Biochemical and Structural Studies of the Carminomycin 4-O-Methyltransferase DnrK.

Structural and functional studies of the carminomycin 4-O-methyltransferase DnrK are described, with an emphasis on interrogating the acceptor substrate scope of DnrK. Specifically, the evaluation of 100 structurally and functionally diverse natural products and natural product mimetics revealed an array of pharmacophores as productive DnrK substrates. Representative newly identified DnrK substrates from this study included anthracyclines, angucyclines, anthraquinone-fused enediynes, flavonoids, pyranonaphthoquinones, and polyketides. The ligand-bound structure of DnrK bound to a non-native fluorescent hydroxycoumarin acceptor, 4-methylumbelliferone, along with corresponding DnrK kinetic parameters for 4-methylumbelliferone and native acceptor carminomycin are also reported for the first time. The demonstrated unique permissivity of DnrK highlights the potential for DnrK as a new tool in future biocatalytic and/or strain engineering applications. In addition, the comparative bioactivity assessment (cancer cell line cytotoxicity, 4E-BP1 phosphorylation, and axolotl embryo tail regeneration) of a select set of DnrK substrates/products highlights the ability of anthracycline 4-O-methylation to dictate diverse functional outcomes.

Analysis of Histomorphologic/Molecular Association and Immune Checkpoint Regulators in Epithelioid Glioblastoma and Pleomorphic Xanthoastrocytoma: Are These Tumors Potential Candidates for Immune Checkpoint Blockade?

Accurate diagnosis of Epithelioid glioblastoma (eGB) and pleomorphic xanthoastrocytoma (PXA) is sometimes challenging owing to overlapping histologic and genetic features. There are limited reports on the immune profile of these tumors. In this study, we assessed 21 PXA [15 PXA Grade 2 (PXAG2); 6 PXA Grade 3 (PXAG3)] and 14 eGB for their histopathological and molecular association. Further, their immune profile was compared with GB, IDH1 wild-type (wt) (n-18). Morphologically, PXAG2 mostly differed from eGB; however, it was occasionally difficult to differentiate PXAG3 from eGB due to their epithelioid pattern and less obvious degenerative features. PXAG2 showed predominantly diffuse, whereas variable positivity for epithelial and glial markers was seen in PXAG3 and eGB. All cases showed retained nuclear ATRX and INI-1 . H3K27M or IDH1 mutation was seen in none. P53 mutation was more common in eGB, followed by PXAG3, and least common in PXAG2. BRAF V600E mutation was observed in 66.67% PXAG2, 33.33% PXAG3, and 50% eGB, with 100% concordance between immunohistochemistry (IHC) and sequencing. Thirty-six percent eGB, 33% PXAG3, and 61% PXAG2 harbored CDKN2A homozygous deletion. EGFR amplification was observed in 14% eGB and 66% of GB, IDH wt. PDL1 and CTLA-4 expression was higher in eGB (71.4% and 57.1%), PXAG3 (66.6% and100%), and PXAG2 (60% & 66.7%) as compared with GB, IDH wt (38.8% and 16.7%). Tumor-infiltrating lymphocytes were also observed in a majority of eGB and PXA (90% to 100%) in contrast to GB, IDH wt (66%). This analysis highlights the homogenous molecular and immune profile of eGB and PXA, suggesting the possibility that histologically and molecularly, these two entities represent 2 ends of a continuous spectrum with PXAG3 lying in between. Higher upregulation of PDL1, CTLA-4, and increased tumor infiltrating lymphocytes in these tumors as compared with GB, IDH wt suggests potential candidature for immunotherapy.

Cancer Stem Cell Markers - CD133 and CD44 - in Paediatric Solid Tumours: A Study of Immunophenotypic Expression and Correlation with Clinicopathological Parameters.

