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Material and Methods:

Study Design 
We conducted a retrospective study to evaluate the diagnostic performance of the Avocado sign as an imaging predictor of mesorectal lymph node metastasis in rectal cancer. Histological examination of surgical specimens served as the reference. Inclusion criteria for this study were a histologically confirmed diagnosis of rectal cancer and a minimum age of 18 years. Patients with unresectable tumors and general contraindications to MRI were excluded.

Study Approval
The study was approved by our institutional review board. Written informed consent was obtained from each patient for contrast-enhanced MRI and data analysis.

Patients and Subgroups
A total of 106 rectal cancer patients are currently enrolled in this study between XX and August 2022. All patients underwent local MRI staging at baseline. We also performed a subgroup analysis and divided the overall patient collective into those who received direct surgery and those who received neoadjuvant radiochemotherapy and an additional preoperative staging MRI after completion.

MRI Protocol
All MRI examinations were performed using a 3.0 T system (MAGNETOM Prisma Fit; Siemens Healthineers, Erlangen, Germany) with a combination of body and spinal phased array coils. The specific imaging protocol for rectal cancer includes T2-weighted turbo spin echo (TSE) sequences acquired in three planes along the main tumor-axis, diffusion-weighted (DWI) sequence in the axial plane, and an axial high-resolution T1-weighted volumetric interpolated gradient echo sequence with Dixon fat-saturation technique (VIBE DIXON) after intravenous administration of a macrocyclic gadolinium chelate contrast agent (Prohance; Bracco, Milan, Italy). The amount of contrast was determined based on the individual patient's body weight, with a prescribed dose of 0.2 mL per kg body weight. A spasmolytic agent (Buscopan) was injected in two fractions at the beginning and the middle of the scan. Detailed sequence parameters are shown in Table X.

Sequence	sagittal T2-TSE	axial T2-TSE	coronal T2-TSE	Diffusion (b100/500/1000)	DIXON-VIBE (contrast enhanced)
TR (ms)	4170	4400	4400	3700	5,8
TE (ms)	72	71	71	59	2,5/3,7
FOV (mm)	220	220	220	220	270
Slice (mm)	3	2	2	2	1,5
Matrix	394/448	380/432	380/432	140/140	326/384
Acquisition time (min)
22:24	4:37	4:50	4:50	3:57	4:10


Morphologic features of the Avocado sign
The avocado sign is a morphologic feature of lymph nodes on contrast-enhanced T1 Dixon VIBE MRI sequence that may indicate lymph node metastases from rectal cancer. It is characterized by a hypointense core in the center of the lymph node surrounded by a hyperintense rim of contrast-enhanced lymphatic tissue. The hypointense core represents the tumor cells infiltrating the lymph node, while the hyperintense rim reflects the preserved lymphatic tissue. The size of the lymph node is not relevant, as metastatic lymph nodes may be smaller than 5 mm or larger than 10 mm. Non-metastatic lymph nodes do not have a hypointense core or a wall-adjacent hypointensity, which is usually the fatty hilus. The avocado sign also differs from other signs of lymph node metastasis, such as irregular shape, spiculated margin, heterogeneous enhancement, or extranodal extension.

Imaging Analysis and Histologic Review
Two independent radiologists with 27 and 5 years of abdominal imaging experience evaluated the latest preoperative MRI images. The readers were blinded to the clinical and histopathologic results during image interpretation. Lymph nodes were graded on a dichotomous scale as malignant if the Avocado sign was present (AS+) and as non-malignant (AS-) if appropriate imaging features were absent. The number and additional standard criteria of malignant lymph nodes were not considered. In case of disagreement between the raters, a third radiologist with 17 years of experience in abdominal imaging was consulted to make the final decision. The findings were compared with the histopathologic results of the resected specimen, where lymph node involvement was graded analogously as N+ if one or more lymph node metastases were confirmed and N- without confirmed lymph node metastases. Histopathologic examination was performed according to standardized criteria.

Statistical Analysis
All statistics were performed with SPSS Statistics 26 (IMB; Armonk, USA).  Values are presented as mean ± standard deviation (SD) for normally distributed variables or median and range for non-normally distributed variables. The prevalence of the avocado sign and the presence of lymph node metastasis for the overall patient population and subgroups were analyzed using descriptive statistics, including frequency data and percentages. Test values such as sensitivity, specificity, accuracy, positive predictive value (PPV) and negative predictive value (NPV) were determined using 2x2 contingency table calculations. Interrater agreement was assessed using Cohen's kappa coefficient. A value of 0.81 to 1.00 was considered almost perfect agreement, 0.61 to 0.80 as substantial, 0.41 to 0.60 as moderate, 0.21 to 0.40 as fair, and less than 0.20 as poor agreement.

Results

Patients
Of the 106 patients enrolled, 80 were male and 26 were female. The mean age was 65 years with a standard deviation of 8.4 years. Treatment management and subgroup distribution are shown in Table X.
Group	Number of Patients
Direct Surgery	29
Neoadjuvant Therapy	77
Overall collective	106

Image assessment 
In the overall patient population, the avocado sign was found on preoperative MRI in 64 patients. Histopathologic examination of surgical specimens confirmed lymph node metastases in 50 patients in this collective, resulting in a sensitivity of 92% (46/50), a specificity of 68% (38/56), an accuracy of 78% (84/106), a PPV of 72% (46/64), and a NPV of 90% (38/42) of the avocado sign for predicting lymph node involvement.
In the subgroup analysis, the avocado-sign had a sensitivity of 100% (16/16), a specificity of 77% (10/13), an accuracy of 87% (26/30), a PPV of 84% (16/19), and a NPV of 100% (10/10) for patients undergoing immediate surgery. 
In the neoadjuvant treated group the test values were calculated as sensitivity 88% (30/34), specificity 65% (28/43), accuracy 74% (58/76), PPV 67% (30/45) and NPV 88% (28/32). 

The frequencies of avocado sign and lymph node metastasis are shown in Table 2. Test characteristics for the overall patient population and subgroups are summarized in Table 3. 


Table 2. Distribution of the Avocado Sign and Histological Findings
Group	AS (+)	AS (-)	N (+)	N (-)	AS (+) /N (+)	AS (+) /N (-)	AS (-) /N (+)	AS (-) /N (-)
Overall	64	42	50	56	46	18	4	38
Directly operated	19	10	16	13	16	3	0	10
Neoadjuvant treated	45	32	34	43	30	15	4	28






Table 3. Test Characteristics of the Avocado Sign for Predicting Lymph Node Involvement
Group	Sensitivity (%)	Specificity (%)	Accuracy (%)	PPV (%)	NPV (%)
Overall	92	68	78	72	90
Directly operated	100	77	87	84	100
Neoadjuvant treated	88	65	74	67	88
PPV denotes positive predictive value, and NPV negative predictive value.