Tongue Carcinoma Research Articles

Imaging of Tongue Carcinoma: About 20 Cases

The tongue enables taste and plays a critical role in formation of food bolus and deglutition. The tongue is also crucial for speech and the earliest sign of tongue paresis is a change in the quality of speech. Given the importance of the tongue, tongue carcinoma should be accurately staged in order to optimise treatment options and preserve organ function. The intent of this review is to familiarise radiologists with the pertinent anatomy of the tongue and the behaviour of tongue carcinoma so as to map malignant infiltration accurately.

Vital Staining Role in Resection and Reconstruction of Tongue Cancers with Free Flap - A Comparative Study

Abstract Introduction: The purpose of this study is to compare and infer the most suitable dye for visualisation of ipsilateral and contralateral sentinel lymph nodes (SLNs), positive, negative outcomes, sensitivity, their specificities and visualisation of anastomosed blood vessels in microvascular reconstruction of tongue cancers. Materials and Methods: This is a prospective in vivo comparative study involving twenty patients diagnosed with squamous cell carcinoma of the tongue treated with wide excision of primary tongue tumour, modified functional neck dissection and microvascular reconstruction with radial forearm free flap. Consecutive random allocation was done into two groups with ten patients each. Patients were injected with methylene blue as the first group and near-infrared fluorescence indocyanine green (ICG) into the second. The patients were evaluated intraoperatively regarding the better dye in methylene blue and ICG for identification of ipsilateral and contralateral SLNs, their positive, negative outcomes, sensitivity and specificities. Apart from these, in cases of group B, after microvascular transfers, ICG is used for visualisation of anastomosed vessels to prevent abnormal perfusions, leaks and post–flap failures. Descriptive statistics, independent samples t-test, and Chi-square were used for statistical analysis. Results: The present study revealed ICG being the better dye in the parameters mentioned. Discussion: Lymph node yield in neck dissection is a prognostic factor in oral cancers. ICG is inferred as the better dye for identification of SLN with 100% outcomes and in blood vessel visualisation after microvascular free tissue transfers. It is also useful in identifying positive contralateral sentinel nodes, which is helpful in undertaking bilateral neck dissections related to tongue carcinoma.

Modular anatomic approach to oral tongue carcinoma: functional outcomes and quality of life.

The purpose of this study is to present the long-term functional swallowing outcomes of various surgical approaches to oral tongue/floor of mouth squamous cell carcinoma (OTFOMSCC) according to a modular and compartment-based concept previously described elsewhere. A retrospective study was conducted on patients undergoing surgery for OTFOMSCC from January 2017 to April 2023 in the Department of Otorhinolaryngology at the Hospital of Bolzano. Functional swallowing outcomes and quality of life (QoL) were assessed through the administration of a scale and questionnaires between 6 months and 1 year postoperatively or after the end of adjuvant therapy, stratifying the results according to the surgical technique used. A total of 92 patients with OTFOMSCC were enrolled: 44 patients underwent transoral anatomically guided surgery (TAGS) for early-stage tumours, and 48 underwent major surgery for advanced stages. Among the latter 48 patients: 35 underwent compartment tongue surgery (CTS), 3 underwent extended glossectomies (EG) Type A, 6 underwent EG Type B, and 4 underwent EG Type C. After TAGS, all patients resumed oral feeding for all consistencies. In the CTS group, only one patient faced challenges with pure liquids, yet all achieved the target of consuming a semisolid and semiliquid diet. EG exhibited a noticeable decline in performance from Type A to total glossectomy (Type C). Following an anatomically-guided approach to the resection of OTFOMSCC allows the surgery to be modulated on the structures involved by the tumour, thus achieving optimal oncological results, while maintaining the possibility to predict functional outcomes and postoperative QoL.

