Performance of Molecular Lymphosonography for Detection and Quantification of Metastatic Involvement in Sentinel Lymph Nodes.

OBJECTIVES: To assess the performance of molecular lymphosonography with dual-targeted microbubbles in detecting and quantifying the metastatic involvement in sentinel lymph nodes (SLNs) using a swine melanoma model. METHODS: Targeted microbubbles were labeled with P-selectin and αV ß3 -integrin antibodies. Control microbubbles were labeled with immunoglobulin G antibodies. First lymphosonography with Sonazoid (GE Healthcare, Oslo, Norway) was used to identify SLNs. Then dual-targeted and control microbubbles were injected intravenously to detect and quantify metastatic disease in the SLNs. Distant non-SLNs were imaged as benign controls. All evaluated lymph nodes (LNs) were surgically removed, and metastatic involvement was characterized by a histopathologic analysis. Two radiologists blinded to histopathologic results assessed the baseline B-mode images of LNs, and the results were compared to the histologic reference standard. The mean intensities of targeted and control microbubbles within the examined LNs were measured and compared to the LN histologic results. RESULTS: Thirty-five SLNs and 34 non-SLNs from 13 Sinclair swine were included in this study. Twenty-one SLNs (62%) were malignant, whereas 100% of non-SLNs were benign. The sensitivity of B-mode imaging for metastatic LN diagnosis for both readers was relatively high (90% and 71%), but the specificity was very poor (50% and 58%). The sensitivity and specificity of molecular lymphosonography for metastatic LN detection were 91% and 67%, respectively. The mean intensities from dual-targeted microbubbles correlated well with the degree of metastatic LN involvement (r = 0.6; P < 0.001). CONCLUSIONS: Molecular lymphosonography can increase the specificity of metastatic LN detection and provide a measure to quantify the degree of metastatic involvement.

CD8+ and FoxP3+ T-Cell Cellular Density and Spatial Distribution After Programmed Death-Ligand 1 Check Point Inhibition.

OBJECTIVES: To analyze CD8+ and FoxP3+ T-cell cellular density (CD) and intercellular distances (ID) in head and neck squamous cell carcinoma (HNSCC) samples from a neoadjuvant trial of durvalumab +/- metformin. METHODS: Paired pre- and post-treatment primary HNSCC tumor samples were stained for CD8+ and FoxP3+. Digital image analysis was used to determine estimated mean CD8+ and FoxP3+ CDs and CD8+-FoxP3+ IDs in the leading tumor edge (LTE) and tumor adjacent stroma (TAS) stratified by treatment arm, human papillomavirus (HPV) status, and pathologic treatment response. A subset of samples was characterized for T-cell related signatures using digital spatial genomic profiling. RESULTS: Post-treatment analysis revealed a significant decrease in FoxP3+ CD and an increase in CD8+ CDs in the TAS between patients receiving durvalumab and metformin versus durvlaumab alone. Both treatment arms demonstrated significant post-treatment increases in ID. Although HPV+ and HPV- had similar immune cell CDs in the tumor microenvironment, HPV+ pre-treatment samples had 1.60 times greater ID compared with HPV- samples, trending toward significance (p = 0.05). At baseline, pathologic responders demonstrated a 1.16-fold greater CD8+ CDs in the LTE (p = 0.045) and 2.28-fold greater ID (p = 0.001) than non-responders. Digital spatial profiling revealed upregulation of FoxP3+ and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) in the TAS (p = 0.006, p = 0.026) in samples from pathologic responders. CONCLUSIONS: Analysis of CD8+ and FoxP3+ detected population differences according to HPV status, pathologic response, and treatment. Greater CD8+-FoxP3+ ID was associated with pathologic response. CD8+ and FoxP3+ T-cell distributions may be predictive of response to immune checkpoint inhibition. CLINICALTRIALS: gov (Identifier NCT03618654). LEVEL OF EVIDENCE: 3 Laryngoscope, 133:1875-1884, 2023.

Disease-free probability after the first primary ductal carcinoma in situ of the breast: a comparison between African-American and White-American women.

