COVID-19 and Early Post-Primary TB: Commonalities of Pathobiology in Pneumonitis and Therapies.

OBJECTIVE: Concurrent infection with COVID-19 and M. tuberculosis has been reported to be more severe than either alone, resulting in increased mortality. Our objective was to define the shared pathobiology of COVID-19 and the developmental stage of TB in the lung and explore adjunctive therapies to treat such commonalities. METHODS: Since morphoproteomics combines the disciplines of histopathology, molecular biology and protein chemistry to paint a portrait of the protein circuitry in diseased cells for the purpose of uncovering targets amenable to specific intervention [1], we used morphoproteomic analyses to study lung tissues of patients with early post-primary tuberculosis or COVID-19 infection. RESULTS: These studies showed co-localization of the COVID-19 virus and M. tuberculosis antigens with cyclo-oxygenase-2 and fatty acid synthase in the reactive alveolar pneumocytes and with programmed death-ligand 1 expression on the alveolar interstitium and alveolar pneumocytes. This was associated with accumulation of pro-infectious M2 polarized macrophages in the alveolar spaces. CONCLUSION: The commonalities in these pathways suggest that they might be susceptible to adjunctive therapies with metformin and vitamin D3. This is supported by published studies that metformin and vitamin D3 could reduce the severity of both COVID-19 and early post-primary TB infections.

Review: Mammalian Target of Rapamycin (mTOR) Pathway Is Critical in Developing Most Renal Cell Tumors.

OBJECTIVE: Various renal cell carcinomas (RCC) are derived from different segments of the renal tubular origin, which determines their morphological and immunohistochemical phenotype and their molecular signaling pathway as a therapeutic target. Most of these tumors utilize the mammalian target of rapamycin (mTOR) pathway to activate pathways involving metabolic and nutritional supplies. METHODS: Overexpressed mTOR signals are reported in more than 90% of the most common types of RCC. Many new renal tumor entities have been reported in recent years. RESULTS: Among them, somatic mutations in tuberous sclerosis complex (TSC) result in loss of its normal inhibitory control over mTOR, thus promoting mTOR-associated proliferative activities in several new renal neoplastic entities including RCC with fibromyomatous stroma (RCCFMS), eosinophilic vacuolated tumor, eosinophilic solid & cystic RCC, and low-grade oncocytic tumor. CONCLUSIONS: This short review provides a comprehensive correlation of tumor morphology and immunohistochemical phenotype with renal tubular differentiation and their shared mTOR. These essential pieces of knowledge are vital in the diagnosis and clinical management of renal cell neoplasms.

Morphoproteomic Features of Pulmonary Influenza A (H1N1) with Therapeutic Implications: A Case Study.

OBJECTIVE: Influenza pandemic of the human lung was caused by the Influenza A (H1N1) over 100 years ago in 1918, but it recurred in pandemic fashion in 2009. Understanding the pathobiology of this infectious agent in the human lung could lead to adjuvant therapies that are relatively non-toxic and reduce the mortality of the human host. Overall, our objective was to apply morphoproteomics to pulmonary lung sections from an autopsied victim so that we may better define its biology from the perspective of its interaction with the host and provide options for therapeutic targets. METHODS: Morphoproteomic analysis from a case study of this Influenza A (H1N1) pulmonary infection included immunohistochemical probes to detect the expressions of fatty acid synthase (FAS), CD163+ (M2 polarized monocytes/macrophages), and programmed death-ligand 1 (PD-L1) expression as part of the host response to interaction with the Influenza A (H1N1) virus. RESULTS: Representative sections of the Influenza A (H1N1) victim's lung showed: cytoplasmic expression of FAS in most of the sloughed and atypical alveolar pneumocytes; abundance of intra-alveolar and alveolar interstitial CD163+ macrophages/monocytes; and PD-L1 expression on occasional macrophages, and focally on collections of alveolar pneumocytes and the alveolar interstitium. CONCLUSION: Morphoproteomics and microanatomical features coincide with the etiopathogenic features of pulmonary Influenza A (H1N1) infection and the host response. This plus data mining of the medical literature suggests that adjunctive, targeted therapy such as metformin and vitamin D3 could address the biology of Influenza A (H1N1) pneumonia, enhance the host immune response, and prevent its progression to a life-threatening, ventilator-dependent clinical situation.

Postmortem study of organ-specific toxicity in glioblastoma patients treated with a combination of temozolomide, irinotecan and bevacizumab.