Upregulation of Apoptosis Related Genes in Clinically Normal Tongue Contralateral to Squamous Cell Carcinoma of the Oral Tongue, an Effort to Maintain Tissue Homeostasis

PurposeThe field cancerization concept indicates the presence of pre-cancerous changes in clinically normal tissue surrounding the tumor. In squamous cell carcinoma of the oral tongue (SCCOT) which is infrequently linked to human papillomavirus infection, we have previously reported that clinically normal tongue contralateral to tumor (NTCT) is molecularly abnormal. Here, combining our transcriptomic and genomic data, we aimed to investigate the contribution of molecular changes in NTCT to cancer development.MethodsMicroarray gene expression data of 14 healthy controls, 23 NTCT and 29 SCCOT samples were investigated to characterize transcriptional profiles in NTCT. Whole exome sequencing and RNA-sequencing data of paired NTCT and tumor samples from 15 SCCOT patients were used to study correlation between copy number variation and differential gene expression.ResultsUsing supervised multivariate partial least squares discriminant analysis, a total of 61 mRNAs that distinguish NTCT from healthy tongue were selected. Functional enrichment analysis of the 22 upregulated genes showed increased “positive regulation of nitrogen compound metabolic process” in NTCT. All 12 genes involved in this process have roles in apoptosis (anti- and/or pro-apoptotic). Compared to healthy controls, Zinc Finger Protein 395 (ZNF395), a pro-apoptotic tumor suppressor located on chromosome 8p, was the only gene showing increased mRNA level in NTCT whereas decreased in SCCOT. Given the frequent loss of chromosome 8p in SCCOT, the impact of ZNF395 copy number variation on gene expression was further examined, revealing a positive correlation between copy number and mRNA level (correlation coefficient = 0.572, p < 0.001).ConclusionNTCT is susceptible to malignant transformation, where tissue homeostasis is maintained at least partly through regulation of apoptosis. Loss of the pro-apoptotic gene ZNF395 could thus initiate cancer development.

Improved ground truth annotation by multimodal image registration from 3D ultrasound to histopathology for resected tongue carcinoma.

This study's objectives are (1) to investigate the registration accuracy from intraoperative ultrasound (US) to histopathological images, (2) to assess the agreement and correlation between measurements in registered 3D US and histopathology, and (3) to train a nnUNet model for automatic segmentation of 3D US volumes of resected tongue specimens. Ten 3D US volumes were acquired, including the corresponding digitalized histopathological images (n = 29). Based on corresponding landmarks, the registrations between 3D US and histopathology images were calculated and evaluated using the target registration error (TRE). Tumor thickness and resection margins were measured based on three annotations: (1) manual histopathological tumor annotation (HTA), manual 3D US tumor annotation, and (2) the HTA registered in the 3D US. The agreement and correlation were computed between the measurements based on the HTA and those based on the manual US and registered HTA in US. A deep-learning model with nnUNet was trained on 151 3D US volumes. Segmentation metrics quantified the model's performance. The median TRE was 0.42mm. The smallest mean difference was between registered HTA in US and histopathology with 2.16mm (95% CI - 1.31; 5.63) and a correlation of 0.924 (p < 0.001). The nnUNet predicted the tumor with a Dice similarity coefficient of 0.621, an average surface distance of 1.15mm, and a Hausdorff distance of 3.70mm. Multimodal image registration enabled the HTA's registration in the US images and improved the agreement and correlation between the modalities. In the future, this could be used to annotate ground truth labels accurately.

Metastatic tongue squamous cell carcinoma at a percutaneous endoscopic gastrostomy site via introducer technique

AbstractBackgroundPercutaneous endoscopic gastrostomy (PEG) site metastases from head and neck cancers have been reported many times using the pull technique, in which a catheter passes through the oral cavity and pharynx.Case presentationA 69‐year‐old man with recurrent tongue squamous cell carcinoma underwent PEG placement using the introducer technique before chemoradiotherapy. After 6 months, the patient developed a metastasis of tongue carcinoma at the PEG site.ConclusionThe risk of PEG site metastasis is not eliminated even with the introducer technique, which is not in direct contact with the tumor.