Compelling evidence about the differences in the biology and behavior of invasive breast cancer between African-American (AA) and White-American (WA) women motivate inquiry into comparing the clinicopathology of non-invasive breast cancer (ductal carcinoma in situ, DCIS). AA and WA women diagnosed with their first primary DCIS between 1990 and 1999 were identified from the institutional tumor registry. Data on method of presentation, treatment, and patient characteristics were retrieved from electronic medical records. Patients were followed up through the medical records until the diagnosis of a subsequent cancer or the last day of contact with the institution. A total of 100 (29.6%) AAs and 236 (70.4%) WAs with the mean age of 60 (SD ± 13) and 57 (SD ± 12), respectively, contributed to this study. DCIS was detected during routine screening mammography for 81% (n = 81) of AAs and 88.4% (n = 206) of WAs (P = 0.073). Differences in the distributions of grade, margin status, necrosis, or treatment modalities were not statistically significant between AAs and WAs. Analysis of competing risks Cox proportional hazard multivariate modeling yielded a significant 8-year cumulative risk of a second cancer for AAs but only in the ipsilateral breast (HR = 3.96, 95% CI 1.42-11.04, P = 0.01). Despite comparable clinical presentation and treatment, 8 years after the initial treatment, AAs experienced a higher risk of second breast cancer in ipsilateral but not in the contralateral breast. The observed excess risk of a second cancer in the ipsilateral breast may suggest of intrinsic differences in the biology of cancer.

Volume Doubling Time of Pulmonary Carcinoid Tumors Measured by Computed Tomography.

INTRODUCTION: Pulmonary carcinoid tumor (PCT) is a rare neuroendocrine lung neoplasm comprising approximately 2% of lung cancer diagnoses. It is classified as either localized low-grade (typical) or intermediate-grade (atypical) subtypes. PCT is known clinically to be a slow-growing cancer, however few studies have established its true growth rate when followed over time by computed tomography (CT). Therefore, we sought to determine the volume doubling time for PCTs as visualized on CT imaging. MATERIALS AND METHODS: We conducted a retrospective analysis of all PCTs treated at our institution between 2006 and 2020. Nodule dimensions were measured using a Picture Archiving and Communication System or retrieved from radiology reports. Volume doubling time was calculated using the Schwartz formula for PCTs followed by successive CT scans during radiographic surveillance. Consistent with Fleischner Society guidelines, tumors were considered to have demonstrated definitive growth by CT only when the interval change in tumor diameter was greater than or equal to 2 mm. RESULTS: The median volume doubling time of 13 typical PCTs was 977 days, or 2.7 years. Five atypical PCTs were followed longitudinally, with a median doubling time of 327 days, or 0.9 years. CONCLUSIONS: Typical pulmonary carcinoid features a remarkably slow growth rate as compared to more common lung cancers. Our analysis of atypical pulmonary carcinoid included too few cases to offer definitive conclusions. It is conceivable that clinicians following current nodule surveillance guidelines may mistake incidentally detected typical carcinoids for benign non-growing lesions when followed for less than 2 years in low-risk patients.

Whole-exome sequencing identifies somatic mutations and intratumor heterogeneity in inflammatory breast cancer.

Inflammatory breast cancer (IBC) is the most aggressive form of breast cancer. Although it is a rare subtype, IBC is responsible for roughly 10% of breast cancer deaths. In order to obtain a better understanding of the genomic landscape and intratumor heterogeneity (ITH) in IBC, we conducted whole-exome sequencing of 16 tissue samples (12 tumor and four normal samples) from six hormone-receptor-positive IBC patients, analyzed somatic mutations and copy number aberrations, and inferred subclonal structures to demonstrate ITH. Our results showed that KMT2C was the most frequently mutated gene (42%, 5/12 samples), followed by HECTD1, LAMA3, FLG2, UGT2B4, STK33, BRCA2, ACP4, PIK3CA, and DNAH8 (all nine genes tied at 33% frequency, 4/12 samples). Our data indicated that PTEN and FBXW7 mutations may be considered driver gene mutations for IBC. We identified various subclonal structures and different levels of ITH between IBC patients, and mutations in the genes EIF4G3, IL12RB2, and PDE4B may potentially generate ITH in IBC.

Metformin Effects on FOXP3+ and CD8+ T Cell Infiltrates of Head and Neck Squamous Cell Carcinoma.

