties. Two SF5 -bearing compounds were found to have greater protein binding than their corresponding CF3 counterparts, but were also less metabolized in human microsomes, resulting in longer half-lives.

SAR and identification of 2-(quinolin-4-yloxy)acetamides as Mycobacterium tuberculosis cytochrome bc1 inhibitors.

A previous phenotypic screen by GSK identified 2-(quinolin-4-yloxy)acetamides as potent growth inhibitors of Mycobacterium tuberculosis (Mtb). We report the results of a preliminary structure-activity relationship (SAR) study of the compound class which has yielded more potent inhibitors. An Mtb cytochrome bd oxidase deletion mutant (cydKO) was found to be hypersensitive to most members of the compound library, while strains carrying single-nucleotide polymorphisms of the qcrB gene, which encodes a subunit of the menaquinol cytochrome c oxidoreductase (bc1) complex, were resistant to the library. These results identify that the 2-(quinolin-4-yloxy)acetamide class of Mtb growth inhibitors can be added to the growing number of scaffolds that target the M. tuberculosis bc1 complex.

Learning curve for endobronchial ultrasound­guided transbronchial needle aspiration: experience of a community­based teaching hospital.

Endobronchial ultrasound­guided transbronchial needle aspiration (EBUS­TBNA) is a minimally invasive procedure. This procedure is useful for nodal staging of lung cancer and evaluating mediastinal lymphoma and granuloma. The present study was a retrospective analysis of our experience when EBUS­TBNA was initially implemented. A total of 112 lymph nodes/masses (51 patients) were divided into two groups: The first and second 8 months. In the first group, 33 lymph nodes/masses (16 patients) were biopsied and tumor diagnoses were made in 9% of the cases (three lymph nodes/masses). The material was adequate to produce a cell block for microscopic analysis in 42% of cases. Subsequent tissue diagnoses were available in 50% of cases. Only one of the three malignant EBUS­TBNA diagnoses (33%) was confirmed by histological examination. In the second 8 months, 79 lymph nodes (35 patients) were sampled. Tumor/granuloma diagnoses were achieved in 27% of the cases (21 nodes) (P=0.045 versus the first 8 months) and the obtained material was adequate for producing a cell block in 90% of cases (P<0.001 versus the first 8 months). Corresponding tissue diagnoses were available in 28% of cases. Correlation of EBUS-TBNA and histological examination for tumor/granuloma diagnosis was 100% (12/12, P=0.029 versus the first 8 months). Immunostains in the cell blocks indicated that all the metastatic adenocarcinomas were thyroid transcription factor­1 (TTF­1)+ and p63­, and that all squamous cell carcinomas were TTF­1­, p63+ and cytokeratin 5/6 (CK5/6)+. Eight granulomata were identified, of which five were positive for Acid­Fast Bacilli (AFB) stain and confirmed by culture or tissue biopsy. The remaining three granulomata were AFB­negative. EGFR/KRAS mutation analysis was conducted in cell blocks of five adenocarcinomas, of which all provided sufficient diagnostic material. The findings showed a steep learning curve when EBUS­TBNA was first adopted, reflected by an increased rate of tumor/granuloma diagnoses as well as an improved sample yield for cell block preparation in the second 8 months. TTF­1, p63 and CK5/6 were useful biomarkers for distinguishing metastatic lung carcinomas.

Respiratory flexibility in response to inhibition of cytochrome C oxidase in Mycobacterium tuberculosis.

We report here a series of five chemically diverse scaffolds that have in vitro activities on replicating and hypoxic nonreplicating bacilli by targeting the respiratory bc1 complex in Mycobacterium tuberculosis in a strain-dependent manner. Deletion of the cytochrome bd oxidase generated a hypersusceptible mutant in which resistance was acquired by a mutation in qcrB. These results highlight the promiscuity of the bc1 complex and the risk of targeting energy metabolism with new drugs.

Concomitant Kaposi sarcoma and multicentric Castleman's disease in a heart transplant recipient.