The study on 4D culture system of squamous cell carcinoma of tongue

Traditional cell culture methods often fail to accurately replicate the intricate microenvironments crucial for studying specific cell growth patterns. In our study, we developed a 4D cell culture model-a precision instrument comprising an electromagnet, a force transducer, and a cantilever bracket. The experimental setup involves placing a Petri dish above the electromagnet, where gel beads encapsulating magnetic nanoparticles and tongue cancer cells are positioned. In this model, a magnetic force is generated on the magnetic nanoparticles in the culture medium to drive the gel to move and deform when the magnet is energized, thereby exerting an external force on the cells. This setup can mimic the microenvironment of tongue squamous cell carcinoma CAL-27 cells under mechanical stress induced by tongue movements. Electron microscopy and rheological analysis were performed on the hydrogels to confirm the porosity of alginate and its favorable viscoelastic properties. Additionally, Calcein-AM/PI staining was conducted to verify the biosafety of the hydrogel culture system. It mimics the microenvironment where tongue squamous cell carcinoma CAL-27 cells are stimulated by mechanical stress during tongue movement. Electron microscopy and rheological analysis experiments were conducted on hydrogels to assess the porosity of alginate and its viscoelastic properties. Calcein-AM/PI staining was performed to evaluate the biosafety of the hydrogel culture system. We confirmed that the proliferation of CAL-27 tongue squamous cells significantly increased with increased matrix stiffness after 5 d as assessed by MTT. After 15 d of incubation, the tumor spheroid diameter of the 1%-4D group was larger than that of the hydrogel-only culture. The Transwell assay demonstrated that mechanical stress stimulation and increased matrix stiffness could enhance cell aggressiveness. Flow cytometry experiments revealed a decrease in the number of cells in the resting or growth phase (G0/G1 phase), coupled with an increase in the proportion of cells in the preparation-for-division phase (G2/M phase). RT-PCR confirmed decreased expression levels of P53 and integrinβ3 RNA in the 1%-4D group after 21 d of 4D culture, alongside significant increases in the expression levels of Kindlin-2 and integrinαv. Immunofluorescence assays confirmed that 4D culture enhances tissue oxygenation and diminishes nuclear aggregation of HIF-1α. This device mimics the microenvironment of tongue cancer cells under mechanical force and increased matrix hardness during tongue movement, faithfully reproducing cell growthin vivo, and offering a solid foundation for further research on the pathogenic matrix of tongue cancer and drug treatments.

Contralateral submental artery island flap for oral tongue reconstruction — a retrospective study in patients with oral tongue squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is a leading cause of cancer-related death in developing countries. The oral tongue is the most common site involved by OSCC. About a third of the patients have neck nodal metastasis at presentation. Reconstruction of the oral tongue after resection for cancer is necessary for proper rehabilitation. For patients who are medically unfit for prolonged surgery like a free tissue transfer, local flaps are employed for tongue reconstruction. The submental flap is a popular option. However, when there is extensive floor of mouth involvement or bulky nodal disease in ipsilateral level 1, harvesting the submental flap based on contralateral facial vessels is possible, without compromising oncological safety. We discuss the feasibility and outcomes of contralateral submental artery island flap for reconstruction after resection of oral tongue carcinoma in a retrospective series of 34 patients followed up for a median of 8.5 months. ResultsOf the 34 oral tongue cancer patients in the study, 16 had neoadjuvant chemotherapy prior to surgery. 33 underwent bilateral neck dissection. Two patients had partial flap loss which was managed conservatively. There were five patients who had either locoregional or distant recurrence, none in the flap site. Three had pathological positive nodes at contralateral level 1b and two had pathologically positive nodes in contralateral level 1b. Tongue motility and speech intelligibility were satisfactory on follow-up. ConclusionIn the properly selected patient, the contralateral submental artery island flap offers an oncologically safe and technically simpler alternative for free flap reconstruction.

Antihypertensive agent losartan promotes tongue squamous cell carcinoma cell proliferation via EGFR/ERK1/2/cyclin D1 signaling axis

ObjectiveTo study the effects of losartan, an angiotensin II receptor blocker, in the SCC4 and SCC25 human tongue squamous cell carcinoma cell lines. MethodsCell proliferation was measured by MTS/PMS activity and trypan blue exclusion assays. The levels of the cell proliferation marker, cyclin D1, were analyzed by western blotting. Apoptosis was assessed by caspase-3 activation and Annexin V-FITC/propidium iodide double staining. Activation of epidermal growth factor receptor (EGFR) and ERK1/2 was validated by western blotting. ResultsModerate concentrations of losartan enhanced the proliferation of SCC4 and SCC25 cells. However, high losartan concentrations induced apoptosis in SCC4 cells. Losartan activated the EGFR/ERK1/2/cyclin D1 signaling axis, which in turn promoted cell proliferation. Afatinib (EGFR inhibitor) and U0126 (ERK1/2 inhibitor) abolished losartan-induced cell proliferation. In contrast, UC2288 (p21 inhibitor) enhanced it. ConclusionsLosartan exhibited dual effects on tongue squamous cell carcinoma cells. Moderate losartan concentrations facilitated cell proliferation, whereas high concentrations induced cytotoxicity in tongue carcinoma cells.

