Towards routine chromosome-scale haplotype-resolved reconstruction in cancer genomics.

Cancer genomes are highly complex and heterogeneous. The standard short-read sequencing and analytical methods are unable to provide the complete and precise base-level structural variant landscape of cancer genomes. In this work, we apply high-resolution long accurate HiFi and long-range Hi-C sequencing to the melanoma COLO829 cancer line. Also, we develop an efficient graph-based approach that processes these data types for chromosome-scale haplotype-resolved reconstruction to characterise the cancer precise structural variant landscape. Our method produces high-quality phased scaffolds on the chromosome level on three healthy samples and the COLO829 cancer line in less than half a day even in the absence of trio information, outperforming existing state-of-the-art methods. In the COLO829 cancer cell line, here we show that our method identifies and characterises precise somatic structural variant calls in important repeat elements that were missed in short-read-based call sets. Our method also finds the precise chromosome-level structural variant (germline and somatic) landscape with 19,956 insertions, 14,846 deletions, 421 duplications, 52 inversions and 498 translocations at the base resolution. Our simple pstools approach should facilitate better personalised diagnosis and disease management, including predicting therapeutic responses.

Semi-automated assembly of high-quality diploid human reference genomes.

The current human reference genome, GRCh38, represents over 20 years of effort to generate a high-quality assembly, which has benefitted society1,2. However, it still has many gaps and errors, and does not represent a biological genome as it is a blend of multiple individuals3,4. Recently, a high-quality telomere-to-telomere reference, CHM13, was generated with the latest long-read technologies, but it was derived from a hydatidiform mole cell line with a nearly homozygous genome5. To address these limitations, the Human Pangenome Reference Consortium formed with the goal of creating high-quality, cost-effective, diploid genome assemblies for a pangenome reference that represents human genetic diversity6. Here, in our first scientific report, we determined which combination of current genome sequencing and assembly approaches yield the most complete and accurate diploid genome assembly with minimal manual curation. Approaches that used highly accurate long reads and parent-child data with graph-based haplotype phasing during assembly outperformed those that did not. Developing a combination of the top-performing methods, we generated our first high-quality diploid reference assembly, containing only approximately four gaps per chromosome on average, with most chromosomes within ±1% of the length of CHM13. Nearly 48% of protein-coding genes have non-synonymous amino acid changes between haplotypes, and centromeric regions showed the highest diversity. Our findings serve as a foundation for assembling near-complete diploid human genomes at scale for a pangenome reference to capture global genetic variation from single nucleotides to structural rearrangements.

Correlation of Nuclear Morphometry with Clinicopathologic Parameters in Malignant Breast Aspirates.

Shilpa GargBackground Nuclear size, shape, chromatin pattern, and nucleolar size and number have all been reported to change in breast cancer. Aim The aim of the study was to quantify nuclear changes on malignant breast aspirates using morphometry and to correlate the morphometric parameters with clinicopathologic features such as cytologic grade, tumor size, lymph node status, mitotic index, and histopathologic grade. Materials and Methods Forty-five cases of carcinoma breast diagnosed on cytology were included in this study. Cytologic grading was performed as per the Robinson's cytologic grading system. Nuclear morphometry was done on Papanicolaou stained smears. One hundred nonoverlapping cells per case were evaluated. Both geometrical and textural parameters were evaluated. Results Comparison of cytologic grades with most morphometric features (nuclear area, perimeter, shape, long axis, short axis, intensity, total run length, and TI homogeneity) was highly significant on statistical analysis. Correlation with tumor size yielded significant results for nuclear area, perimeter, long and short axes, and intensity with p < 0.05. The study of lymph node status and morphometry showed a highly significant statistical association with all the parameters. Mitotic count was significantly associated with all the geometric parameters and one textural parameter (total run length). On correlation of ductal carcinoma in situ and histopathological Grades 1 to 3 with morphometry, it was found that all the parameters except long-run emphasis were highly significant with p < 0.001. Conclusion Morphometry as a technique holds immense promise in prognostication in breast carcinoma.

Somatic structural variant formation is guided by and influences genome architecture.