PURPOSE: Systemic chemotherapy including monotherapy with temozolomide (TMZ) or bevacizumab (BEV); two-drug combinations, such as irinotecan (IRI) and BEV, TMZ and BEV and a three-drug combination with TMZ, IRI and BEV (TIB) have been used in treating patients with progressive high-grade gliomas including glioblastoma (GBM). Most patients tolerated these regimens well with known side effects of hypertension, proteinuria, and reversible clinical myelosuppression (CM). However, organ- or system- specific toxicities from chemotherapy agents have never been examined by postmortem study. This is the largest cohort used to address this issue in glioma patients. METHODS: Postmortem tissues (from all major systems and organs) were prospectively collected and examined by standard institution autopsy and neuropathological procedures from 76 subjects, including gliomas (N = 68, 44/M, and 24/F) and brain metastases (N = 8, 5/M, and 3/F) between 2009 and 2019. Standard hematoxylin and eosin (H&E) were performed on all major organs including brain specimens. Electronic microscopic (EM) study was carried out on 14 selected subject's kidney samples per standard EM protocol. Medical records were reviewed with adverse events (AEs) analyzed and graded according to the Common Terminology Criteria for Adverse Events (CTCAE), version 4.03. A swimmer plot was utilized to visualize the timelines of patient history by treatment group. The binary logistic regression models were performed to explore any associations between treatment strategies and incident myelosuppression. RESULTS: Twenty-four glioma subjects were treated with TIB [median: 5.5 (range: 1-25) cycles] at tumor recurrence. Exposure to IRI significantly increased the frequency of CM (p = 0.05). No unexpected adverse events clinically, or permanent end-organ damage during postmortem examination was identified in glioma subjects who had received standard or prolonged duration of BEV, TMZ or TIB regimen-based chemotherapies except rare events of bone marrow suppression. The most common causes of death (COD) were tumor progression (63.2%, N = 43) followed by aspiration pneumonia (48.5%, N = 33) in glioma subjects. No COD was attributed to acute toxicity from TIB. The study also demonstrated that postmortem kidney specimen is unsuitable for studying renal ultrastructural pathological changes due to autolysis. CONCLUSION: There is no organ or system toxicity by postmortem examinations among glioma subjects who received BEV, TMZ or TIB regimen-based chemotherapies regardless of durations except for occasional bone marrow suppression and reversible myelosuppression clinically. IRI, but not the extended use of TMZ, significantly increased CM in recurrent glioma patients. COD most commonly resulted from glioma tumor progression with infiltration to brain stem and aspiration pneumonia.

Morphoproteomics Identifies SIRT1, EZH2 and CXCR4 Pathways in Diffuse Large B-Cell Lymphoma: Therapeutic Implications.

OBJECTIVE: Diffuse large B-cell lymphoma (DLBCL) is the most aggressive form of non-Hodgkin's lymphoma. METHODS: We applied morphoproteomics to a tissue microarray of DLBCL cases to uncover commonalities in its biology with therapeutic implications. Morphoproteomic analysis of 9 individual cases of classic DLBCL included immunohistochemical probes to detect silent mating type information regulation 2 homolog 1 (SIRT1), enhancer of Zeste homolog 2 (EZH2) and C-X-C chemokine receptor 4 (CXCR4) and their cellular compartmentalization by bright field microscopy. RESULTS: We detected the expression of SIRT1 in the majority (>50%) of the tumoral nuclei of 8 of 9 cases of DLBCL and of EZH2 expression in the majority (>50%) of the tumoral nuclei in 9 of the 9 cases; and the expression of the tumoral stem cell marker, CXCR4 in the cytoplasmic and/or plasmalemmal compartment at >50% of the tumor cells in all 9 cases of DLBCL. The morphoproteomic findings of SIRT1 and EZH2 expression in DLBCL, for the most part, parallel the morphoproteomic findings in B-cell acute lymphoblastic leukemia. This concordance has pharmacogenomic and therapeutic implications. Similarly, the fact that EZH2 can enhance the expression of tumoral stem cell marker, CXCR4 implies that there is a block in differentiation in DLBCL. CONCLUSION: By targeting the Sirt1, EZH2 and CXCR4 pathways using relatively non-toxic adjuvant therapeutic agents such as metformin, melatonin, curcumin, sulforaphane, vitamin D3 and plerixafor, we should be able to target the biology of DLBCL.

Commentary: Morphoproteomics and Data Mining of the Medical Literature Define the Pathobiology of COVID-19 Pneumonitis in Humans and Provide Adjuvant Therapeutic Options.

