Development of a Single Molecule Counting Assay to Differentiate Chromophobe Renal Cancer and Oncocytoma in Clinics.

Malignant chromophobe renal cancer (chRCC) and benign oncocytoma (RO) are two renal tumor types difficult to differentiate using histology and immunohistochemistry-based methods because of their similarity in appearance. We previously developed a transcriptomics-based classification pipeline with "Chromophobe-Oncocytoma Gene Signature" (COGS) on a single-molecule counting platform. Renal cancer patients (n = 32, chRCC = 17, RO = 15) were recruited from Augusta University Medical Center (AUMC). Formalin-fixed paraffin-embedded (FFPE) blocks from their excised tumors were collected. We created a custom single-molecule counting code set for COGS to assay RNA from FFPE blocks. Utilizing hematoxylin-eosin stain, pathologists were able to correctly classify these tumor types (91.8%). Our unsupervised learning with UMAP (Uniform manifold approximation and projection, accuracy = 0.97) and hierarchical clustering (accuracy = 1.0) identified two clusters congruent with their histology. We next developed and compared four supervised models (random forest, support vector machine, generalized linear model with L2 regularization, and supervised UMAP). Supervised UMAP has shown to classify all the cases correctly (sensitivity = 1, specificity = 1, accuracy = 1) followed by random forest models (sensitivity = 0.84, specificity = 1, accuracy = 1). This pipeline can be used as a clinical tool by pathologists to differentiate chRCC from RO.

The Role of Serial Liquid Biopsy in the Management of Metastatic Non-Small Cell Lung Cancer (NSCLC).

Lung cancer is the leading cause of cancer-related deaths. Surgery remains the best option to treat lung cancer when feasible. However, many cases are diagnosed beyond the initial stages. There has been tremendous progress in the treatment of lung cancer over the last few years. Studies have shown that biomarker-driven targeted therapies lead to better outcomes. Due to the technical difficulties and significant procedural risk associated with repeated tissue biopsies, analysis of tumor constituents circulating in the blood, such as circulating tumor DNA (ctDNA) and various proteins, is becoming more widely recognized as an alternative method of tumor sampling, i.e., liquid biopsy. Liquid biopsy is superior to tissue biopsy, as it is minimally invasive and easily repeatable. Given the recent data on changes in mutations as the disease progresses or responds to treatment, liquid biopsies can help monitor the changes and guide us in giving targeted drugs. Here we present a case of advanced NSCLC who was initially started on Alectinib based on positivity for ALK gene rearrangement found in the FISH study. At the time of progression, molecular profiling liquid biopsy was obtained, which revealed KRAS-p.G12C mutation. Thus, the patient's therapy was later on changed to sotorasib after the FDA approved a KRAS-p.G12C mutation inhibitor.

Anaplastic Lymphoma Kinase (ALK)-Negative Inflammatory Myofibroblastic Tumor of the Kidney in a Nine-Month-Old Girl.

An inflammatory myofibroblastic tumor (IMT) is an uncommon, benign tumor of myofibroblastic spindle cells. An IMT can occur in any part of the body. However, involving kidney is exceedingly rare. When this rare entity occurs in children, it becomes incredibly challenging to distinguish this rare entity from other malignancies such as Wilms tumor. Although imaging studies of the abdomen and pelvis add to the diagnosis, however, histological examination and immunohistochemical staining remain the gold standard for the precise diagnosis of this rare entity. To the best of our knowledge, only 48 cases of renal IMT have been published in the medical literature so far. We report the case of a nine-month-old girl who was brought with complaints of hematuria, and later, imaging and histological confirmation revealed an anaplastic lymphoma kinase (ALK)-negative IMT of the kidney.

Oncocytic Papillary Cystadenoma, an Unusual Variant Presenting as a Laryngeal Ventricular Cyst.

Cystadenoma arising from the larynx is a rare benign minor salivary gland tumor that can show mucinous or papillary morphology. The epithelial lining of the salivary gland tumor can present with oncocytic features, which is attributed to an increased number of mitochondria. We present a rare case of oncocytic papillary cystadenoma (OPC) of the larynx which has a combination of these features. The WHO defines OPC tumors as entities which closely resemble Warthin tumor, but lack its classic lymphoid component. The immunohistochemical profile and molecular genetic features are largely unknown. We present an 84-year-old female, former smoker, who presented with progressive dysphonia, dysphagia, and shortness of breath. Laryngoscopy revealed a large, smooth mass originating from the ventricle of the right vocal fold. Subsequent biopsy demonstrated cyst wall fragments lined by a bilayer of large columnar to cuboidal oncocytic cells that had granular eosinophilic cytoplasm, round to oval nuclei with finely dispersed chromatin, and small but distinct nucleoli. The surrounding stroma was slightly fibrotic with scant lymphoid elements. No nuclear pleomorphism, increased mitosis, or necrosis was identified. In the larynx, benign salivary gland tumors are rare and less frequent than malignant neoplasms. Awareness of rare benign entities like OPC help ensure proper management and aid in avoiding unnecessary therapy.

Label-free ferrohydrodynamic cell separation of circulating tumor cells.