The prognostic role of MRI-based radiomics in tongue carcinoma: a multicentric validation study

PurposeRadiomics is an emerging field that utilizes quantitative features extracted from medical images to predict clinically meaningful outcomes. Validating findings is crucial to assess radiomics applicability. We aimed to validate previously published magnetic resonance imaging (MRI) radiomics models to predict oncological outcomes in oral tongue squamous cell carcinoma (OTSCC).Materials and methodsRetrospective multicentric study on OTSCC surgically treated from 2010 to 2019. All patients performed preoperative MRI, including contrast-enhanced T1-weighted (CE-T1), diffusion-weighted sequences and apparent diffusion coefficient map. We evaluated overall survival (OS), locoregional recurrence-free survival (LRRFS), cause-specific mortality (CSM). We elaborated different models based on clinical and radiomic data. C-indexes assessed the prediction accuracy of the models.ResultsWe collected 112 consecutive independent patients from three Italian Institutions to validate the previously published MRI radiomic models based on 79 different patients. The C-indexes for the hybrid clinical-radiomic models in the validation cohort were lower than those in the training cohort but remained > 0.5 in most cases. CE-T1 sequence provided the best fit to the models: the C-indexes obtained were 0.61, 0.59, 0.64 (pretreatment model) and 0.65, 0.69, 0.70 (posttreatment model) for OS, LRRFS and CSM, respectively.ConclusionOur clinical-radiomic models retain a potential to predict OS, LRRFS and CSM in heterogeneous cohorts across different centers. These findings encourage further research, aimed at overcoming current limitations, due to the variability of imaging acquisition, processing and tumor volume delineation.

Functional outcome of carcinoma tongue after primary surgical treatment

Functional outcome of carcinoma tongue after primary surgical treatment

Postoperative radiotherapy for pT1-2N0 oral tongue carcinoma: time to reconsider?

Postoperative radiotherapy for pT1-2N0 oral tongue carcinoma: time to reconsider?

Assessment of oncologic and cosmetic outcomes of robotic elective neck dissection in early-stage tongue cancer

BackgroundRobotic neck dissection is emerging as an alternative to conventional open neck dissection. However, the oncologic safety of robotic elective neck dissection (END) and its indications in early-stage tongue cancer are unclear. MethodsWe retrospectively reviewed the data of 78 patients who underwent transoral excision for T1, T2 squamous cell carcinoma of tongue with simultaneous ipsilateral END. Patients were assigned to two groups: the robotic group (n = 32)—postauricular face-lift —and the conventional group (n = 46)— transcervical incision. We compared the survival, clinical, pathologic and cosmetic outcomes of the two groups, and evaluated the number of retrieved lymph nodes and robot console time in the robotic group. ResultsThe mean age was lower in the robotic group (43.6 ± 12.8 vs. 55.8 ± 14.0, p < 0.001) and the conventional group had more T2 patients (p = 0.01). The mean operation time was significantly longer in the robotic group than the conventional group (178.81 ± 33.9 vs. 92.28 ± 16.7, p < 0.001). The mean number of retrieved lymph nodes was not significantly different between the two groups (19.22 ± 8.51 vs. 20.7 ± 11.4, p = 0.41). The 5-year disease-free survival rate was not significantly different between the two groups (93.6 % vs. 82.9 %, p = 0.59). Overall scar satisfaction assessed by VAS score, the robotic group showed significantly better results compared to the conventional group (8.38 vs. 5.86, p = 0.033). ConclusionRobotic END by a postauricular facelift approach is a feasible and safe approach for early-stage tongue cancer.

Assessment of quality of life in head and neck cancer patients visiting the Palliative Care outpatient department in a tertiary care oncology centre in India.