Paediatric solid tumours account for about 30% of all the paediatric malignancies. They differ from adult tumours in various aspects like incidence, etiopathogenesis, biology, response rate and outcome. Immunohistochemical markers such as CD133, CD44, CD24, CD90, CD34, CD117, CD20 and ALDH 1 (aldehyde dehydrogenase-1) have been proposed to detect cancer stem cells in tumours. CD133 is a marker of tumour initiating cells in many human cancers and therefore, it may be possible to develop future therapies by targeting cancer stem cells via this marker. CD44 is a transmembrane glycoprotein also known as homing cell adhesion molecule. It is a multifunctional cell-adhesion molecule and plays an important role in cell-cell interaction, lymphocyte homing, tumour progression and metastasis. In the present study, we assessed the expression of CD133 and CD44 in paediatric solid tumours and correlated their expression with clinico-pathological parameters in paediatric solid tumours. This study was a cross-sectional observational study conducted in the department of pathology at a tertiary care centre. All the histologically diagnosed paediatric solid tumours for a period of one year and four months were retrieved from the archives. The cases were reviewed and included in the study after obtaining informed consent. Immunohistochemistry using the monoclonal antibodies for CD133 and CD44 was performed in the representative tissue sections of all the cases. Immuno-scores were assessed, and the results were compared using Pearson's chi-square test. The present study included 50 cases of paediatric solid tumours. The majority (34%) of the patients were in the age group of less than 5 years, with male preponderance (M:F = 2.3:1). The tumours included were Wilms tumour, yolk sac tumour, rhabdomyosarcoma, lymphoma, neuroblastoma, hepatoblastoma, gastrointestinal stromal tumour (GIST), medulloblastomas, pilocytic astrocytomas, ependymomas and glioblastoma. On immunohistochemical analysis, high expression of CD133 and CD44 was found. A significant association between the expression of CD133 and various tumour groups was observed (p = 0.004). However, CD44 showed variable expression in different tumour groups. Both CD133 and CD44 identified cancer stem cell in paediatric solid tumours. A further validation is warranted to investigate their potential role in therapy and prognosis.

A discrete intermediate for the biosynthesis of both the enediyne core and the anthraquinone moiety of enediyne natural products.

The enediynes are structurally characterized by a 1,5-diyne-3-ene motif within a 9- or 10-membered enediyne core. The anthraquinone-fused enediynes (AFEs) are a subclass of 10-membered enediynes that contain an anthraquinone moiety fused to the enediyne core as exemplified by dynemicins and tiancimycins. A conserved iterative type I polyketide synthase (PKSE) is known to initiate the biosynthesis of all enediyne cores, and evidence has recently been reported to suggest that the anthraquinone moiety also originates from the PKSE product. However, the identity of the PKSE product that is converted to the enediyne core or anthraquinone moiety has not been established. Here, we report the utilization of recombinant E. coli coexpressing various combinations of genes that encode a PKSE and a thioesterase (TE) from either 9- or 10-membered enediyne biosynthetic gene clusters to chemically complement ΔPKSE mutant strains of the producers of dynemicins and tiancimycins. Additionally, 13C-labeling experiments were performed to track the fate of the PKSE/TE product in the ΔPKSE mutants. These studies reveal that 1,3,5,7,9,11,13-pentadecaheptaene is the nascent, discrete product of the PKSE/TE that is converted to the enediyne core. Furthermore, a second molecule of 1,3,5,7,9,11,13-pentadecaheptaene is demonstrated to serve as the precursor of the anthraquinone moiety. The results establish a unified biosynthetic paradigm for AFEs, solidify an unprecedented biosynthetic logic for aromatic polyketides, and have implications for the biosynthesis of not only AFEs but all enediynes.

Extracranial Schwannomas of the Head and Neck: A Literature Review and Audit of Diagnosed Cases Over a Period of Eight Years.

