Lithium isotopic evidence for enhanced reverse weathering during the Early Triassic warm period.

Elevated temperatures persisted for an anomalously protracted interval following pulsed volcanic carbon release associated with the end-Permian mass extinction, deviating from the expected timescale of climate recovery following a carbon injection event. Here, we present evidence for enhanced reverse weathering-a CO2 source-following the end-Permian mass extinction based on the lithium isotopic composition of marine shales and cherts. We find that the average lithium isotopic composition of Lower Triassic marine shales is significantly elevated relative to that of all other previously measured Phanerozoic marine shales. Notably, the record generated here conflicts with carbonate-based interpretations of the lithium isotopic composition of Early Triassic seawater, forcing a re-evaluation of the existing framework used to interpret lithium isotopes in sedimentary archives. Using a stochastic forward lithium cycle model, we demonstrate that elevated reverse weathering is required to reproduce the lithium isotopic values and trends observed in Lower Triassic marine shales and cherts. Collectively, this work provides direct geochemical evidence for enhanced reverse weathering in the aftermath of Earth's most severe mass extinction.

Diagnostic accuracy of FNA to determine HPV status in HPV-associated oropharyngeal squamous cell carcinoma.

PURPOSE/OBJECTIVE(S): Accurate diagnosis of human papillomavirus (HPV) status in oropharyngeal squamous cell carcinoma (OPSCC) affects prognosis and can alter the treatment plan. We evaluated the diagnostic accuracy of FNA biopsies to determine malignancy and HPV status in OPSCC at our institution. METHODS: Pathology samples from consecutive patients with pathologically confirmed HPV-associated OPSCC who underwent FNA of a cervical lymph node during initial diagnostic work-up were retrospectively analyzed between November 2015 and August 2021. RESULTS: Initial FNA was diagnostic for malignancy in 109/148 (73.6%) patients and non-diagnostic in 39/148 (26.4%). P16 staining of FNAs positive for malignancy showed: 54/109 (49.5%) p16 positive, 6/109 (5.5%) p16 negative, 49/109 (45.0%) p16 indeterminate. In patients with an initial non-diagnostic sampling or p16 indeterminate, repeat FNA was performed in 30/88 (34.1%) patients. Of the 30 repeat FNAs: 23/30 (76.7%) were diagnostic of malignancy and 7/30 (23.3%) remained non-diagnostic for malignancy. Of the 23 repeat FNAs diagnostic of malignancy: 16/23 (69.6%) were p16 positive and 7/23 (30.4%) were p16 indeterminate. In summary, 88/148 (59.5%) initial FNAs and 14/30 (46.7%) of repeat FNAs were non-diagnostic of malignancy or p16 indeterminate. Final yield of FNA biopsies (initial and first repeat FNA) to diagnose malignancy and p16 status was 70/148 (47.3%). CONCLUSIONS: Fine needle aspirations of lymph nodes in patients with HPV-associated OPSCC are frequently non-diagnostic for malignancy or indeterminate for p16 status, requiring repeat FNA or biopsy of the primary site. This can potentially cause treatment delay and increase morbidity and cost to the patient.

Qualitative and quantitative methods in post-chemoradiation PET for head and neck cancer.

OBJECTIVE: The aim of this study was to determine whether quantitative methods could aid in the evaluation of post-treatment head and neck scans, particularly taking human papillomavirus status into account. METHODS: Clinical readings of positron emission tomography/computed tomography scans as well as standardized uptake value (SUV)max (and other metrics) of nodes visible on PET conducted on a total of 172 patients with head and neck squamous cell cancer were examined. Locoregional recurrence at 2 years was assessed. In total 88 of these patients had close enough follow-up to determine whether individual nodes were positive or negative, and 233 nodes on these patients were compared to surgical pathology notes or follow-up (if no path was available). RESULTS: General negative predictive value (NPV) of complete response was 93% and an equivocal response was 89%; focusing on nodal recurrence, NPV was found to be 97% and positive predictive value (PPV) 46% if equivocal reads were treated as negative and NPV 98% and PPV 16% if equivocal reads were treated as positive. Using SUVmax of the hottest node with a cutoff of 3.4 gave NPV 97% and PPV 26%; a direct re-read (using 2 observers) gave NPV 98% and PPV 32% if equivocal reads were treated as negative, and NPV 99% and PPV 18% if equivocal reads were treated as positive. Using other first-order radiomics data such as SD and skewness did not improve this. CONCLUSIONS: Quantitative data such as SUVmax does not show additional value over qualitative evaluation of response to chemoradiation in head and neck tumors.