Patients with head and neck cancers (HNCs) are at an increased risk of developing functional symptoms associated with eating, speaking, and breathing along with symptoms caused by a fungating tumour (e.g., cosmetic change, malodor, and bleeding). These may substantially reduce their physical functioning and quality of life (QoL). This observational study aimed to find out the QoL in patients with HNC in a tertiary care oncology centre. A prospective observational study was conducted in adult patients diagnosed with HNC. The patients were divided depending upon their disease extent into early, advanced, and very advanced local disease. The physical, cognitive, emotional, financial and social domains were assessed using a validated Hindi version of the European Organisation for Research and Treatment of cancer (EORTC)- QoL 30 and EORTC H&N 35 at baseline and 3 months. A total of 100 patients were assessed with a mean age of 49.3 ± 12.4 years. Most of the patients had carcinoma buccal mucosa (42%) followed by carcinoma tongue (17%). The patients experienced difficulties with physical functioning and deterioration in emotional functioning. Pain and fatigue were the major problematic symptoms especially in advanced disease patients resulting in poor QoL. There was a significant improvement in various domains of QoL at 3 months follow-up in those with advanced disease. The fatigue scores at baseline and follow-up showed a positive correlation with other symptoms. Patients with HNC have a high symptom burden that leads to poor QoL. Appropriate palliative care interventions help to decrease symptom burden and prevent deterioration of their QoL in patients with HNC.

"Rare Encounter: Pneumoparotid in Clinical Practice" - A Case Report

Pneumoparotid is a rare condition which is frequently observed in the patients with parotid gland infection or during maneuver leading to increase in intraoral pressure. The common maneuver being performed is puff-cheek technique for diagnostic computed tomography (CT). It is always important to distinguish the etiology of pneumoparotid being pathological or due to maneuver to proceed for further management. Here we present a case of a 56 year old man with suspicion of carcinoma of tongue evaluated by CT using puff cheek technique. On evaluating the images, a finding of bilateral pneumoparotid a case report was made.

Effects of α‑solanine on human head and neck squamous cell carcinoma cells and human umbilical vein endothelial cells in vitro.

α-solanine is a glycoalkaloid that is commonly found in nightshades (Solanum) and has a toxic effect on the human organism. Among other things, it is already known to inhibit tumor cell proliferation and induce apoptosis in tumor cell lines. Due to its potential as a tumor therapeutic, the current study investigated the effect of α-solanine on head and neck squamous cell carcinoma (HNSCC). In addition, genotoxic and antiangiogenic effects on human umbilical vein endothelial cells (HUVECs) were evaluated at subtoxic α-solanine concentrations. Cytotoxicity and apoptosis rates were measured in two human HNSCC cell lines (FaDu pharynx carcinoma cells and CAL-33 tongue carcinoma cells), as well as in HUVECs. MTT and Annexin V analyses were performed 24 h after α-solanine treatment at increasing doses up to 30 µM to determine cytotoxic concentrations. Furthermore, genotoxicity at subtoxic concentrations of 1, 2, 4 and 6 µM in HUVECs was analyzed using single-cell gel electrophoresis (comet assay). The antiangiogenic effect on HUVECs was evaluated in the capillary tube formation assay. The MTT assay indicated an induction of concentration-dependent viability loss in FaDu and CAL-33 cancer cell lines, whereas the Annexin V test revealed α-solanine-induced cell death predominantly independent from apoptosis. In HUVECs, the cytotoxic effect occurred at lower concentrations. No genotoxicity or inhibition of angiogenesis were detected at subtoxic doses in HUVECs. In summary, α-solanine had a cytotoxic effect on both malignant and non-malignant cells, but this was only observed at higher concentrations in malignant cells. In contrast to existing data in the literature, tumor cell apoptosis was less evident than necrosis. The lack of genotoxicity and antiangiogenic effects in the subtoxic range in benign cells are promising, as this is favorable for potential therapeutic applications. In conclusion, however, the cytotoxicity in non-malignant cells remains a severe hindrance for the application of α-solanine as a therapeutic tumor agent in humans.

Evaluation of Depth of Invasion as a Histologic Predictor of Neck Node Metastasis in Squamous Cell Carcinoma of the Oral Tongue

Evaluation of Depth of Invasion as a Histologic Predictor of Neck Node Metastasis in Squamous Cell Carcinoma of the Oral Tongue

Naringenin-induced Oral Cancer Cell Apoptosis Via ROS-mediated Bid and Bcl-xl Signaling Pathway.