Due to the resurgence of COVID-19, understanding the biology of SARS-CoV-2 is an opportunity to develop adjuvant therapies that could target its pathobiology and lessen the severity of the COVID-19 infection so that our patients could survive. This commentary serves to accomplish this by using published morphoproteomic findings with data mining of the medical literature to define the pathobiology of COVID-19 pneumonitis and provide combinatorial and relatively non-toxic adjuvant therapies that have been successful against this viral infection.

Early Lesion of Post-Primary Tuberculosis: Subclinical Driver of Disease and Target for Vaccines and Host-Directed Therapies.

The characteristic lesion of primary tuberculosis is the granuloma as is widely studied in human tissues and animal models. Post-primary tuberculosis is different. It develops only in human lungs and begins as a prolonged subclinical obstructive lobular pneumonia that slowly accumulates mycobacterial antigens and host lipids in alveolar macrophages with nearby highly sensitized T cells. After several months, the lesions undergo necrosis to produce a mass of caseous pneumonia large enough to fragment and be coughed out to produce a cavity or be retained as the focus of a post-primary granuloma. Bacteria grow massively on the cavity wall where they can be coughed out to infect new people. Here we extend these findings with the demonstration of secreted mycobacterial antigens, but not acid fast bacilli (AFB) of M. tuberculosis in the cytoplasm of ciliated bronchiolar epithelium and alveolar pneumocytes in association with elements of the programmed death ligand 1 (PD-L1), cyclo-oxygenase (COX)-2, and fatty acid synthase (FAS) pathways in the early lesion. This suggests that M. tuberculosis uses its secreted antigens to coordinate prolonged subclinical development of the early lesions in preparation for a necrotizing reaction sufficient to produce a cavity, post-primary granulomas, and fibrocaseous disease.

A Brief History, the Progress in the Variants of Therapies against Metastatic Neoplasms, and the Role of Pathologists.

Chemotherapy originated in the early 1960s. The initial chemotherapeutic agents focused on blocking metabolic pathways and found substantial success in certain types of tumors, but they are generally considered toxic to all normal and tumor cells, and they have significant side effects. As more scientific studies began to identify many new, specific targets and mutations, along with a multitude of growth pathways in tumor cells, new agents targeting cell growth pathways began to emerge in the late 1990s and early 2000s. In 2003, a method called morphoproteomics was developed to evaluate the immunohistochemical protein expressions of target markers in tumors, and it has been considered a pioneering method for guiding targeted therapy. Subsequently, many genomic techniques have been established for identifying specific mutations and tumor markers in order to guide the targeted therapy. More recently, immuno-oncology therapy targeting specific immune markers has been rapidly developed, and the immunohistochemical evaluation of specific immune markers such as PD-L1 demonstrates further expansion of oncologic morphoproteomics. This brief review will focus on the role of pathologists in developing various techniques and guiding targeted therapies during the era of personalized medicine.

Commentary: Pharmacologic Correlates that Address the Clinicopathobiology of COVID-19.

COVID-19 could remain a pandemic until we get a SARS-CoV-2 vaccine to immunize the world population. In the interim, we have the opportunity to consider its clinicopathobiology in the context of the pharmacologic correlates available from the existing medical literature to prevent the COVID-19 infected patient from progressing into a fatal stage. This commentary serves as a forum for that end and suggests relatively non-toxic therapies that could be applied in a combinatorial fashion for consideration by the physicians and their patients with COVID-19 infection.

Morphoproteomics Identifies the Foamy Alveolar Macrophage as an M2 Phenotype with PD-L1 Expression in the Early Lesion of Post-Primary Tuberculosis: Implications for Host Immune Surveillance and Therapy.

Post-primary tuberculosis (TB) disease is characterized by paucibacillary necrosis of the early lesion, tuberculous pneumonia, in the adult human lung. The mechanism is speculated to be a strong localized delayed type hypersensitive response (DTH). However, up to this date, no one has been able to identify the source of the large accumulation of MTB antigens required for the DTH response. Although it is known and accepted that the pathogen, Mycobacterium tuberculosis (MTB), significantly affects macrophage function and activity, few studies have focused on macrophages at the site of the early lesion of developing post-primary MTB in human lungs. In vitro studies have examined the effect of MTB on skewing the macrophage phenotype, specifically the dynamic of the M1 and M2 differentiation. Additionally, it is also well documented that MTB infection induces macrophages to become foamy, accumulating host, and potentially MTB, lipids in the cytoplasm. The foamy macrophage is necessary for prolonging MTB survival in the infected lung. Using autopsy derived lung samples from untreated TB diseased individuals, this report, by applying morphoproteomics, demonstrates that the alveolar macrophages present in the early lesion of TB are primarily of the M2 phenotype. The M2 foamy alveolar macrophages (FAM) are also loaded with MTB antigens by immunohistochemistry and are paucibacillary. Moreover, the M2 alveolar macrophages predominately express PD-L1, leading to suppression of PD-1+ lymphocytes and host immunosurveillance. These morphoproteomic analyses indicate that early lesion of MTB in the adult human lung leads to a skewed M2 foamy alveolar macrophage phenotype that creates a protective microenvironment that accumulates high concentrations of MTB antigens, which when released can lead to necrosis and eventual cavitation.