Does Timing Matter? Surgical Outcomes in High-Grade Sarcomas after Neoadjuvant Radiation Therapy.

BACKGROUND: The impact of time to surgical resection after neoadjuvant external beam radiation therapy (EBRT) in the high-grade soft tissue and retroperitoneal sarcomas has not been well established. We aimed to evaluate how surgical timing from EBRT affects oncologic and perioperative outcomes. METHODS: We performed a single institution retrospective cohort study of patients with biopsy-proven, high-grade sarcoma who completed neoadjuvant EBRT and resection from January 1, 1999 to September 1, 2018. We collected demographic and clinicopathologic variables, stratifying patients by time interval between EBRT and surgery: <6, 6-8, 8-10, and >10 wk. Primary outcomes collected were as follows: disease-free survival, overall survival, and perioperative complications. RESULTS: Of the 269 patients identified, 146 met inclusion criteria. The median follow-up was 24 mo. Overall and local recurrence were 37% (n = 54) and 14.4% (n = 21), respectively. Time to surgery did not affect recurrence (P = 0.82) or survival (P = 0.88). Positive margins (odds ratio 2.7, confidence interval 1.14, 6.51, P < 0.05) were predictive of recurrence. Primary tumor location, surgical timing, histology, and intraoperative radiation therapy were not associated with differences in recurrence. The overall complication rate was 28%, with 63% from wound infections. Fewer postoperative complications occurred in the < 6-wk cohort versus > 6-wk cohort (15% versus 38%, P < 0.05). CONCLUSIONS: We found no difference in oncologic outcomes associated with the timing of surgical resection after EBRT. Patients undergoing resection >6 wk were at higher risk for all complications without impacting wound complication rates. Future studies may include preoperative optimization of patients requiring delays in surgical planning to decrease perioperative complication rates.

Alternatives to Surgery for Early-Stage Non-Small Cell Lung Cancer: Stereotactic Radiotherapy.

Lung cancer is the leading cause of cancer death worldwide. Diagnosis of early-stage disease is becoming more common. In an aging population, more and more patients have substantial comorbidities that might limit feasibility of surgical management of early-stage disease. Stereotactic body radiotherapy (SBRT) enables delivery of high-dose, precisely delivered radiation to early-stage lung cancers without surgical risk. This technique has rates of local control similar to surgery and can be considered in medically operable patients who refuse surgery. This article details the technique of SBRT, the data for its efficacy, as well as the potential toxicities of treatment.

PIK3CA Mutation in HPV-Associated OPSCC Patients Receiving Deintensified Chemoradiation.

PIK3CA is the most frequently mutated gene in human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OPSCC). Prognostic implications of such mutations remain unknown. We sought to elucidate the clinical significance of PIK3CA mutations in HPV-associated OPSCC patients treated with definitive chemoradiation (CRT). Seventy-seven patients with HPV-associated OPSCC were enrolled on two phase II clinical trials of deintensified CRT (60 Gy intensity-modulated radiotherapy with concurrent weekly cisplatin). Targeted next-generation sequencing was performed. Of the 77 patients, nine had disease recurrence (two regional, four distant, three regional and distant). Thirty-four patients had mutation(s) identified; 16 had PIK3CA mutations. Patients with wild-type-PIK3CA had statistically significantly higher 3-year disease-free survival than PIK3CA-mutant patients (93.4%, 95% confidence interval [CI] = 85.0% to 99.9% vs 68.8%, 95% CI = 26.7% to 89.8%; P = .004). On multivariate analysis, PIK3CA mutation was the only variable statistically significantly associated with disease recurrence (hazard ratio = 5.71, 95% CI = 1.53 to 21.3; P = .01). PIK3CA mutation is associated with worse disease-free survival in a prospective cohort of newly diagnosed HPV-associated OPSCC patients treated with deintensified CRT.

Rapid Clearance Profile of Plasma Circulating Tumor HPV Type 16 DNA during Chemoradiotherapy Correlates with Disease Control in HPV-Associated Oropharyngeal Cancer.