Schwannoma is a benign, slow growing, usually solitary and encapsulated tumor derived from Schwann cells of the nerve sheath. Schwannomas can be divided into central, or intraosseous, and peripheral lesions. The etiology is unknown, but it is postulated that lesions arise by the proliferation of Schwann cells at one point inside the perineurium. Schwannomas may mimic other diseases of the head and neck, such as infection, tumor or metastasis. Extracranial schwannomas are rare; in this study we review a series of 22 cases of schwannomas originating in the head and neck region over a period of eight years. All tumors were benign and well-encapsulated. Tumor size ranged from 0.5 to 9 cm. The age range of patients studied was 15-74 years with a mean age of 35 years and a male predilection (M:F, 2.6:1)was noted. Four cases of schwannomas occurred in the tongue (18.18%) and lower lip (18.18%), three in the nasal cavity (13.64%), two each (9.09%) in the buccal mucosa, parapharyngeal space (9.09%), and eyebrow (9.09%), and one each in the upper lip (4.55%), lateral canthus of the eye (4.55%), intraorbital region (4.55%), submandibular gland (4.55%), and ear (4.55%). Schwannomas can present in a wide variety of sites within the head and neck. The tumor is benign and tend to be asymptomatic for long periods of time. Histopathology is the gold standard for diagnosis. Our study describes the clinicopathologic features of extracranial head and neck schwannomas, highlights the histopathologic features, and discusses pertinent findings with correlation to the present literature. It is important that both clinicians and pathologists be familiar with the uncommon sites of occurrence and the potential pitfalls associated with the diagnosis and management of these tumors.

Comparative evaluation of squash smear and frozen section in the intraoperative diagnosis of central nervous system tumours.

BACKGROUND: The squash smear technique was introduced into intra-operative neurosurgical diagnosis as early as 1930. It is becoming increasingly popular in the diagnosis of central nervous system (CNS) lesions, and is fairly accurate even with a small sample. The current study assesses the accuracy and utility of the squash smear and frozen section (FS) techniques in intraoperative consultations. Correlations with histopathological diagnoses are presented. AIMS: To compare two intra-operative diagnostic techniques-squash smear cytology and FS examination-in the context of central nervous system tumours. MATERIALS AND METHODS: A total of 53 cases of CNS tumours were included in the study, and all were subjected to squash smear examination. FS examinations were conducted for 39 of these samples. The results of the two techniques were compared and correlated with histopathological diagnoses. The observed results were then analysed using SPSS software. RESULTS: The most common primary CNS tumours were gliomas and meningiomas (28.3% each). The sensitivity, specificity, positive predictive value and negative predictive value for squash cytology were 86.67%, 87.5%, 81.25% and 91.3%, and for FS were 91.67%, 93.10%, 91.67%, 93.10%, respectively, with a corresponding comparative P-value of 0.56 (insignificant). Cytological diagnosis showed complete correlation with histopathological diagnosis in 39 cases (73.58%), partial correlation in eight cases (15.1%) and no correlation in six cases (11.32%). FS diagnosis showed complete correlation in 29 cases (74.35%), partial correlation in eight cases (20.5%) and no correlation in two cases (5.1%). CONCLUSION: Squash smear is a rapid, self-sufficient and cost-effective method for the intraoperative diagnosis of CNS tumours. The squash smear and FS techniques are complementary procedures that assist the pathologist in reaching a diagnosis.

Enzymatic Cß-H Functionalization of l-Arg and l-Leu in Nonribosomally Derived Peptidyl Natural Products: A Tale of Two Oxidoreductases.

Muraymycins are peptidyl nucleoside antibiotics that contain two Cß-modified amino acids, (2S,3S)-capreomycidine and (2S,3S)-ß-OH-Leu. The former is also a component of chymostatins, which are aldehyde-containing peptidic protease inhibitors that─like muraymycin─are derived from nonribosomal peptide synthetases (NRPSs). Using feeding experiments and in vitro characterization of 12 recombinant proteins, the biosynthetic mechanism for both nonproteinogenic amino acids is now defined. The formation of (2S,3S)-capreomycidine is shown to involve an FAD-dependent dehydrogenase:cyclase that requires an NRPS-bound pathway intermediate as a substrate. This cryptic dehydrogenation strategy is both temporally and mechanistically distinct in comparison to the biosynthesis of other capreomycidine diastereomers, which has previously been shown to proceed by Cß-hydroxylation of free l-Arg catalyzed by a member of the nonheme Fe2+- and α-ketoglutarate (αKG)-dependent dioxygenase family and (eventually) a dehydration-mediated cyclization process catalyzed by a distinct enzyme(s). Contrary to our initial expectation, the sole nonheme Fe2+- and αKG-dependent dioxygenase candidate Mur15 encoded within the muraymycin gene cluster is instead demonstrated to catalyze specific Cß hydroxylation of the Leu residue to generate (2S,3S)-ß-OH-Leu that is found in most muraymycin congeners. Importantly, and in contrast to known l-Arg-Cß-hydroxylases, the Mur15-catalyzed reaction occurs after the NRPS-mediated assembly of the peptide scaffold. This late-stage functionalization affords the opportunity to exploit Mur15 as a biocatalyst, proof of concept of which is provided.