Oral cancer is a malignant tumor with a high impact and poor prognosis. Naringenin, a flavonoid found in citrus fruits and its anti-inflammatory and antioxidant properties offer potential therapeutic benefits. However, limited studies have been conducted on the impact of naringenin on human tongue carcinoma CAL-27 cells. This study aims to elucidate the correlation between naringenin and tongue cancer, thereby identifying a potential therapeutic candidate for drug intervention against tongue cancer. The effect of naringenin on the apoptosis of CAL-27 cells and its mechanism were studied by cell counting kit-8, mitochondrial membrane potential assay with JC-1, Annexin V-- FITC apoptosis detection, cell cycle, and apoptosis analysis, Reactive Oxygen Species assay and Western blot. The results showed that naringenin significantly induced apoptosis in CAL-27 cells in a dose-dependent manner. Mechanistically, naringenin-induced apoptosis was mediated through the upregulation of Bid and downregulation of Bcl-xl, which led to increased generation of ROS. The findings suggested that naringenin may represent a promising candidate for the treatment of oral cancer by inducing apoptotic cell death via modulation of the Bid and Bcl-xl signaling pathways.

In Vitro Assessment of the Cytotoxic Effect of 5-Fluorouracil, Thymoquinone and their Combination on Tongue Squamous Cell Carcinoma Cell Line.

Tongue cancer is the most prevalent type of oral cancer. Recently, natural compounds have been considered important resources for several anticancer drugs. Thymoquinone (TQ) exhibits a potent anti-cancer effect. 5-Fluorouracil (5-FU) is a chemotherapeutic drug that has been utilized in the treatment of cancer. Recently, combination therapy has gained popularity as a treatment option for patients with cancer. The present study was carried out to assess the cytotoxic effect of 5-Fluorouracil (5-FU), Thymoquinone (TQ), and their combination on tongue squamous cell carcinoma cell line (HNO-97). Tongue carcinoma cell line (HNO-97) was maintained in cultured flasks and the cells were divided into four groups; group Ι: control untreated group, group ΙΙ: HNO-97-treated cells with different concentrations of 5-FU from 0.5 µM/ml to 3µM/ml, group ΙIΙ: HNO-97-treated cells with different concentrations of TQ from 7.25µM/ml to 23.05µM/ml, and group ΙV: HNO-97-treated cells with both 5-FU and TQ in serial concentrations till (IC50) in a dose of 27.44 µM/ml. Determination of the cytotoxic effect of the tested agents on the HNO-97 cell line was done using methyl thiazole tetrazolium assay, nuclear morphometric analysis, microscopic examination, and annexin-v/ propidium iodide staining assay. The findings revealed that the cytotoxic effect of 5-FU, TQ, and their combination on tongue squamous cell carcinoma cell line (HNO-97) was dose-dependent. The microscopic examination revealed that 5-FU, TQ alone, or their combination induced apoptotic cell death. P-value < 0.05 was statistically significant. The combination of 5-FU and TQ produced a marked cytotoxic effect on HNO-97 cells.

Tumor thickness and depth of invasion in squamous cell carcinoma of tongue as indicators of the loco-regional spread of the disease: A preliminary study

BackgroundMagnetic resonance imaging (MRI) is a routinely used imaging modality for pre-treatment radiologic evaluation of tongue carcinoma, providing accurate information regarding the extent of the disease. Aims and objectivesTo investigate the role of MRI-derived depth of invasion and tumor thickness evaluation in squamous cell carcinoma of the tongue, and to assess if any correlation exists between depth of invasion, tumor thickness, nodal metastasis, muscles, and space involved. Materials and methodsThirty-three patients with oral squamous cell carcinoma of the tongue who had undergone pre-treatment MRI and excisional biopsy were included. The tumor thickness (TT) and depth of invasion (DOI) were evaluated on MRI and histopathologic images. ResultThe relation between different methodologies for assessing showed a very high correlation for the tumor tissue thickness (r = 0.99, p < 0.05) and depth of invasion (r = 0.82, p < 0.05). The tumor thickness and the depth of invasion increased with the loss of differentiation in the carcinoma histopathologically. As the depth of invasion increases, the extent of the spread of the carcinoma to tongue musculature, lingual septum, and spaces also increases. ConclusionThe present study has depicted a high correlation between the tumor thickness and the depth of invasion between MRI and histopathological findings and is the first of its kind to correlate DOI to the invasiveness of the disease.