Post-transplant human herpes virus -8 (HHV-8)/Kaposi sarcoma herpes virus (KSHV) infection is associated with neoplastic and non-neoplastic diseases. Kaposi sarcoma (KS), multicentric Castleman's disease (MCD), and primary effusion lymphomas (PEL) are the most common HHV-8-associated neoplastic complications described in solid organ transplant (SOT) patients. Concurrent KS and MCD have been previously described after transplantation only twice - once after liver transplantation and once after renal transplantation. We describe a unique heart transplant patient who also developed concurrent KS and MCD. To our knowledge this is the first documented case of a heart transplant recipient presenting with these two HHV-8-mediated complications at the same time.

Follow-up of breast papillary lesion on core needle biopsy: experience in African-American population.

BACKGROUND: The optimal course of clinical follow-up after a diagnosis of breast papillary lesion on a core needle biopsy (CNB) remains elusive. In particular, no reports in literature have addressed this question in African-American population. We describe our experience with breast papillary lesions in a primarily African-American population. METHODS: A search of our database for breast papillary lesions diagnosed on CNB between September 2002 and September 2012 was conducted. Cases were categorized into benign, atypical, and malignant. CK5/6 and CK903 stains were performed when necessary. RESULTS: A total of 64 breast papillary lesions were diagnosed on CNB, including 55 (86%) benign papillary lesions, 6 (9%) atypical lesions, and 3 (5%) intraductal papillary carcinomas. Of these 64 patients, 29 patients (25 African-Americans, 3 Hispanics, 1 Asian American) underwent lumpectomy within 6 months after CNB. Pathology of the lumpectomy showed: five of the 25 (20%) benign papillary lesions on needle biopsy were upgraded to intraductal or invasive papillary carcinoma; 2 of the 3 atypical papillary lesion cases on core biopsy were upgraded (67%), one into intraductal papillary carcinoma, the other invasive papillary carcinoma; the only case of malignant papillary lesion on CNB remained as intraductal papillary carcinoma on lumpectomy. The rate of upgrade in lumpectomy/mastectomy was 25%. CK5/6 and CK903 immunostains were performed on all seven core needle biopsies that were later upgraded. CONCLUSIONS: In our predominantly African-American urban population, 25% of benign or atypical papillary lesions diagnosed on CNB was upgraded in the final excisional examination. Early excision of all papillary lesions diagnosed on CNB may be justified in this patient population. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/7950117821177201.

The brain microenvironment preferentially enhances the radioresistance of CD133(+) glioblastoma stem-like cells.

Brain tumor xenografts initiated from glioblastoma (GBM) CD133(+) tumor stem-like cells (TSCs) are composed of TSC and non-TSC subpopulations, simulating the phenotypic heterogeneity of GBMs in situ. Given that the discrepancies between the radiosensitivity of GBM cells in vitro and the treatment response of patients suggest a role for the microenvironment in GBM radioresistance, we compared the response of TSCs and non-TSCs irradiated under in vitro and orthotopic conditions. As a measure of radioresponse determined at the individual cell level, γH2AX and 53BP1 foci were quantified in CD133(+) cells and their differentiated (CD133(-)) progeny. Under in vitro conditions, no difference was detected between CD133(+) and CD133(-) cells in foci induction or dispersal after irradiation. However, irradiation of orthotopic xenografts initiated from TSCs resulted in the induction of fewer γH2AX and 53BP1 foci in CD133(+) cells compared to their CD133(-) counterparts within the same tumor. Xenograft irradiation resulted in a tumor growth delay of approximately 7 days with a corresponding increase in the percentage of CD133(+) cells at 7 days after radiation, which persisted to the onset of neurologic symptoms. These results suggest that, although the radioresponse of TSCs and non-TSCs does not differ under in vitro growth conditions, CD133(+) cells are relatively radioresistant under intracerebral growth conditions. Whereas these findings are consistent with the suspected role for TSCs as a determinant of GBM radioresistance, these data also illustrate the dependence of the cellular radioresistance on the brain microenvironment.

Synthetic lethal screen identifies NF-κB as a target for combination therapy with topotecan for patients with neuroblastoma.

