The prognostic factors for breast cancer include pretreatment staging, nodal and distant metastasis, histopathological type, hormone receptor status, ki67 proliferation index, and molecular subtypes. For adequate treatment, staging and metastatic workup are two important considerations. Histopathological examination (HPE) and immunohistochemistry (IHC) markers are considered the gold standard for diagnosing breast cancer histology and further receptor status. Hormone receptors for breast cancer are the Estrogen receptor (ER), Progesterone receptor (PgR), and Her2neu receptor (Her2), which are further used to characterize molecular subtypes as Luminal A, Luminal B, Her2 Enriched, and Triple negative.

Knowledge of the receptor status and imaging characteristics like size, morphology, and vascularity is vital in guiding the treatment plan. Previously, the use of 18-Fluorodeoxyglucose positron emission tomography / computed tomography ([18F]FDG PET/CT) was limited to pre-treatment, interim, and post-treatment response assessment for cancers. However, the role of PET/CT can be expanded to predict the molecular subtype based on PET/CT uptake values. In the current era, PET/CT is used to prognosticate the affected individuals by correlating it with the HPE and IHC markers. 12 This study aims to find the association between metabolic activity of primary tumors as Standardized uptake value (SUV) on PET/CT and prognostic factors such as ER, PgR, and Her2neu receptor status, molecular subtypes, ki67 index, and nodal/distant metastasis. This correlation will help in the management of patients concerning treatment planning, prediction of response, survival, and recurrence rates of breast cancer.

Under a waiver of consent by the institutional ethics committee, a retrospective observational study was done at the Department of Radio-diagnosis in our tertiary care institute. The study recruited 228 breast cancer patients from March 2017 to April 2021. The cases under study were either pathologically proven cases of breast cancer who were referred to the radiology department for whole-body PET/CT imaging or were suspicious on imaging for breast cancer and later underwent biopsy/surgical management.

Patients who were undergoing primary breast cancer evaluation with PET/CT (i.e., with no prior treatment for presenting breast complaints) and those with complete IHC panels were included in this study. Patients who obtained any form of treatment before undergoing PET/CT investigation or those without required IHC data were excluded.

All the PET/CT imaging was done using Siemens Biograph Horizon TRUE V PET/CT scanner and Syngo.via version VB30A software. In all the studies, 18F-Fluorodeoxyglucose (dose: 185-555MBq) radiotracer and Iohexol-350mg/dl (dose: 1.5mg/kg) IV contrast agents were used. Post IV contrast administration low dose whole body CT scan with 16 slice CT scanner was performed to acquire slices with 5mm thickness which were reconstructed into 1.5mm thickness slices. Approximately, 1-2 minutes was the routine time to acquire the CT data. PET count acquisition was done in 6 to 7 beds of ~20cm length with exposure time lasting ~2 minutes for each. Detector sensitivity was 7.6cps/kBq at the rate of 435keV with a resolution of 4.2mm. The routine time taken for PET count acquisition was 7-8 minutes. The PET and CT image fusion was done by the software (syngo.via) in the Siemens PET/CT workstation. For measuring the [18F]FDG uptake in a primary lesion, lymph nodes, and distant metastasis, a region of interest (ROI) iso contour was drawn, including the entire lesion. The system calculated and displayed the SUV max of the lesion under consideration. The staging was done according to the AJCC Staging Manual (8th edition).

Histological assessment and interpretation of data were done, and an appropriate block was chosen. Sections of 3µ thickness were cut and immunohistochemical staining was performed using monoclonal antibodies (MAB) at a dilution of 1:100 to the Estrogen Receptor (ER) {rabbit MAB -Alpha (clone EP1) (BioGenex, CA)}, PgR {mouse MAB to PgR (clone PR88) (BioGenex, CA)}, Her2 {rabbit MAB (BioGenex, CA)} and Ki67% {mouse MAB (clone MIB-1) (BioGenex, CA)}. Tumors with >1% nuclear staining were defined as ER and PgR positive (+). Tumors with 2+/3+ positivity were defined as Her2 positive and confirmed by FISH (Fluro in-situ Hybridization). For Ki67%, scoring was done relatively as a percentage of tumor cells positivity with a cut-off of 15%. St. Gallen International Expert Consensus on the primary Therapy of Early Breast Cancer 2011 was used to define the molecular subtypes.