OBJECTIVES: Alterations of cellular metabolism have been implicated in immune dysfunction in the tumor microenvironment (TME) of head and neck squamous cell carcinoma (HNSCC). Metformin has recently emerged as a candidate of interest for combination with immunotherapy in HNSCC. This study investigated the effect of metformin on immune cell infiltrates of HNSCC. METHODS: Retrospective analysis of T cell infiltrates in primary tumor specimens from patients enrolled in a clinical window of opportunity trial of presurgical metformin. Metformin was titrated to a standard diabetic dose (2000 mg/day) for a minimum of 9 days (mean 13.6 days) prior to surgical resection. Pre and posttreatment surgical specimens from 36 patients (16 HPV+ , 20 HPV- ) were comparatively analyzed. FOXP3+ and CD8+ immune cell infiltrates in the tumor and peritumoral stroma of pre and posttreatment HNSCC specimens were quantified by digital image analysis using Visiopharm software. RESULTS: Metformin treatment was associated with a 41.4% decrease in FOXP3+ T cells in intratumor regions of interest (P = .004) and a 66.5% increase in stromal CD8+ T cells at the leading edge of the tumor (P = .021) when compared to pretreatment biopsies. This was reflected in increased CD8+ /FOXP3+ cell ratios within the tumor (P < .001) and stromal compartments (P < .001). The effects of metformin occurred independently of HPV status. CONCLUSION: Metformin treatment may favorably alter the immune TME in HNSCC independent of HPV status. LEVEL OF EVIDENCE: 1b. This study is most accurately described as a non-randomized controlled trial and therefore may reflect a level of evidence below 1b but above 2a from the provided "levels of evidence" chart. Laryngoscope, 130:E490-E498, 2020.

Discordant Responses Between Primary Head and Neck Tumors and Nodal Metastases Treated With Neoadjuvant Nivolumab: Correlation of Radiographic and Pathologic Treatment Effect.

PD-1 blockade represents a promising treatment in patients with head and neck squamous cell carcinoma (HNSCC). We analyzed results of a neoadjuvant randomized window-of-opportunity trial of nivolumab plus/minus tadalafil to investigate whether immunotherapy-mediated treatment effects vary by site of involvement (primary tumor, lymph nodes) and determine how radiographic tumor shrinkage correlates with pathologic treatment effect. PATIENTS AND METHODS: Forty-four patients enrolled in trial NCT03238365 were treated with nivolumab 240 mg intravenously on days 1 and 15 with or without oral tadalafil, as determined by random assignment, followed by surgery on day 31. Radiographic volumetric response (RVR) was defined as percent change in tumor volume from pretreatment to posttreatment CT scan. Responders were defined as those with a 10% reduction in the volume of the primary tumor or lymph nodes (LN). Pathologic treatment effect (PTE) was defined as the area showing fibrosis or lymphohistiocytic inflammation divided by total tumor area. RESULTS: Sixteen of 32 patients (50%) with pathologic evidence of LN involvement exhibited discordant PTE between primary sites and LN. In four patients with widely discordant adjacent LN, increased PTE was associated with increased infiltration of tumor CD8+ T cells and CD163+ macrophages, whereas stromal regulatory T cells were associated with low nodal PTE. RVR correlated with PTE at both primary tumor (slope = 0.55, p < 0.001) and in LN (slope = 0.62, p < 0.05). 89% (16/18) of radiographic non-responders with T1-T3 primary sites had no (n = 7) or minimal PTE (n = 9), whereas 15/17 (88%) of radiographic responders had moderate (n = 12) or complete (n = 3) PTE. CONCLUSION: Nivolumab often induces discordant treatment effects between primary tumor sites and metastatic lymph nodes within subjects. This treatment discordance was also demonstrated in adjacent lymph nodes, which may correlate with local immune cell makeup. Finally, although these data were generated by a relatively small population size, our data support the use of early radiographic response to assess immunotherapy treatment effect in HNSCC.

Review of Cytology Practice at Thomas Jefferson University Hospital before and after High-Risk Human Papillomavirus Testing.

OBJECTIVE: We performed a retrospective review of Papanicolaou (Pap) testing to assess whether the cytology practice in our institution was affected by the introduction of high-risk (HR) human papillomavirus (HPV) assays over time. STUDY DESIGN: Cytology, HPV and histopathology records were retrieved from our laboratory information system from 2003 to 2015. Records for Digene Hybrid Capture 2®, Hologic Cervista® and Roche Cobas® HPV assays were obtained. A 3-month follow-up for HPV detected cases was performed, and results were correlated with cytology and biopsies. A 1-year follow-up of HPV 16/18 and other HR HPV detected cases was also performed. RESULTS: From 2008 to 2015, a noticeable decrease in Pap testing volume occurred, from 11,792 to 4,664, while the percentage of HPV testing increased from 19 to 59%. Similar HPV detection rates and follow-up results for both reflex and cotesting were observed in the 3 HPV assays. CONCLUSIONS: The decrease in Pap testing was due to the lengthening of the test interval when cotesting results were negative. Practitioners adhering to guidelines accounts for increased molecular testing volume. A trend towards higher-grade cervical intraepithelial neoplasia in the follow-up of detected HPV 16/18 was noted. So far there has been no demand for HPV as a stand-alone test.