Morphoproteomics and Etiopathogenic Features of Pulmonary COVID-19 with Therapeutic Implications: A Case Study.

OBJECTIVE: The COVID-19 pandemic has challenged the world economically and medically. Understanding and defining the biology of this specific coronavirus infection may lead to targeted therapies to lessen its virulence and expand the host resistance. This study's objective was to apply morphoproteomics to pulmonary lung sections from a forensic autopsy of an untreated COVID-19 victim, so that we may better define its biology from the perspective of its interaction with the host and provide options for therapeutic targets. DESIGN: Morphoproteomic analysis from a case study of this COVID-19 pulmonary infection included immunohistochemical probes to detect phosphorylated p-STAT3 (Tyr 705), as part of the interleukin (IL)-6 pathway; cyclooxygenase (COX)-2, CD8+ cytotoxic lymphocytes, Programmed Death (PD)-1 receptor+ lymphoid cells, CD56+ NK lymphoid cells, CD163+ (M2 polarized monocytes/macrophages), and programmed death-ligand 1 (PD-L1) expression as part of the host response to interaction with the COVID-19 virus. RESULTS: Representative sections of the COVID-19 victim's lung showed: nuclear expression of p-STAT3 (Tyr 705) in many of the alveolar pneumocytes and in occasional endothelial cells; COX-2 expression in the alveolar pneumocytes; a relative paucity of CD8+ cytotoxic lymphocytes; absence of CD56+ NK lymphoid cells; abundance of intra-alveolar and alveolar interstitial CD163+ macrophages/monocytes; PD-L1 expression on occasional macrophages, focally on collections of alveolar pneumocytes, and on cells in the alveolar interstitium; and rare PD-1+ lymphocytes in similar regions as CD8+ lymphocytes. CONCLUSION: Morphoproteomics and microanatomical features coincide with the etiopathogenic features of pulmonary coronavirus infection and the host response. This suggests that a targeted therapy could address the biology of COVID-19 pneumonia, enhance the host immune response and prevent its progression to a life-threatening, ventilator-dependent clinical situation.

Morphoproteomics Identifies CXCR4 in Undifferentiated Colorectal Cancer: A Case Study with Therapeutic Implications.

Undifferentiated colorectal cancer (UCRC) has a well-documented CD44 cancer stem cell (CSC) component. CXCR4 (C-X-C motif receptor 4) also is a CSC marker and with its ligand, stromal-derived factor (SDF)-1 alpha has been shown to act in concert with CD44 to promote cancer cell invasion. To date, CXCR4 has not been reported in the MEDLINE (PubMed) literature with respect to UCRC. Morphoproteomic analysis, utilizing bright field microscopy, can visualize the intensity and cell location of protein analytes with a patient's tumor including CXCR4 expression and was applicable to this case study. Using morphoproteomic analysis we identified chromogenic expression of both CD44 on the plasmalemmal aspect of the majority of the undifferentiated colorectal cancer foci and CXCR4 expression on the plasmalemmal or cytoplasmic aspect in up to 50% of the tumor cells in some regions and also on the intratumoral endothelial cells in the patient's UCRC. Moreover, both CSC markers were retained in the foci showing incomplete mucinous differentiation. In the context of data mining of the scientific literature, these findings suggest a collaboration between CXCR4 and CD44 in promoting tumor invasion and angiodependent metastasis in UCRC. Therapies designed to inhibit both the CD44 and the CXCR4 pathways are available and could reduce the metastatic potential of UCRC.

Genomics, Morphoproteomics, and Treatment Patterns of Patients with Alveolar Soft Part Sarcoma and Response to Multiple Experimental Therapies.