The collected data were analyzed with IBM SPSS Statistics for Windows, Version 23.0 (Armonk, NY: IBM Corp). Descriptive statistics frequency analysis, and percentage analysis were used for categorical variables, and the mean and standard deviation (SD) were used for continuous variables. SUV max differences were calculated by the Kruskal-Wallis test. Spearman's rho was used to Correlate the Ki-67 index and SUV max. Unpaired sample t-test was used to find the significant difference between the bivariate samples in independent groups. For skewed data, the normality of data was verified with Shapiro-Wilk's Test. Median values with interquartile range (IQR) were also calculated for the data. For multivariate analysis, the one-way ANOVA with Tukey's PostHoc test was used. In the above statistical tools, the probability value 0.05 was considered significant.

The mean SUV uptake values were found to be significantly higher for ER/PgR negative and triple negative IHC receptor status. However, no significant association was found for isolated Her2neu receptor status (P-value = 0.17 > 0.05) ( Table 1).

A significant association was found for a higher Ki67 index (>15%) with higher [18F]FDG uptake (P<0.001). (Table 1). The mean SUV max uptake for nodal metastasis was found to be higher in patients with Her2 enriched and TNBC variants compared to the Luminal A and B subtypes (Table 3). On multiple intergroup comparisons of mean SUV max uptake for nodal metastasis, statistical difference was found between Luminal A versus TNBC (P= 0.02). However, no statistical difference was found for Luminal A and Luminal B (P=0.7), Luminal A versus HER2 (P=0.06), Luminal B versus HER2 (P=0.18), and TNBC versus Luminal B (P= 0.07) and HER2 (P= 0.9)

The median SUV max uptake for distant metastasis was almost similar in all the molecular subtypes. On multiple intergroup comparisons of mean distant metastasis SUV max uptake, no statistical difference could be found (P-value-0.26 >0.05) between the various molecular subtypes of breast cancer (Table 3).

According to our study, we found that as the [18F]FDG uptake increases in the primary breast lesion, it tends to become more aggressive pathologically. In a study conducted by Brock et al. 1 , it was found that tumors with high [18F]FDG uptake are more aggressive and therefore [18F]FDG uptake on PET/CT scans can be utilized for assessing the prognosis of the disease. According to the study conducted by Oshida et al., 2 the lesions with lower SUV uptake have a better disease-free survival rate, which fits the criteria of a better prognosis of breast cancer.

In studies conducted by Mauri et al. 3 and Clahsen et al. 4 ER positivity status was shown to have a higher disease-free survival rate in the affected patients. In the current study, we found a significant association of ER-negative status with increased [18F]FDG uptake on PET/CT scan (Table. 1) as was also demonstrated in studies conducted by Ekmekcioglu et al. 5 , Kitajima et al. 6 , Koolen et al. 7 , Ueda et al. 8 , Abubakar et al. 9 , Groheux et al. 10 , Mavi et al. 11 , Osborne et al. 12 , Keam et al. 13 , Ugurluer et al. 14 and Dondi et al. 15 (Table 4). However, no significant association between ER-negative status and [18F]FDG uptake was found by Buck et al. 16 , Heudel et al. 17 , and Utech et al. 18 (Table 4).

The current study found a significant association between PgR negative hormone status with increased [18F]FDG uptake on PET/CT imaging (Table 1) which is in concordance with the results of Ekmekcioglu et al. 5 , Kitajima et al. 6 , Koolen et al. 7 , Abubakar et al. 9 , Groheux et al. 10 , Ugurluer et al. 14 , Dondi et al. 15 and Heudel et al. 17 (Table 4). In comparison to this, studies conducted by Buck et al. 16 (Table 4), Mavi et al. 11 , and Keam et al. 13 did not find a significant association between PgR negative status and SUV max uptake in breast cancer patients.