PURPOSE: To identify a profile of circulating tumor human papilloma virus (HPV) DNA (ctHPVDNA) clearance kinetics that is associated with disease control after chemoradiotherapy (CRT) for HPV-associated oropharyngeal squamous cell carcinoma (OPSCC). EXPERIMENTAL DESIGN: A multi-institutional prospective biomarker trial was conducted in 103 patients with (i) p16-positive OPSCC, (ii) M0 disease, and (iii) receipt of definitive CRT. Blood specimens were collected at baseline, weekly during CRT, and at follow-up visits. Optimized multianalyte digital PCR assays were used to quantify ctHPVDNA (types 16/18/31/33/35) in plasma. A control cohort of 55 healthy volunteers and 60 patients with non-HPV-associated malignancy was also analyzed. RESULTS: Baseline plasma ctHPVDNA had high specificity (97%) and high sensitivity (89%) for detecting newly diagnosed HPV-associated OPSCC. Pretreatment ctHPV16DNA copy number correlated with disease burden, tumor HPV copy number, and HPV integration status. We define a ctHPV16DNA favorable clearance profile as having high baseline copy number (>200 copies/mL) and >95% clearance of ctHPV16DNA by day 28 of CRT. Nineteen of 67 evaluable patients had a ctHPV16DNA favorable clearance profile, and none had persistent or recurrent regional disease after CRT. In contrast, patients with adverse clinical risk factors (T4 or >10 pack years) and an unfavorable ctHPV16DNA clearance profile had a 35% actuarial rate of persistent or recurrent regional disease after CRT (P = 0.0049). CONCLUSIONS: A rapid clearance profile of ctHPVDNA may predict likelihood of disease control in patients with HPV-associated OPSCC patients treated with definitive CRT and may be useful in selecting patients for deintensified therapy.

Digging a little deeper: the stages of invadopodium formation and maturation.

Invadopodia are actin-rich protrusions that degrade the extracellular matrix and are required for penetration through the basement membrane, stromal invasion and intravasation. Invadopodia are enriched in actin regulators, such as cortactin, cofilin, N-WASp, Arp2/3 and fascin. Much of the work to date has centered around identifying the proteins involved in regulating actin polymerization and matrix degradation. Recently, there have been significant advances in characterization of the very early stages of invadopodium precursor assembly and the role of adhesion proteins, such as ß1 integrin, talin, FAK and Hic-5, in promoting invadopodium maturation. This review summarizes these findings in the context of our current model of invadopodial function and highlights some of the important unanswered questions in the field.

Talin regulates moesin-NHE-1 recruitment to invadopodia and promotes mammary tumor metastasis.

Invadopodia are actin-rich protrusions that degrade the extracellular matrix and are required for stromal invasion, intravasation, and metastasis. The role of the focal adhesion protein talin in regulating these structures is not known. Here, we demonstrate that talin is required for invadopodial matrix degradation and three-dimensional extracellular matrix invasion in metastatic breast cancer cells. The sodium/hydrogen exchanger 1 (NHE-1) is linked to the cytoskeleton by ezrin/radixin/moesin family proteins and is known to regulate invadopodium-mediated matrix degradation. We show that the talin C terminus binds directly to the moesin band 4.1 ERM (FERM) domain to recruit a moesin-NHE-1 complex to invadopodia. Silencing talin resulted in a decrease in cytosolic pH at invadopodia and blocked cofilin-dependent actin polymerization, leading to impaired invadopodium stability and matrix degradation. Furthermore, talin is required for mammary tumor cell motility, intravasation, and spontaneous lung metastasis in vivo. Thus, our findings provide a novel understanding of how intracellular pH is regulated and a molecular mechanism by which talin enhances tumor cell invasion and metastasis.

An in vitro one-dimensional assay to study growth factor-regulated tumor cell-macrophage interaction.

Growth factor-dependent pairing and motility between tumor cells and tumor-associated macrophages on extracellular matrix (ECM) fibers of the tumor microenvironment have been shown to enhance intravasation and metastatic spread of breast carcinomas. We describe an in vitro motility assay that combines time-lapse wide-field microscopy and micro-patterned linear adhesive substrates to reconstitute the in vivo behavior between macrophages, tumor cells, and ECM fibers in orthotopic rodent tumor models observed by intravital imaging. Commercially available linear stripes of 650 nm dye-labeled fibronectin microlithographed onto glass cover slips are sequentially plated with fluorescently labeled MTLn3 tumor cells and bone marrow-derived macrophages and time-lapse imaged for up to 8 h. Incubation with pharmacological inhibitors during the assay can identify important paracrine or autocrine signaling pathways involved in the macrophage-tumor cell interaction. This high-resolution motility assay will lead to a more detailed description of immune cell-tumor cell behavior as well as interrogating additional cell types within the tumor microenvironment which use cytokine/growth factor paracrine signaling interactions to facilitate intravasation and metastasis.

Filamin A interacting protein 1-like inhibits WNT signaling and MMP expression to suppress cancer cell invasion and metastasis.