Overexpression of transcription factor 3 in alveolar soft part sarcoma(ASPS) results in upregulation of cell proliferation pathways. No standard treatment algorithm exists for ASPS; multikinase inhibitors[tyrosine kinase inhibitor (TKI)] and immune checkpoint inhibitors (ICI) have shown clinical benefit. To date, no studies have reported on management strategies or sequencing of therapy. We evaluated ASPS treatment patterns and responses in an experimental therapeutics clinic. Genomic and morphoproteomic analysis was performed to further elucidate novel targets. We retrospectively reviewed patients with ASPS treated on clinical trials. Demographic and clinical next-generation sequencing (NGS) profiles were collected. AACR GENIE database was queried to further evaluate aberrations in ASPS. Morphoproteomic analysis was carried out to better define the biology of ASPS with integration of genomic and proteomic findings. Eleven patients with ASPS were identified; 7 received NGS testing and mutations in CDKN2A (n = 1) and hepatocyte growth factor (n = 1) were present. Ten patients were treated with TKIs with stable disease as best response and 4 patients with ICI (three partial responses). Within GENIE, 20 patients were identified harboring 3 called pathogenic mutations. Tumor mutation burden was low in all samples. Morphoproteomic analysis confirmed the expression of phosphorylated c-Met. In addition, fatty acid synthase and phosphorylated-STAT3 were detected in tumor cell cytoplasm and nuclei. Patients with ASPS have a quiescent genome and derive clinical benefit from VEGF-targeting TKIs. Morphoproteomic analysis has provided both additional correlative pathways and angiogenic mechanisms that are targetable for patients with ASPS. Our study suggests that sequential therapy with TKIs and immune checkpoint inhibitors is a reasonable management strategy.

M2 Macrophages in Crystal Storing Histiocytosis Associated with Plasma Cell Myeloma.

Crystal Storing Histiocytosis (CSH) is a rare entity occurring in association with underlying lymphoproliferative disorders and plasma cell neoplasms. It denotes accumulation of immunoglobulin crystals in the histiocyte cytoplasm. In this study, we report a patient with plasma cell myeloma who presented with bilateral comminuted femur fractures. Histological examination of fracture tissue revealed hypercellular (~100%) marrow with extensive involvement by sheets of histiocytes with abundant eosinophilic cytoplasm admixed with scattered plasma cells. Intracytoplasmic diamond and rhomboid crystals within histiocytes were demonstrated by electron microscopy. Immunohistochemistry highlighted monotypic plasma cells with kappa restriction, representing 20-30% of marrow cellularity; however, non-polarizable cytoplasmic striations in histiocytes were negative for light chain expression. A diagnosis of crystal-storing histiocytosis associated with plasma cell myeloma was rendered. Further evaluation of these macrophages is positive for CD163 and COX2 and shows pSTAT3 with variable nuclear staining in some histiocytes. This case demonstrates that numerous M2 macrophages are present as crystal storing histiocytosis; and this knowledge might convey prognostic and therapeutic significance for the patients with crystal storing histiocytosis.

Morphoproteomics and Biomedical Analytics Identify the c-Myc, EZH2, Sirt1 and CXCR4 Pathways as Potential Blocks to Differentiation Leading to Proliferation and Metastatic Potential in Sinonasal Undifferentiated Carcinoma (SNUC) and Provide Therapeutic Options: A Case Study.

Sinonasal undifferentiated carcinoma (SNUC) is a highly malignant tumor in the nasal cavity or paranasal sinuses. Morphoproteomics has defined its biology to some degree, allowing the identification of targeted therapeutic options with clinical efficacy [1]. This study's objective was to identify putative SNUC pathways that are known to pose a block in differentiation both in early embryogenesis and in tumorigenesis or that might promote metastasis and recurrent disease. DESIGN: Morphoproteomic analysis of SNUC from a case study of this patient included immunohistochemical probes to detect c-Myc, EZH2, Sirt1 and CXCR4 protein analytes. Biomedical analytics schematically showed the interactions of these analytes with the morphoproteomic findings and illustrated targeted therapeutic options. RESULTS: Representative sections of this patient's tumor displayed plasmalemmal expression for CXCR4 and nuclear immunopositivity for c-Myc, EZH2, and Sirt1. This coincided with their block in differentiation and their proliferative state with progression into the mitotic phase. Biomedical analytics integrated the morphoproteomic findings with the undifferentiated and proliferative state of SNUC and depicted pharmacogenomic and other related factors that target the c-Myc, EZH2, Sirt1 and CXCR4 pathways. CONCLUSION: Morphoproteomics and biomedical analytics have identified c-Myc, EZH2, Sirt1 and CXCR4 pathways that collectively could contribute to the block in differentiation and increase the propensity for recurrence and metastasis in SNUC. This suggests that combinatorial therapies modulating these pathways could be used in a maintenance mode to minimize the risk of recurrent disease.