Koo et al. 19 stated that HER2-positive breast cancers are characterized by HER2 gene overexpression, which promotes tumor growth and progression. According to the study conducted by Osborne et al.,12 Her2 overexpression is associated with poor prognosis and aggressive disease, which shows comparatively higher uptake of [18F]FDG as compared to ER and PR hormone-positive receptor status. Kitajima et al. 6 (P=0.0002) and Ueda et al. 8 (P=0.006) found a significant association between Her2 overexpression and increased [18F]FDG uptake ( Table 4). As opposed to this, no significant association between the two factors could be determined by our study (Table 1) as well as studies conducted by Ekmekcioglu et al. 5 , Koolen et al. 7 , Abubakar et al. 9 , Mavi et al. 11 , Osborne et al. 12 , Buck et al. 16 , Berriolo-Riedinger et al. 20 , Dondi et al. 15 and Keam et al. 13 (Table 4). Groheux et al. 10 suggested that HER2 has no major role to play in glycolysis, which can be a reason why its overexpression is not significantly associated with higher [18F]FDG uptake.

In a study conducted by Kumar et al. 21 , no significant relationship was found between PET results and tumor type, Her2, and ER/PgR receptors.

The current study found a significant association between PgR negative hormone status with increased [18F]FDG uptake on PET/CT imaging (Table 1) which is in concordance with the results of Ekmekcioglu et al. 5 , Kitajima et al. 6 , Koolen et al. 7 , Abubakar et al. 9 , Groheux et al. 10 , Ugurluer et al. 14 , Dondi et al. 15 and Heudel et al. 17 (Table 4). In comparison to this, studies conducted by Buck et al. 16 (Table 4), Mavi et al. 11 , and Keam et al. 13 did not find a significant association between PgR negative status and SUV max uptake in breast cancer patients.

Koo et al. 19 stated that HER2-positive breast cancers are characterized by HER2 gene overexpression, which promotes tumor growth and progression. According to the study conducted by Osborne et al.,12 Her2 overexpression is associated with poor prognosis and aggressive disease, which shows comparatively higher uptake of [18F]FDG as compared to ER and PR hormone-positive receptor status. Kitajima et al. 6 (P=0.0002) and Ueda et al. 8 (P=0.006) found a significant association between Her2 overexpression and increased [18F]FDG uptake ( Table 4). As opposed to this, no significant association between the two factors could be determined by our study (Table 1) as well as studies conducted by Ekmekcioglu et al. 5 , Koolen et al. 7 , Abubakar et al. 9 , Mavi et al. 11 , Osborne et al. 12 , Buck et al. 16 , Berriolo-Riedinger et al. 20 , Dondi et al. 15 and Keam et al. 13 (Table 4). Groheux et al. 10 suggested that HER2 has no major role to play in glycolysis, which can be a reason why its overexpression is not significantly associated with higher [18F]FDG uptake. In a study conducted by Kumar et al. 21 , no significant relationship was found between PET results and tumor type, Her2, and ER/PgR receptors.

In the current study, significant differences were found in [18F]FDG uptake on PET/CT imaging for Luminal type A (Fig.1a,1b and 1c, Fig.2) and Luminal B (Fig.3a, 3b and 3c, Fig.4) molecular subtypes (p<0.001) ( Table 2). These findings were in agreement with the studies conducted by Koolen et al. 7 , Abubakar et al. 9 , and Kitajima et al. 6 Table 5. The comparison between current and previous studies regarding mean/ median SUV uptake values on PET/CT for different molecular subtypes with sample size (n) in each study.

The Her2-enriched subtype is associated with higher recurrence rates and mortality. The randomized control trial by Gianni et al. 22 concluded that targeted treatment with Trastuzumab monoclonal antibodies has increased the survival rates in such patients. In our study, we found a significantly increased [18F]FDG uptake (P<0.002) in Her2 enriched subtype (Fig. 5a, 5b and 5c, Fig. 6) in comparison to hormone receptor-positive subtypes (Table 2). Similar results (with higher uptake in Her 2 enriched subtype) were demonstrated by Ueda et al. 8 (P <0.001), Bitencourt et al. 23 (P <0.006), Koolen et al. 7 (P <0.0001) and Keam et al. 13 (Table 5). However, the study conducted by Abubakar et al. 9 showed no significant difference in the Her 2 enriched subtype (Table 5). In a study by Kitajima et al. 6 , the Her2 subtype had maximum [18F]FDG uptake (P =0.0002) followed by the triple-negative subtype (P =0.0003) ( Table 5).