The occurrence and formation of genomic structural variants (SVs) is known to be influenced by the 3D chromatin architecture, but the extent and magnitude have been challenging to study. Here, we apply Hi-C to study chromatin organization before and after induction of chromothripsis in human cells. We use Hi-C to manually assemble the derivative chromosomes following the occurrence of massive complex rearrangements, which allows us to study the sources of SV formation and their consequences on gene regulation. We observe an action-reaction interplay whereby the 3D chromatin architecture directly impacts the location and formation of SVs. In turn, the SVs reshape the chromatin organization to alter the local topologies, replication timing, and gene regulation in cis We show that SVs have a strong tendency to occur between similar chromatin compartments and replication timing regions. Moreover, we find that SVs frequently occur at 3D loop anchors, that SVs can cause a switch in chromatin compartments and replication timing, and that this is a major source of SV-mediated effects on nearby gene expression changes. Finally, we provide evidence for a general mechanistic bias of the 3D chromatin on SV occurrence using data from more than 2700 patient-derived cancer genomes.

Correlation of Nuclear Morphometry with Clinicopathologic Parameters in Malignant Breast Aspirates.

Objectives The primary objective of this study was to correlate nuclear morphometric parameters with clinicopathologic features such as cytologic grade, tumor size, lymph node status, mitotic index, and histopathologic grade. Secondary objective was to quantify nuclear changes on malignant breast aspirates using morphometry. Material and Methods Forty-five cases of carcinoma breast diagnosed on cytology were included in this study. These were graded into cytologic grades 1, 2, and 3 as per Robinson's cytologic grading system. Nuclear morphometry was done in all cases on smears stained with Papanicolaou stain. Clinicopathologic parameters including cytological grade, tumor size, lymph node status, mitotic count, and histological grade were correlated with nuclear morphometric parameters, namely, area, perimeter, shape, long axis, short axis, intensity, long-run emphasis, total run length, and T1 homogeneity. Results There were 9 cases in cytologic grade 1, 26 in grade 2, and 10 cases in cytologic grade 3. Histopathology showed 42 cases of infiltrating duct carcinoma, not otherwise specified (IDC, NOS) and 3 cases (6.7%) of ductal carcinoma in situ (DCIS). IDC (NOS) included 6, 27, and 9 cases in grades 1, 2, and 3, respectively. Majority of our cases had a tumor size less than 5 cm ( n = 38, 84.4%) and had positive nodes ( n = 30, 66.7%). Correlation of cytologic and histopathologic grades (including DCIS) with all morphometric features except long-run emphasis was statistically significant. Correlation of morphometry with tumor size yielded significant results for nuclear area, perimeter, long and short axes, and intensity with p < 0.05. Study of lymph node status (positive/negative) versus morphometry showed a highly significant statistical association with all the geometric as well as textural parameters. Mitotic count was significantly associated with all the geometric parameters and one textural parameter (total run length). Statistics Continuous variables were presented as mean ± standard deviation and compared using the two-tailed, independent sample t -test and one-way analysis of variance test. Tests were performed at significance level of 0.05. Conclusion Morphometry is an objective technique which holds immense promise in prognostication in breast carcinoma.

Computational methods for chromosome-scale haplotype reconstruction.

High-quality chromosome-scale haplotype sequences of diploid genomes, polyploid genomes, and metagenomes provide important insights into genetic variation associated with disease and biodiversity. However, whole-genome short read sequencing does not yield haplotype information spanning whole chromosomes directly. Computational assembly of shorter haplotype fragments is required for haplotype reconstruction, which can be challenging owing to limited fragment lengths and high haplotype and repeat variability across genomes. Recent advancements in long-read and chromosome-scale sequencing technologies, alongside computational innovations, are improving the reconstruction of haplotypes at the level of whole chromosomes. Here, we review recent and discuss methodological progress and perspectives in these areas.

Unusual Clinical Presentations in Early-Onset Childhood Sarcoidosis: A Correlation or Coincidence?

Sarcoidosis is a multisystem granulomatous disease which frequently affects young adults. Because of its rarity, the exact incidence and prevalence of childhood sarcoidosis is not known. It mostly affects children of older age group i.e., 13-15 years. Early onset sarcoidosis (<5 years) is characterized by a triad of arthritis, uveitis and rash. Late onset sarcoidosis present with a multisystem disease similar to adults, with frequent pulmonary infiltrations and lymphadenopathy. Herein, we report a case of early-onset childhood sarcoidosis in a four-year-old female along with uncommon clinical features like cutaneous ulceration, onycholysis and geographical tongue and its rarity in the literature.

Sclerosing Stromal Tumor: A Rare Ovarian Neoplasm.