BACKGROUND: Despite aggressive multimodal treatments the overall survival of patients with high-risk neuroblastoma remains poor. The aim of this study was to identify novel combination chemotherapy to improve survival rate in patients with high-risk neuroblastoma. METHODS: We took a synthetic lethal approach using a siRNA library targeting 418 apoptosis-related genes and identified genes and pathways whose inhibition synergized with topotecan. Microarray analyses of cells treated with topotecan were performed to identify if the same genes or pathways were altered by the drug. An inhibitor of this pathway was used in combination with topotecan to confirm synergism by in vitro and in vivo studies. RESULTS: We found that there were nine genes whose suppression synergized with topotecan to enhance cell death, and the NF-κB signaling pathway was significantly enriched. Microarray analysis of cells treated with topotecan revealed a significant enrichment of NF-κB target genes among the differentially altered genes, suggesting that NF-κB pathway was activated in the treated cells. Combination of topotecan and known NF-κB inhibitors (NSC 676914 or bortezomib) significantly reduced cell growth and induced caspase 3 activity in vitro. Furthermore, in a neuroblastoma xenograft mouse model, combined treatment of topotecan and bortezomib significantly delayed tumor formation compared to single-drug treatments. CONCLUSIONS: Synthetic lethal screening provides a rational approach for selecting drugs for use in combination therapy and warrants clinical evaluation of the efficacy of the combination of topotecan and bortezomib or other NF-κB inhibitors in patients with high risk neuroblastoma.

Systematic proteome analysis identifies transcription factor YY1 as a direct target of miR-34a.

MicroRNA 34a (miR-34a) is a potential tumor suppressor gene and has been identified as a miRNA component of the p53 network. To better understand the biological pathways involved in miR-34a action, a parallel global protein and mRNA expression profiling on miR-34a treated neuroblastoma cells (IMR32) was performed using isotope-coded affinity tags (ICAT) and Affymetrix U133plus2 microarray, respectively. Global profiling showed that miR-34a causes much smaller mRNA expression changes compared to changes at the protein level. A total of 1495 proteins represented by two or more peptides were identified from the quantitative ICAT analysis, of which 143 and 192 proteins are significantly up- or down-regulated by miR-34a, respectively. Pathway analysis of these differentially expressed proteins showed the enrichment of apoptosis and cell death processes in up-regulated proteins but DNA replication and cell cycle processes in the down-regulated proteins. Ribosomal proteins are the most significant set down-regulated by miR-34a. Additionally, biological network analysis to identify direct interactions among the differentially expressed proteins demonstrated that the expression of the ubiquitous transcription factor YY1, as well as its downstream proteins, is significantly reduced by miR-34a. We further demonstrated that miR-34a directly targets YY1 through a miR-34a-binding site within the 3' UTR of YY1 using a luciferase reporter system. YY1 is a negative regulator of p53, and it plays an essential role in cancer biology. Therefore, YY1 is another important direct target of miR-34a which closely regulates TP53 activities.

TIMP-2 modulates VEGFR-2 phosphorylation and enhances phosphodiesterase activity in endothelial cells.

In this study, we examine the effects of tissue inhibitor of metalloproteinases-2 (TIMP-2) on the phosphorylation status of specific phosphotyrosine residues on the vascular endothelial cell growth factor receptor-2 (VEGFR-2) cytoplasmic tail and examine the effects on associated downstream signaling pathways. To focus on metalloproteinase-independent mechanisms, we used the TIMP-2 analog known as Ala+TIMP-2 that is deficient in matrix metalloproteinase-inhibitory activity. Our experiments are designed to compare the effects of VEGF-A stimulation with or without Ala+TIMP-2 pretreatment, as well as basal responses in human microvascular endothelial cells. Our results show that Ala+TIMP-2 selectively alters the phosphorylation pattern of VEGFR-2 after VEGF-A stimulation and disrupts the downstream activation of PLC-gamma, Ca(+2) flux, Akt, and eNOS, as well as decreasing cGMP levels. Moreover, we observed an Ala+TIMP-2-induced reduction in cGMP levels typically elevated by exogenous NO donors implicating Ala+TIMP-2 in the direct activation of an isobutylmethylxanthine (IBMX)-sensitive cGMP phosphodiesterase activity. TIMP-2 suppression of endothelial mitogenesis and angiogenesis involves at least two mechanisms, one mediated by protein tyrosine phosphatase inhibition of VEGFR-2 activation as well as downstream signaling and a second mechanism involving direct activation of an IBMX-sensitive phosphodiesterase activity.

