Expression of ALDH1A1 & Nanog in squamous cell carcinoma of cervix

Sir,

Cervical cancer is fourth most common cause for cancer mortality and is frequently diagnosed cancer in all females¹. Annual incidence is around 120,000 cases. India contributes to 15.2 per cent of global cervical cancer-related deaths². Among all cervical cancers majority (80%) are squamous cell carcinomas (SCC)³. The prudent risk factor for cancer cervix is persistent infectivity with oncogenic stains of Human Papilloma Virus (HPV)¹. Cancer cells have a subpopulation called cancer stem cells (CSCs) having abilities of self-renewal and survival after cytotoxic treatment⁴. CSCs are capable of initiating tumour and often responsible for relapses, arising either from differentiated cells acquiring stem-like properties or tissue-resident stem cells during tumourigenesis. Numerous biomarkers associated with CSCs have been identified, linked to cancer diagnosis, treatment response, and prognosis⁵. These CSCs are present in various tumours. Cell surface markers including aldehyde dehydrogenase (ALDH) 1 and transcription factors like Nanog, have been employed to separate and concentrate CSCs from many tumours, which includes cancer cervix. The given biomarkers of CSCs might function as a target molecule improving effectiveness of chemotherapy along with lessening its harmful effects in cases of cervical cancer associated with high-risk HPV⁶. ALDH is a detoxifying enzyme. ALDH1 is a CSC marker, strongly involved in cancer aggressiveness along with auto-renewal characteristics in various tumours⁷. ALDH1 expression is seen in carcinoma cervix primary tissues⁸. Nanog is an emerging biomarker with prognostic and predictive value as increased expression of Nanog is observed in malignancy with later tumour stages, poor prognosis, less differentiation along with reduced responsiveness to therapy⁹.

This study was prospective, performed at tertiary healthcare facility, Departments of Pathology, and Obstetrics and Gynaecology, Maulana Azad Medical College, New Delhi, India, from November 2023 to October 2024. Ethical clearance was obtained prior to the start of the study from Institutional Ethics Committee. A total of 60 cervical carcinoma cases and 30 healthy controls participated in the study. Those with prior chemotherapy and radiotherapy for SCC cervix were excluded. Clinical details of all participants were noted and staged as per FIGO (International Federation of Gynaecology and Obstetrics) clinical stages with the help of MRI imaging. Sections were routinely processed and paraffin encased. Haematoxylin and eosin staining was done. Histopathological typing was done as per 2022 WHO (World Health Organization) 5th edition (Fig. A)¹⁰. About 3-4 sections were taken on poly-L-lysine coated slides and staining for immunohistochemistry was done. For ALDH1A1, Mouse Monoclonal antibody, Clone: EP168 and for NANOG, Bio SB^TM, Mouse Monoclonal antibody, Clone: EP225 (both from PathnSitu Biotechnologies^TM, USA) were used. Breast carcinoma and embryonal carcinoma of testis were used as positive control for ALDH1A1 and NANOG, respectively. Healthy cervical tissue was taken as negative control for both markers. ALDH1A1 expressed cytoplasmic positivity and Nanog expressed cytoplasmic as well as nuclear positivity^11,12. Immunohistochemical expression of ALDH1A1 and Nanog was evaluated semi quantitatively. For ALDH1A1, total score was Positivity × Intensity (0 = negative, 1 = weakly positive, 2 = positive, ≥3 = strongly positive; Figs A-C)¹¹. For Nanog, total score was Positivity × Intensity (0 = negative, ≤4 = low, ≥6 = high expression; Figs D-F)^12,13.

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Figure.

(A) ALDH1A1 showing complete absence of staining, indicating negative expression; score 0 (IHC X200). (B) ALDH1A1 showing positive expression, with focal cytoplasmic staining in tumour cells; score 2 (IHC X200). (C) ALDH1A1 showing strong positive expression, with diffuse and intense cytoplasmic reactivity; score 3 (IHC X200). (D) Nanog showing negative expression, with no detectable nuclear staining; score 0 (IHC X200). (E) Nanog showing low expression, with weak and patchy nuclear staining in a minority of tumour cells; score ≤4 (IHC X200). (F) Nanog showing high expression, with strong and diffuse nuclear positivity in the majority of tumour cells; score ≥6 (IHC X400).

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The association of ALDH1A1 and Nanog expression with clinicopathological parameters was calculated. SPSS software version 25 was utilised for data analysis, statistically. Chi-square test/Fisher’s exact test was used for P value calculation. Statistical significance was defined as a P value <0.05. Demographic data and clinicopathological parameters were noted. ALDH1A1 was observed to be expressed in 54 (90%) out of 60 cases. Higher ALDH1A1 expression was found in cases compared to negative controls (Table).

Nanog was observed to be expressed in 53 (88.3%) out of the 60 cases with high expression observed in 37 cases (61.7%). No significant correlation was obtained for expression of Nanog with clinicopathological parameters. Higher expression of Nanog was found among cases compared to negative controls (Table). No significant association was obtained between ALDH1A1 and Nanog expression (P-0.515). Reserve cells of cervix located in suprabasal layer may be converted to cervical CSCs¹⁴. ALDH1A1 is the fundamental isozyme of ALDH associated with stem cell (SC) subsets. It serves as a crucial biomarker of SCs and CSCs¹⁵. Nanog has emerged as protumourigenic factor that may play a crucial role for cancer diagnosis and represent a therapeutic target predicted to ablate CSCs¹⁶. The combined detection of ALDH1A1 and Nanog markers during circulation in blood, expression of protein and transcription level might enhance validity as well as selectivity pertaining to biomarker profiling, providing assistance in evaluation for patient outcomes, establishing diagnosis, and observing treatment response of cervical cancer¹⁴. In this context, significant associations between ALDH1A1 expression and SCC and small cell cancer of lung have been reported previously¹⁷ where ALDH1A1 overexpression with unfavourable histological subtypes and advanced tumour grades in patients with lung cancer was reported¹⁷. According to Zhang et al¹⁸, the differences were not statistically significant for ALDH1-positive groups in tumour size and clinical staging. Yao et al⁷ (2011), Liu et al⁸ (2013) and Tulake et al¹⁴ (2018) also studied ALDH expression in cervical cancer. Han et al¹⁹ (2020) concluded that Nanog overexpression could be a potential indicator for the chemoradiation sensitivity prediction. Gu et al²⁰ and a meta-analysis by Zhao et al²¹ on solid tumours including cervical cancer showed increased expression of Nanog correlated to higher T stage. Chopra et al²² found insignificant Nanog expression with cervical cancer staging. Javed et al²³ observed that ALDH1 overexpression in the biopsies taken before initiating therapy showed a worsened course.

The present study highlights the significant association of ALDH1A1 expression with advanced tumour stage in SCC of cervix, suggesting its role in tumour advancement and predicting the course of cervical cancer. Additionally, the co-expression of Nanog supports their potential as prognostic markers. This was a limited study in a minority of SCC cervix patients that focused on correlation of ALDH1A1 and Nanog expression with clinicopathological parameters. The current study findings augment the growing literature that concluded ALDH1A1 and Nanog expression together might have crucial function in the development of carcinoma cervix, advocate further exploration of ALDH1A1 and Nanog as therapeutic targets and prognostic markers for improved patient outcomes using larger sample size over longer study period to validate these findings.