A study of epithelial-mesenchymal transition immunohistochemical markers in primary oral squamous cell carcinoma.

OBJECTIVE: To analyze markers of epithelial-mesenchymal transition (EMT) in oral squamous cell carcinoma (SCC) using E-cadherin, epithelial membrane antigen (EMA), vimentin, and smooth muscle actin (SMA), and their association with tumor metastasis and grade. STUDY DESIGN: This was a retrospective study, which included 45 diagnosed cases of primary oral SCC with known lymph node status, taken from the archives of the Department of Pathology along with their clinical profile. Histomorphologic evaluation and immunohistochemical (IHC) analysis for E-cadherin, EMA, vimentin, and SMA were performed. IHC expression of these markers was compared with tumor differentiation as well as lymph node metastasis. RESULTS: We observed that reduced E-cadherin expression and positive expression of SMA were significantly higher in tumors having lymph node metastasis and loss of tumor differentiation, respectively. Reduced EMA expression was associated with the presence of lymph node metastasis but not with the histologic differentiation. Vimentin positivity did not show any correlation with lymph node metastasis or histologic differentiation. CONCLUSIONS: This study emphasizes the importance of EMT in the pathogenesis of oral SCC. The use of biomarkers like E-cadherin, EMA, and SMA might be a valuable tool for predicting patient outcomes and therapy.

Encapsulated papillary variant of medullary carcinoma of thyroid with extensive cystic change: an extremely rare presentation.

Papillary variant of medullary carcinoma of the thyroid is an unusual subtype with many diagnostic challenges. The authors report a case of papillary variant of thyroid medullary carcinoma in a 37-year-old female, who presented with complaints of pain in the thyroid nodule for the latter two months. Contrast enhanced computed tomography (CECT) neck revealed an enlarged and heterogeneously enhancing left lobe of thyroid. This was followed by hemithyroidectomy for suspicion of colloid goitre. Gross examination of the cut surface of the thyroid parenchyma had a sponge like appearance. On histopathology a diagnosis of encapsulated papillary variant of medullary carcinoma thyroid was made with the help of special stains and immunohistochemistry (IHC).

Epithelioid inflammatory myofibroblastic sarcoma: the youngest case reported.

Epithelioid inflammatory myofibroblastic sarcoma (EIMS) is a rare variant of the inflammatory myofibroblastic tumor. It has an aggressive clinical course and a high rate of recurrence. EIMS primarily affects children and young adults. Hereby, we report this entity in a 4-month-old infant who presented with an abdominal mass. Imaging studies revealed a large hypodense mesentery-based lesion involving the right half and mid-region of the abdomen. The mass with an attached segment of the small bowel was excised in toto. Grossly, a large encapsulated tumor was identified arising from the mesentery of the small bowel. The histological examination showed a tumor consisting of epithelioid to spindle cells loosely arranged in a myxoid background with numerous blood vessels and lymphoplasmacytic inflammatory infiltrate. On immunohistochemistry, the tumor cells showed positivity for ALK1 (nuclear), desmin, SMA, CD68, and focal positivity for CD30. A final diagnosis of EIMS of the small intestine was rendered. To the best of our knowledge, this case is the youngest reported case in literature.

Giant virilising adrenal cortical carcinoma.

Androgen secreting adrenocortical carcinoma (ACC) is a very rare disease with a poor prognosis. Approximately 80% of tumors are functional, most commonly secreting glucocorticoids. We herewith report a case of a huge functional ACC of the right adrenal gland in a 33-year-old female who presented with complaints of hirsutism, amenorrhea and an abdominal lump. On abdominal examination a large lump was palpable in the right hypochondrium reaching up to the umbilicus. Contrast-enhance computed tomography (CECT) revealed a mass in the right suprarenal region. The tumor measured 29 cm × 20 cm × 12 cm and weighed 7.8 kg, the largest reported case of ACC in the world to the best of our knowledge.