Circulating tumor cells (CTCs) have significant implications in both basic cancer research and clinical applications. To address the limited availability of viable CTCs for fundamental and clinical investigations, effective separation of extremely rare CTCs from blood is critical. Ferrohydrodynamic cell separation (FCS), a label-free method that conducted cell sorting based on cell size difference in biocompatible ferrofluids, has thus far not been able to enrich low-concentration CTCs from cancer patients' blood because of technical challenges associated with processing clinical samples. In this study, we demonstrated the development of a laminar-flow microfluidic FCS device that was capable of enriching rare CTCs from patients' blood in a biocompatible manner with a high throughput (6 mL h-1) and a high rate of recovery (92.9%). Systematic optimization of the FCS devices through a validated analytical model was performed to determine optimal magnetic field and its gradient, ferrofluid properties, and cell throughput that could process clinically relevant amount of blood. We first validated the capability of the FCS devices by successfully separating low-concentration (∼100 cells per mL) cancer cells using six cultured cell lines from undiluted white blood cells (WBCs), with an average 92.9% cancer cell recovery rate and an average 11.7% purity of separated cancer cells, at a throughput of 6 mL per hour. Specifically, at ∼100 cancer cells per mL spike ratio, the recovery rates of cancer cells were 92.3 ± 3.6% (H1299 lung cancer), 88.3 ± 5.5% (A549 lung cancer), 93.7 ± 5.5% (H3122 lung cancer), 95.3 ± 6.0% (PC-3 prostate cancer), 94.7 ± 4.0% (MCF-7 breast cancer), and 93.0 ± 5.3% (HCC1806 breast cancer), and the corresponding purities of separated cancer cells were 11.1 ± 1.2% (H1299 lung cancer), 10.1 ± 1.7% (A549 lung cancer), 12.1 ± 2.1% (H3122 lung cancer), 12.8 ± 1.6% (PC-3 prostate cancer), 11.9 ± 1.8% (MCF-7 breast cancer), and 12.2 ± 1.6% (HCC1806 breast cancer). Biocompatibility study on H1299 cell line and HCC1806 cell line showed that separated cancer cells had excellent short-term viability, normal proliferation and unaffected key biomarker expressions. We then demonstrated the enrichment of CTCs in blood samples obtained from two patients with newly diagnosed advanced non-small cell lung cancer (NSCLC). While still at its early stage of development, FCS could become a complementary tool for CTC separation for its high recovery rate and excellent biocompatibility, as well as its potential for further optimization and integration with other separation methods.

Multiple Discordant Histology After Nephrectomy: Descriptive Analysis and Outcomes.

BACKGROUND: While RCC is the most common primary renal neoplasm, few cases of ipsilateral renal lesions of different RCC histologic subtypes have been described. The objective of this study was to evaluate our experience with synchronous, histologically unique, primary renal neoplasms within the same kidney. PATIENTS AND METHODS: We retrospectively analyzed 2 institutional nephrectomy databases from 2000 to 2013. The study cohort comprised 15 patients with multiple, discordant renal histology after partial or radical nephrectomy. Demographic data, immunohistochemical analysis, and clinical course were assessed and analyzed. RESULTS: Eight patients (53%) were black, 10 (60%) were male, and 5 (36%) were tobacco users. Median follow-up time was 13 months (range, 1-62 months), and 9 patients (56%) underwent radical nephrectomy. Among 36 tumors, the median tumor size was 2.3 cm (range, 0.4-9 cm). The most common combination of discordant tumor histology among patients with ≥ 2 tumors was clear-cell (cc) renal-cell carcinoma (RCC) with chromophobe RCC (3 cases, 19%). In 3 patients (19%), a single tumor was noted to have 2 distinct patterns; all patients had ccRCC with papillary RCC. Three (20%) of 15 patients developed metastatic disease. The median cancer-free survival time for patients with metastasis was 2 months. CONCLUSION: Multiple, discordant renal pathology represents a rarely reported entity in patients receiving nephrectomy. We introduce the largest cohort of synchronous renal tumors, of which ccRCC/chromophobe RCC was the most common pairing.

6p22.3 amplification as a biomarker and potential therapeutic target of advanced stage bladder cancer.

Genetic and epigenetic alterations have been identified as to contribute directly or indirectly to the generation of transitional cell carcinoma of the urinary bladder (TCC-UB). In a comparative fashion much less is known about copy number alterations in TCC-UB, but it appears that amplification of chromosome 6p22 is one of the most frequent changes. Using fluorescence in situ hybridization (FISH) analyses, we evaluated chromosomal 6p22 amplification in a large cohort of bladder cancer patients with complete surgical staging and outcome data. We have also used shRNA knockdown candidate oncogenes in the cell based study. We found that amplification of chromosome 6p22.3 is significantly associated with the muscle-invasive transitional cell carcinoma of the urinary bladder (TCC-UB) (22%) in contrast to superficial TCC-UB (9%) (p=7.2-04). The rate of 6p22.3 amplification in pN>1 patients (32%) is more than twice that in pN0 (16%) patients (p=0.05). Interestingly, we found that 6p22.3 amplification is as twice as high (p=0.0201) in African American (AA) than European American (EA) TCC-UB patients. Moreover, we showed that the expression of some candidate genes (E2F3, CDKAL1 and Sox4) in the 6p22.3 region is highly correlated with the chromosomal amplification. In particular, knockdown of E2F3 inhibits cell proliferation in a 6p22.3-dependent manner, whereas knockdown of CDKAL1 and Sox4 has no effect on cell proliferation. Using gene expression profiling, we further identified some common as well as distinctive subset targets of the E2F3 family members. In summary, our data indicate that E2F3 is a key regulator of cell proliferation in a subset of bladder cancer and the 6p22.3 amplicon is a biomarker of aggressive phenotype in this tumor type.