According to Haffty et al. 25 , TNBC is associated with the most aggressive clinical course and poor prognosis. In our study, we found that the primary pretreated cancers with very high [18F]FDG uptake (Fig. 7a, 7b, and 7c) correlated with TNBC subtypes on immunohistochemistry (Fig. 8) ( Table 2). The findings of our study were supported by studies conducted by Basu et al. 24 , Koolen et al. 7 , Ekmekcioglu et al. 5 , Bitencourt et al. 23 , Groheux et al. 10 and Keam et al. 13 (Table 5). Koolen et al. 7 pointed out that the most beneficial role of PET/CT is in assessing the TNBC since they show high uptake. In a study conducted by Basu et al. 24 , the percentage change in SUV uptake was calculated for TNBC versus non-TNBC, and 100% PET sensitivity was found for TNBC with increased [18F]FDG uptake when compared to the hormone receptor-positive subtypes. Ueda et al. 8 24 suggested, PET/CT can be an important marker for tumor activity and treatment response in breast cancer patients.

This is a proliferation marker for cancerous cells within the breast parenchyma. As the proliferation index value increases, the grade of carcinoma increases owing to the increased proliferation of malignant cells within the tissue. The cutoff for low and high ki67 has been variable in different studies (e.g., 14 / 15 or 20%) but in our studies, a value <14 % was taken to be a low index and a value ≥14 % as a high index. This marker has invariably shown a positive association with increased [18F]FDG uptake on PET/CT imaging including our study (Table 1) (Fig. 2,4,6 and 8 25 , Abubakar et al. 9 and Keam et al. 13 (Table 4).

Lymph node involvement is the prognostic factor with the most variable findings according to previous studies. The study by Heudel et al. 17 found no significant association between morphological and histological characteristics of axillary nodal involvement and the primary subtype of breast cancer (Table 4). However, increased sensitivity and specificity for the detection of nodal involvement were seen on PET/CT. Shimoda et al. 25 determined only a weak correlation between the two factors which corroborates with the findings of the current study (Table 3). According to Wahl et al. 26 , [18F]FDG PET/CT has moderate accuracy for detecting axillary nodal involvement except for small and few axillary nodal metastases. Ekmekcioglu et al. 5 , Abubakar et al. 9 Dondi et al. 15 found a significant association between the high SUV max uptake and nodal enlargement whereas Koolen et al. 7 and Buck et al. 16 found no significant association between the two factors (Table 4). Kumar et al. 21 found no significant relationship between PET results with metastasis for sentinel or distant lymph nodes. Inoue et al. 27 found that the increased [18F]FDG uptake with nodal involvement is a predictor for poor prognosis in breast carcinoma. Veronesi et al. 28 concluded that PET imaging has high specificity, so patients with PET-positive axilla should have an axillary lymph node dissection rather than a sentinel node biopsy for axillary staging similar to the results of the study by Heudel et al. 17 However, due to the low sensitivity of [18F]FDG PET/CT for axillary metastases, PET-negative axillae should undergo sentinel node biopsy, which is also supported by Heusner et al. 29

Few studies have considered and commented on the correlation between [18F]FDG uptake in distant metastasis involvement along with primary breast cancers. In our study, we found that distant metastasis has no significant association with a higher mean SUV max uptake of the primary breast lesion (P >0.05) ( Table 3). These results are in concordance with the study conducted by Abubakar et al. 9 , in which no significant association was seen between distant metastasis and increased FDG uptake (n = 219, P = 0.086) ( Table 4). A study conducted by Dondi et al. 15 stated that 2 out of 46 cases had distant metastasis on PET/CT imaging with higher SUV max. However, the P -value was 0.2 (insignificant). In contrast, the study conducted by Koolen et al. 7 suggested that metastasis is seen in patients with ER-negative status and higher grades of primary breast cancer (n=214, P <0.006) (Table 4). However, further studies are required to establish a more confident correlation between the two factors.

In conclusion, significantly higher SUV max was seen in tumors with ER, PgR negative, triple negative receptor status, and high Ki67 index which is likely to predict poor prognosis in the patient. However, only a weak correlation was found for axillary nodal spread and no significant correlation was seen for distant metastasis.

The values for different molecular subtypes can be calculated and used as Mean or Median SUV uptake. However, owing to data skewing in practical scenarios, we suggest the use of median values (with interquartile range) can be applied for predicting the molecular subtypes of breast cancer on PET/CT Imaging.

Luminal A -Median SUV -3.9 (IQR -3.

The authors declare that they have no competing interests.

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.