Screening a panel of drugs with diverse mechanisms of action yields potential therapeutic agents against neuroblastoma.

Neuroblastoma (NB) is the most common extracranial solid tumor in children. Despite current aggressive therapy, the survival rate for high risk NB remains less than 40%. To identify novel effective chemo-agents against NB, we screened a panel of 96 drugs against two NB cell lines, SK-N-AS and SH-SY5Y. We found 30 compounds that were active against NB cell lines at < or =10 microM concentration. More interestingly, 17 compounds are active at < or =1 microM concentration, and they act through a wide spectrum of diverse mechanisms such as mitotic inhibition, topoisomerase inhibition, targeting various biological pathways, and unknown mechanisms. The majority of these active compounds also induced caspase 3/7 by more than 2-fold. Of these 17 active compounds against NB cell lines at sub-micromolar concentration, eleven compounds are not currently used to treat NB. Among them, nine are FDA approved compounds, and three agents are undergoing clinical trials for various malignancies. Furthermore, we identified four agents active against these NB cell lines that have not yet been tested in the clinical setting. Finally we demonstrated that Cucurbitacin I inhibits neuroblastoma cell growth through inhibition of STAT3 pathway. These drugs thus represent potential novel therapeutic agents for patients with NB, and further validation studies are needed to translate them to the clinic.

Identification of FGFR4-activating mutations in human rhabdomyosarcomas that promote metastasis in xenotransplanted models.

Rhabdomyosarcoma (RMS) is a childhood cancer originating from skeletal muscle, and patient survival is poor in the presence of metastatic disease. Few determinants that regulate metastasis development have been identified. The receptor tyrosine kinase FGFR4 is highly expressed in RMS tissue, suggesting a role in tumorigenesis, although its functional importance has not been defined. Here, we report the identification of mutations in FGFR4 in human RMS tumors that lead to its activation and present evidence that it functions as an oncogene in RMS. Higher FGFR4 expression in RMS tumors was associated with advanced-stage cancer and poor survival, while FGFR4 knockdown in a human RMS cell line reduced tumor growth and experimental lung metastases when the cells were transplanted into mice. Moreover, 6 FGFR4 tyrosine kinase domain mutations were found among 7 of 94 (7.5%) primary human RMS tumors. The mutants K535 and E550 increased autophosphorylation, Stat3 signaling, tumor proliferation, and metastatic potential when expressed in a murine RMS cell line. These mutants also transformed NIH 3T3 cells and led to an enhanced metastatic phenotype. Finally, murine RMS cell lines expressing the K535 and E550 FGFR4 mutants were substantially more susceptible to apoptosis in the presence of a pharmacologic FGFR inhibitor than the control cell lines expressing the empty vector or wild-type FGFR4. Together, our results demonstrate that mutationally activated FGFR4 acts as an oncogene, and these are what we believe to be the first known mutations in a receptor tyrosine kinase in RMS. These findings support the potential therapeutic targeting of FGFR4 in RMS.

Therapeutic implications of T-cell clonopathy of unknown significance in chronic immune thrombocytopenic purpura.

Immune thrombocytopenic purpura (ITP) is typically considered an autoimmune disorder related to the production of autoantibodies; however, recent evidence indicates that cell-mediated cytotoxicity may be important pathogenetically in some cases. We describe seven patients with chronic ITP and concurrent T-cell clonopathy of unknown significance (TCUS), who failed various treatment regimens for ITP, including steroids, gamma globulins, splenectomy and rituximab, but had anecdotal success with azathioprine. These observations support the notion that ITP has heterogeneous biologic mechanisms, and that patients with persistent chronic ITP should be evaluated for T-cell clonality and considered for treatment options that are directed against cytotoxic T-cells.