A clinicopathological study of peripheral schwannomas.

AIM AND OBJECTIVE: Schwannomas are benign neoplasms of neural origin with sporadic or syndromic occurence. They are commonly seen in cranial nerves. Peripheral schwannomas occur rarely and may have unique presentations. The aim of this study is to evaluate the clinico-pathological characteristics of peripheral schwannomas. METHODS: A retrospective cross sectional study of peripheral schwannomas excluding head neck region was conducted. The study group consisted of 18 cases which were recorded over a period of seven years. The corresponding data were collected from the archives of the Department of Pathology. RESULTS: Male to female ratio was 1:1. The average age of the cases was 47 years. The most common site was the upper limbs (55.55%) followed by lower limbs, chest and penis. The lesions mostly presented as painless swellings (62%). Histopathological examination revealed classic features of schwannoma. Secondary changes included cystic degeneration, foam cells, epitheloid cells, hyalinization, microcystic change and collection of plasma cells. All cases were confirmed by positive S100 staining. CONCLUSION: Peripheral schwannomas may be missed due to its rarity and atypical presentations. Both clinicians and pathologists should be aware of this common entity at unusual sites for the proper management of the patients. Surgery is usually the treatment of choice.

Pure yolk sac tumor of testis with lung metastasis in an adult patient - case report.

Pure yolk sac tumor is the most frequently seen testicular tumor in childhood; however it is usually a component of mixed germ cell tumor in adults. Pure yolk sac tumors are quite rare in adults. Pure yolk sac tumor in adults has been categorized as yolk sac tumor, post-pubertal type. We report a case of pure yolk sac tumor of right testis with lung metastasis in a 37-year-old man who was successfully treated by high inguinal orchidectomy and BEP combination chemotherapy.

Concurrent Thoracic Spinal Intradural Extramedullary Epidermoid and Neurenteric Cyst in a Spinal Dysraphism Child.

INTRODUCTION: Intraspinal epidermoid cysts are congenital or acquired in origin; whereas intraspinal neurenteric cysts (NECs) are of congenital origin. Their individual association with spinal dysraphism and vertebral segmentation anomalies is very well known. CASE PRESENTATION: We hereby report a case of concurrent intradural extramedullary epidermoid and NEC at adjacent vertebral levels in a spinal dysraphism child, not reported in English Literature till now. CONCLUSION: Multiple spinal lesions related to any/all of the 3 germ layers can coexist at same or adjacent vertebral levels in the same patient and surgical planning shown to be done accordingly.

Cutaneous Histoplasmosis in HIV Seronegative Patients: A Clinicopathological Analysis.

BACKGROUND: Diagnosis of extrapulmonary histoplasmosis in HIV seronegative and immunocompetent patients is often challenging, so a high index of suspicion is required. Cutaneous manifestation of infection shows a wide spectrum of lesions including erythematous plaques; maculopapules; crusted, verrucous, or desquamative papules and nodules; abscesses; and mucocutaneous ulcers among others. Due to the variations in its clinical presentation, histopathology plays a very important role in the detection of spores and the confirmation of diagnosis. OBJECTIVES: The aim of our study was to analyze clinicopathological characteristics of cutaneous manifestations of biopsy-proven histoplasmosis in HIV seronegative individuals. We also examined the utility of Fite stain for the diagnosis of Histoplasma capsulatum on tissue biopsy sections. METHODS: This was a retrospective, observational study on 7 patients who were HIV seronegative and clinically manifested with isolated cutaneous lesions or disseminated disease. Skin biopsy from the lesions was performed on all 7 patients. In addition to H&E staining and special stains for detecting fungus, Fite staining was performed on all of the cases to study its utility in detecting H. capsulatum spores. RESULTS: The skin lesions were widely disseminated in all patients and the most common cutaneous lesions were papules, present in all 7 patients. On review of the H&E-stained slides, the most common pattern was histiocytic lobular panniculitis-like infiltrate observed in 4 cases. Fite stain highlighted the yeast as magenta-colored spores on a blue background in all cases, except for 1 with a granulomatous pattern. CONCLUSION: A primary cutaneous manifestation of H. capsulatum infection in non-HIV-infected individuals is extremely rare. Fite stain could aid in differentiating the spores of H. capsulatum from those of other fungi, Cryptococcus and Candida in particular.