Ovarian sex cord-stromal tumors are relatively infrequent neoplasms that account for approximately 8% of all primary ovarian neoplasm. Sex cord-stromal tumors of the ovary include granulosa cell tumors, fibrothecomas, Sertoli-Leydigcell tumors, steroid cell tumors, and sclerosing stromal tumors (SST). Sclerosing stromal tumors account for 2% to 6% of ovarian stromal tumors. Despite the rarity of this particular neoplasm, it is not always possible to predict the presence of this tumor preoperatively on the basis of clinical and sonographic findings. Histopathological and immunohistochemical (IHC) examinations confirm the diagnosis. Herein, the clinical findings and histopathological features of SST are described in a 24-year-old female.

Monthly rifampicin, ofloxacin, and minocycline therapy for generalized and localized granuloma annulare.

BACKGROUND: The localized form of granuloma annulare is usually self-limiting, resolving within 2 years. Generalized granuloma annulare, on the other hand, runs a protracted course, with spontaneous resolution being rare. It is also characterized by a later age of onset, an increased incidence of diabetes mellitus, poor response to therapy, and an increased prevalence of HLA Bw35. OBJECTIVE: To assess the efficacy of monthly pulsed rifampicin, ofloxacin, and minocycline (ROM) therapy in the management of granuloma annulare. METHODS: Six biopsy proven patients of granuloma annulare were included in the study, five of the generalized variety, and one localized. Three of these patients were resistant to standard modalities of treatment. All six patients were treated with pulses of once monthly ROM till complete resolution of all lesions. Results were analyzed in terms of complete resolution of lesions and side effects. Presence of comorbid conditions was noted. RESULT: All six patients were successfully treated with 4-8 pulses of monthly ROM. None of the patients reported any adverse effects. LIMITATIONS: Small sample size and the lack of a control group are limitations. CONCLUSION: Treatment with pulses of once monthly ROM caused complete resolution of lesions in both localized and generalized granuloma annulare, even in cases recalcitrant to conventional therapy. There were no side effects in any of the patients. Larger trials are needed to substantiate the efficacy of monthly ROM in granuloma annulare.

Surgical scar revision: an overview.

Scar formation is an inevitable consequence of wound healing from either a traumatic or a surgical intervention. The aesthetic appearance of a scar is the most important criteria to judge the surgical outcome. An understanding of the anatomy and wound healing along with experience, meticulous planning and technique can reduce complications and improve the surgical outcome. Scar revision does not erase a scar but helps to make it less noticeable and more acceptable. Both surgical and non-surgical techniques, used either alone or in combination can be used for revising a scar. In planning a scar revision surgeon should decide on when to act and the type of technique to use for scar revision to get an aesthetically pleasing outcome. This review article provides overview of methods applied for facial scar revision. This predominantly covers surgical methods.

Combination therapy in the management of atrophic acne scars.

BACKGROUND: Atrophic acne scars are difficult to treat. The demand for less invasive but highly effective treatment for scars is growing. OBJECTIVE: To assess the efficacy of combination therapy using subcision, microneedling and 15% trichloroacetic acid (TCA) peel in the management of atrophic scars. MATERIALS AND METHODS: Fifty patients with atrophic acne scars were graded using Goodman and Baron Qualitative grading. After subcision, dermaroller and 15% TCA peel were performed alternatively at 2-weeks interval for a total of 6 sessions of each. Grading of acne scar photographs was done pretreatment and 1 month after last procedure. Patients own evaluation of improvement was assessed. RESULTS: Out of 16 patients with Grade 4 scars, 10 (62.5%) patients improved to Grade 2 and 6 (37.5%) patients improved to Grade 3 scars. Out of 22 patients with Grade 3 scars, 5 (22.7%) patients were left with no scars, 2 (9.1%) patients improved to Grade 1and 15 (68.2%) patients improved to Grade 2. All 11 (100%) patients with Grade 2 scars were left with no scars. There was high level of patient satisfaction. CONCLUSION: This combination has shown good results in treating not only Grade 2 but also severe Grade 4 and 3 scars.

Intralesional immunotherapy for difficult to treat warts with Mycobacterium w vaccine.