Osteocutaneous radial forearm reconstruction of large partial cricotracheal defects.

BACKGROUND: Single-stage procedures for reconstruction of large cricotracheal defects have been limited in success and malignant immature teratomas in the larynx of an adult have never been reported. METHODS AND RESULTS: This was a case report and literature review. A 27-year-old man presented with 2 weeks of new-onset stridor and was found to have a mass obstructing 80% of the subglottis and trachea. He underwent a noncircumferential partial cricotracheal resection with a resultant 7-cm luminal defect, followed by osteocutaneous radial forearm flap semi-rigid reconstruction. A novel technique was used to allow a functional and patent airway, which may be applied to other partial luminal reconstructive defects. Postoperatively, the patient was decannulated and has good speech quality and swallowing function. This work was performed at the Henry Ford Health System. This project was reviewed and was granted full approval by the Henry Ford Health System Institutional Review Board. CONCLUSIONS: This is the first reported case of malignant immature teratoma to involve the larynx, trachea, and recurrent laryngeal nerve in an adult patient, and the first single-stage technique described to use free tissue transfer without prefabrication for a 7-cm airway defect.

Aurora A is differentially expressed in gliomas, is associated with patient survival in glioblastoma and is a potential chemotherapeutic target in gliomas.

Aurora A is critical for mitosis and is overexpressed in several neoplasms. Its overexpression transforms cultured cells, and both its overexpression and knockdown cause genomic instability. In transgenic mice, Aurora A haploinsufficiency, not overexpression, leads to increased malignant tumor formation. Aurora A thus appears to have both tumor-promoting and tumor-suppressor functions. Here, we report that Aurora A protein, measured by quantitative protein gel blotting, is differentially expressed in major glioma types in lineage-specific patterns. Aurora A protein levels in WHO grade II oligodendrogliomas (n=16) and grade III anaplastic oligodendrogliomas (n=16) are generally low, similar to control epilepsy cerebral tissue (n=11). In contrast, pilocytic astrocytomas (n=6) and ependymomas (n=12) express high Aurora A levels. Among grade II to grade III astrocytomas (n=7, n=14, respectively) and grade IV glioblastomas (n=31), Aurora A protein increases with increasing tumor grade. We also found that Aurora A expression is induced by hypoxia in cultured glioblastoma cells and is overexpressed in hypoxic regions of glioblastoma tumors. Retrospective Kaplan-Meier analysis revealed that both lower Aurora A protein measured by quantitative protein gel blot (n=31) and Aurora A mRNA levels measured by real-time quantitative RT-PCR (n=58) are significantly associated with poorer patient survival in glioblastoma. Furthermore, we report that the selective Aurora A inhibitor MLN8237 is potently cytotoxic to glioblastoma cells, and that MLN8237 cytotoxicty is potentiated by ionizing radiation. MLN8237 also appeared to induce senescence and differentiation of glioblastoma cells. Thus, in addition to being significantly associated with survival in glioblastoma, Aurora A is a potential new drug target for the treatment of glioblastoma and possibly other glial neoplasms.

The usefulness of IgG and IgM immunostaining of periportal inflammatory cells (plasma cells and lymphocytes) for the distinction of autoimmune hepatitis and primary biliary cirrhosis and their staining pattern in autoimmune hepatitis-primary biliary cirrhosis overlap syndrome.

Autoimmune hepatitis (AIH)-primary biliary cirrhosis (PBC) overlap syndrome (OS) is a vaguely defined entity demonstrating features of AIH and PBC. We investigated the usefulness of IgG and IgM immunostaining for the distinction of AIH and PBC and their staining pattern in cases of possible OS. The approximate quantity of IgG+ and IgM+ periportal inflammatory cells in immunohistochemical analysis were compared in cases of AIH, PBC, and OS. AIH cases showed predominant IgG immunostaining of periportal inflammatory cells. A significant number of PBC cases also demonstrated IgG predominance rather than IgM. Six OS cases had IgG predominance, 4 had IgM predominance, and 1 was equivocal. The usefulness of IgG and IgM immunostaining is limited in PBC cases with IgM predominance for excluding AIH. IgG predominance is not specific for AIH. OS does not demonstrate either IgG or IgM predominance (P > .2) and does not help classify OS into either category.