A Prospective Study Examining the Effect of Selected Topical and Systemic Drugs on Pruritus Grading System Score and STAT 6 Expression in Patients of Prurigo Nodularis.

Background: Prurigo nodularis (PN) is a chronic dermatologic condition presenting as multiple papulonodular lesions occurring with intense pruritus. Though numerous agents (topical, systemic, phototherapy and biological drugs) have been tried, the outcomes are variable. Objectives: The aim of this study was to assess the role of topical and systemic therapies in primary PN by comparing the Pruritus Grading System (PGS) score at baseline and 1 month post-therapy. Materials and Methods: Of 86 diagnosed cases of PN, 49 cases of primary PN were clinically graded by Pruritus Grading System Score (PGSS), and assessed histopathologically by IHC staining (STAT-1, 3, and 6). Apart from topical agents, oral nortriptyline (mild grade), methotrexate (moderate grade) and thalidomide (severe grade) were administered, whereas doxepin was administered for itching. The PGSS was assessed after 1 month of therapy. Results: Among 49 patients of PN, the majority of patients showed a significant decrease in PGSS (P = <0.001) in 1 mont, which correlated with STAT-6 expression. The combination of different topical and oral agents resulted in a statistically significant change in severity, though individual drugs did not achieve statistically significant results. Conclusion: A combination of selected oral and topical agents can effectively control the severity of PN within one month, and this was found to correlate with STAT 6 expression.

Epithelioid inflammatory myofibroblastic sarcoma: the youngest case reported

Epithelioid inflammatory myofibroblastic sarcoma (EIMS) is a rare variant of the inflammatory myofibroblastic tumor. It has an aggressive clinical course and a high rate of recurrence. EIMS primarily affects children and young adults. Hereby, we report this entity in a 4-month-old infant who presented with an abdominal mass. Imaging studies revealed a large hypodense mesentery-based lesion involving the right half and mid-region of the abdomen. The mass with an attached segment of the small bowel was excised in toto. Grossly, a large encapsulated tumor was identified arising from the mesentery of the small bowel. The histological examination showed a tumor consisting of epithelioid to spindle cells loosely arranged in a myxoid background with numerous blood vessels and lymphoplasmacytic inflammatory infiltrate. On immunohistochemistry, the tumor cells showed positivity for ALK1 (nuclear), desmin, SMA, CD68, and focal positivity for CD30. A final diagnosis of EIMS of the small intestine was rendered. To the best of our knowledge, this case is the youngest reported case in literature.

Giant virilising adrenal cortical carcinoma

Androgen secreting adrenocortical carcinoma (ACC) is a very rare disease with a poor prognosis. Approximately 80% of tumors are functional, most commonly secreting glucocorticoids. We herewith report a case of a huge functional ACC of the right adrenal gland in a 33-year-old female who presented with complaints of hirsutism, amenorrhea and an abdominal lump. On abdominal examination a large lump was palpable in the right hypochondrium reaching up to the umbilicus. Contrast-enhance computed tomography (CECT) revealed a mass in the right suprarenal region. The tumor measured 29 cm × 20 cm × 12 cm and weighed 7.8 kg, the largest reported case of ACC in the world to the best of our knowledge.

Adrenocortical Carcinoma in a 6-Month-Old Infant.

Adrenocortical carcinoma (ACC) is a rare malignant tumor arising from the adrenal cortex and it is even rarer in infancy. These tumors are usually hormonally active. We report a case of ACC in a 6-month-old infant who was brought by her mother with complaints of hirsutism and abnormally looking genitalia. Serum 17-alpha-hydroxyprogesterone and serum cortisol levels were raised. Subsequently, imaging studies revealed a large well-circumscribed soft-tissue mass in the right suprarenal region. The tumor was excised, and on histopathology in conjunction with immunohistochemistry, a diagnosis of ACC was given. Only four other cases of ACC have been reported till date in the literature, with an age of 6 months or younger. Hereby, we present a case of ACC in a young infant.