Identifying key mediators of cancer invasion and metastasis is crucial to the development of new and more effective therapies. We previously identified FILamin A Interacting Protein 1-Like (FILIP1L) as an important inhibitor of cell migration and invasion. FILIP1L expression was inversely correlated with the invasive potential of ovarian tumors. In our study, we established an orthotopic ovarian cancer model, wherein FILIP1L expression can be regulated in vivo. Using this model, we observed that expression of FILIP1L in ovarian cancer cells inhibited spontaneous lung metastasis. Experimental lung metastases (established via tail vein injection of cancer cells) as well as the extravasation step of metastasis were not inhibited by FILIP1L, suggesting that FILIP1L inhibits the earlier steps of metastasis such as invasion and intravasation. FILIP1L inhibited matrix metalloproteinase (MMP)-dependent invasion in vivo. MMP3, -7 and -9 were transcriptionally downregulated, and MMP9 protein expression and activity were inhibited in FILIP1L-expressing tumors. Importantly, overexpression of MMP9 compensated for the anti-invasive activity of FILIP1L. Furthermore, our studies suggest that FILIP1L regulates invasion and metastasis by inhibiting components of the WNT signaling pathway. FILIP1L expression reduced the induction of WNT target genes such as MMP3, -7 and -9, and ß-catenin-directed transcriptional activity, suggesting inhibition of the canonical WNT pathway. Nuclear ß-catenin, an indicator of an active canonical WNT pathway, was reduced in FILIP1L-expressing tumors. Overall, these findings suggest that FILIP1L reduces ß-catenin levels, which may lead to the transcriptional downregulation of WNT target genes such as MMPs, resulting in inhibition of metastasis. Modulation of FILIP1L expression has the potential to be a target for cancer therapy.

ß1 integrin regulates Arg to promote invadopodial maturation and matrix degradation.

ß1 integrin has been shown to promote metastasis in a number of tumor models, including breast, ovarian, pancreatic, and skin cancer; however, the mechanism by which it does so is poorly understood. Invasive membrane protrusions called invadopodia are believed to facilitate extracellular matrix degradation and intravasation during metastasis. Previous work showed that ß1 integrin localizes to invadopodia, but its role in regulating invadopodial function has not been well characterized. We find that ß1 integrin is required for the formation of mature, degradation-competent invadopodia in both two- and three-dimensional matrices but is dispensable for invadopodium precursor formation in metastatic human breast cancer cells. ß1 integrin is activated during invadopodium precursor maturation, and forced ß1 integrin activation enhances the rate of invadopodial matrix proteolysis. Furthermore, ß1 integrin interacts with the tyrosine kinase Arg and stimulates Arg-dependent phosphorylation of cortactin on tyrosine 421. Silencing ß1 integrin with small interfering RNA completely abrogates Arg-dependent cortactin phosphorylation and cofilin-dependent barbed-end formation at invadopodia, leading to a significant decrease in the number and stability of mature invadopodia. These results describe a fundamental role for ß1 integrin in controlling actin polymerization-dependent invadopodial maturation and matrix degradation in metastatic tumor cells.

Reconstitution of in vivo macrophage-tumor cell pairing and streaming motility on one-dimensional micro-patterned substrates.

In mammary tumors, intravital imaging techniques have uncovered an essential role for macrophages during tumor cell invasion and metastasis mediated by an epidermal growth factor (EGF) / colony stimulating factor-1 (CSF-1) paracrine loop. It was previously demonstrated that mammary tumors in mice derived from rat carcinoma cells (MTLn3) exhibited high velocity migration on extracellular matrix (ECM) fibers. These cells form paracrine loop-dependent linear assemblies of alternating host macrophages and tumor cells known as "streams." Here, we confirm by intravital imaging that similar streams form in close association with ECM fibers in a highly metastatic patient-derived orthotopic mammary tumor (TN1). To understand the in vivo cell motility behaviors observed in streams, an in vitro model of fibrillar tumor ECM utilizing adhesive 1D micropatterned substrates was developed. MTLn3 cells on 1D fibronectin or type I collagen substrates migrated with higher velocity than on 2D substrates and displayed enhanced lamellipodial protrusion and increased motility upon local interaction and pairing with bone marrow-derived macrophages (BMMs). Inhibitors of EGF or CSF-1 signaling disrupted this interaction and reduced tumor cell velocity and protrusion, validating the requirement for an intact paracrine loop. Both TN1 and MTLn3 cells in the presence of BMMs were capable of co-assembling into linear arrays of alternating tumor cells and BMMs that resembled streams in vivo, suggesting the stream assembly is cell autonomous and can be reconstituted on 1D substrates. Our results validate the use of 1D micropatterned substrates as a simple and defined approach to study fibrillar ECM-dependent cell pairing, migration and relay chemotaxis as a complementary tool to intravital imaging.