BACKGROUND: Immunotherapy is an evolving therapeutic modality for the treatment of warts. We conducted a study to assess the efficacy and safety of intralesional Mycobacterium w vaccine for the treatment of warts at sites that were difficult to treat. MATERIALS AND METHODS: Thirty patients with at least one wart present on either the plantar surface of their feet, palms, volar aspect of their fingers, or periungual or subungual region, were treated with 0.1 ml of killed Mycobacterium w vaccine given intralesionally in a single wart, without any prior sensitisation dose. Thereafter, a single injection of 0.1 ml of vaccine was given at intervals of four weeks in a single wart till there was complete resolution of the warts or a maximum of 10 injections. Treatment was stopped if there was no response after three injections. The patients were followed up for at least six months. RESULTS: Out of the 30 patients, 28 (93.33%) patients had complete resolution of their warts, both at the injected and distant sites. The mean (SD) time for complete clearance of warts was 43.71(32.82) days and the mean (SD) dose of vaccine that was required for complete clearance of warts was 0.186 ml (0.101). Four patients (14.28%) had a recurrence of warts. The treatment was well-tolerated and the side effects were reversible in the majority of the patients. CONCLUSION: In comparison to the earlier studies using Mycobacterium w vaccine for the treatment of warts, our study was different in the following aspects: No sensitisation dose was given, only a single wart was injected at a time and the duration between the period of injections was increased to four weeks. With all these changes we eliminated the complications due to the sensitisation dose and achieved good results. This study provides new insight into the dose and schedule of treatment of this evolving therapeutic modality.

Generalized granuloma annulare treated with monthly rifampicin, ofloxacin, and minocycline combination therapy.

Granuloma annulare (GA) is a disease characterized by granulomatous inflammation of the dermis. A variant form of the disease, generalized granuloma annulare (GGA), can be observed in 15% of affected patients. Localized GA is likely to resolve spontaneously within months or a few years, whereas GGA can persist for decades. There are various therapies for treating GGA. Monthly combination therapy of rifampicin 600 mg, ofloxacin 400 mg, and minocycline 100 mg (ROM) is used for treating paucibacillary leprosy which shares both clinical and histopathologic similarities with GA. Therefore, we decided to evaluate the possible efficacy of monthly ROM in a patient with GGA.

Correlation of gallstone characteristics with mucosal changes in gall bladder.

BACKGROUND AND AIM: Gallstones are known to produce diverse histopathological changes in the gall bladder. Our aim was to correlate various gallstone characteristics (number, size, weight, volume and morphological type) with the type of mucosal response in gall bladder (inflammation, hyperplasia, metaplasia and carcinoma). METHODS: The study was conducted on 330 open cholecystectomy specimens with complete gallstones. The stones were assessed for various parameters i.e. number, size, weight, volume and morphological type. For microscopy, sections were obtained from the fundus, body and neck of the gallbladder. Additional sections were taken from abnormal looking areas. RESULTS: Out of the 330 cases, 194 (59%) had mixed stones, 84 (25%) combined, 30 (9%) pigment and 22 (7%) had cholesterol stones. Number of stones varied from a single calculus in 131 (39.6%) cases, double in 29 (8.8%) and multiple in the remaining 170 (51.6%) cases. Cholecystitis, hyperplasia, metaplasia and carcinoma were more commonly seen with mixed and multiple stones. The average weight of calculi in cholecystitis was 2.551 gm, in hyperplasia 3.619 gm, metaplasia4.549 gm and 17.96 gm in cases with carcinoma. Similarly, average volume of the stone(s) was 2.664 ml in cholecystitis, 3.742 ml in hyperplasia, 4.532 ml in metaplasia and 19.178 ml in carcinoma. The average calculus size (2.147 cm) was found to be maximum in cases with carcinoma, followed by hyperplasia (1.187 cm), metaplasia (1.145 cm) and cholecystitis (1.136 cm). CONCLUSION: As the weight, volume and size of the stone increases the changes in the gall bladder mucosa changes from cholecystitis, hyperplasia, metaplasia, dysplasia, to carcinoma.

Sebaceous carcinoma of the eyelid diagnosed on fine needle aspiration cytology.

Sebaceous carcinoma of the ocular adnexa is a malignant neoplasm which has aggressive local behavior and can metastasize to regional lymph nodes and distant organs. It is a malignant neoplasm known to masquerade as other benign and less malignant lesions, resulting in delay in diagnosis and relatively high morbidity and mortality. Aspiration cytological features of this neoplasm have not been well characterized in the literature. We report a case of this tumor diagnosed on fine needle aspiration. Clinically, a diagnosis of chalazion was made and fine needle aspiration cytology (FNAC) was performed. Cytological diagnosis of a malignant tumor with closest resemblance to sebaceous carcinoma was suggested which was confirmed on histopathology. Eyelid reconstruction was done after histopathological confirmation of tumor-free margins. The article highlights the role of FNAC in early diagnosis and subsequent appropriate surgical management of eyelid sebaceous gland carcinoma to prevent recurrence and metastasis.