Magnetic resonance imaging downstaging, pathological response, and microsatellite instability status in patients with signet-ring cell carcinoma rectum undergoing preoperative long-course chemoradiation

B Rajkrishna; Saikat Das; Dipti Masih; Tharani Putta; Rajat Raghunath; Thomas Samuel Ram

doi:10.4103/cmi.cmi_23_22

ORIGINAL ARTICLE

Year : 2022 | Volume : 20 | Issue : 3 | Page : 154-160

Magnetic resonance imaging downstaging, pathological response, and microsatellite instability status in patients with signet-ring cell carcinoma rectum undergoing preoperative long-course chemoradiation

B Rajkrishna¹, Saikat Das¹, Dipti Masih², Tharani Putta³, Rajat Raghunath⁴, Thomas Samuel Ram¹
¹ Department of Radiation Oncology, Ida B Scudder Cancer Centre, Christian Medical College, Vellore, Tamil Nadu, India
² Department of General Pathology, Christian Medical College, Vellore, Tamil Nadu, India
³ Department of Radiology, Christian Medical College, Vellore, Tamil Nadu, India
⁴ Department of Colorectal Surgery, Christian Medical College, Vellore, Tamil Nadu, India

Date of Submission	21-Feb-2022
Date of Decision	22-Apr-2022
Date of Acceptance	12-May-2022
Date of Web Publication	01-Aug-2022

Correspondence Address:
Dr. Thomas Samuel Ram
Department of Radiation Oncology, Ida B Scudder Cancer Centre, Christian Medical College, Vellore - 632 004, Tamil Nadu
India

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/cmi.cmi_23_22

Abstract

Aim and Objective: To assess the magnetic resonance imaging (MRI) downstaging, pathological response, and the relationship between microsatellite instability (MSI) and radiotherapy response in signet-ring cell carcinoma rectum. Materials and Methods: Twenty two patients were recruited prospectively and retrospectively in this observational study. Six weeks following radiotherapy, the response was assessed using an MRI pelvis, and patients who were operable underwent total mesorectal excision followed by adjuvant chemotherapy. The outcome of radiotherapy was correlated with post radiation MRI downstaging, pathological response, and MSI status. Results: The post radiotherapy response assessment MRI showed tumor regression grading (TRG) 5 in 5 patients. TRG 4 seen in 12, TRG 3 in 3, and TRG 2 in 1 patient. Fifteen patients were operable and post-operativ histopathology showed that 40% had pathological complete response (pCR) and 26.7% had near-complete response. Even those who had no response in MRI had pCR. MSI done 17 patients were stable. Of the 6 patients who had complete pathological response, two were MRI TRG 5, three were MRI TRG 4, and one was MRI TRG 3. The median survival was 23 months. The 2-year and 3-year disease-free survival was 46% and 38%, respectively. Conclusion: The predictive value of MRI downstaging in these tumors following neoadjuvant long-course chemoradiation therapy is not often in concurrence with the histopathological response and needs to be interpreted carefully. Even though the pCR rate seen in this cohort is encouraging, this needs to be evaluated in studies with large cohorts.

Keywords: Long course chemoradiation, microsatellite instability, signet-ring cell carcinoma rectum, tumor regression grade

How to cite this article:
Rajkrishna B, Das S, Masih D, Putta T, Raghunath R, Ram TS. Magnetic resonance imaging downstaging, pathological response, and microsatellite instability status in patients with signet-ring cell carcinoma rectum undergoing preoperative long-course chemoradiation. Curr Med Issues 2022;20:154-60

How to cite this URL:
Rajkrishna B, Das S, Masih D, Putta T, Raghunath R, Ram TS. Magnetic resonance imaging downstaging, pathological response, and microsatellite instability status in patients with signet-ring cell carcinoma rectum undergoing preoperative long-course chemoradiation. Curr Med Issues [serial online] 2022 [cited 2023 Mar 22];20:154-60. Available from: https://www.cmijournal.org/text.asp?2022/20/3/154/352971

Introduction

Signet ring-cell carcinoma rectum is a rare subtype that constitutes about 1% of all rectal cancers.^[1] Its prognosis is poor with a median survival of 20–30 months, a 5-year survival rate between 10% and 35% and a higher chance of distant metastasis.^{[1],[2],[3],[4],[5],[6],[7],[8],[9],[10]} Many randomized studies have shown that neoadjuvant chemoirradiation followed by total mesorectal excision (TME) is the recommended treatment for locally advanced rectal cancer.^{[11],[12],[13],[14]} Neoadjuvant chemoirradiation helps in effective downstaging of the primary tumor and 15%–27% pathological complete response (p CR).^[15] After preoperative chemoirradiation, high-resolution magnetic resonance imaging (MRI) will help to assess both primary and lymph node downstaging.^[16],[17] However, its role in predicting the survival has not been established. The specific data on the role of neoadjuvant chemoirradiation and its response in signet-ring cell carcinoma rectum are less studied.^[18]

Microsatellites are tandemly repeated short deoxyribonucleic acid (DNA) sequences and their instability will lead to impaired DNA mismatch repair.^[19] Fifteen percent of colorectal cancers can have microsatellite instability (MSI).^[20] Studies have shown that colorectal cancer with MSI has increased sensitivity to radiotherapy.^[21],[22]

Hence, given the paucity of data on radiological and pathological response to radiation therapy and MSI status in signet-ring cell carcinoma of the rectum, we planned to undertake this study. This study aimed to assess the MRI downstaging, pathological response, and the relationship between MSI and radiotherapy response in signet-ring cell carcinoma rectum.

Materials and Methods

Study design

This was an observational study with patients recruited prospectively and retrospectively. Eight patients were added prospectively after informed written consent and 14 patients were added retrospectively.

Setting

We conducted this study in the Department of Radiation Oncology at a Tertiary Care Cancer Center of South India from a period of 2013–2016. Clearance was obtained from the Institution Review Board (IRB Number: 9777 dated December 03, 2015).

Study participants

Patients who were diagnosed to have non metastatic signet-ring cell carcinoma rectum between the age group 18 and 70 years, who were planned for neoadjuvant chemoirradiation were included in the study. Pregnant women, patients who had prior pelvic malignancies, prior radiation to abdomen and pelvis, and those with hematological malignancies were excluded from the study.

Study protocol

All patients with signet-ring cell carcinoma rectum had undergone standard workup which included biopsy, colonoscopy, MRI pelvis, and carcinoma embryonic antigen (CEA). Distant metastatic work-up was done either with computed tomography thorax and abdomen or with chest X-ray and ultrasound abdomen and pelvis. Non metastatic patients received preoperative long-course chemoradiotherapy. Tablet Capecitabine (825 mg/m2) twice daily was given as concurrent chemotherapy on days of radiation therapy. Radiation therapy was delivered either by two-dimensional (2D) four-field box technique, three-dimensional conformal radiotherapy (3DCRT), or by intensity-modulated radiation therapy (IMRT). Patients who received 2D four field box and 3DCRT were planned for 50.4 Gy in 28 fractions (Phase 1: 45 Gy in 25 fractions and Phase II: 5.4 Gy in 3 fractions) and patients who received IMRT were planned for either 50.4 Gy in 28 fractions (Phase 1: 45 Gy in 25 fractions and Phase II: 5.4 Gy in 3 fractions) or received a simultaneous integrated boost of 45 Gy in 25 fractions to clinical target volume (CTV) 45 (primary tumor + mesorectum + draining lymphatics) and 50.4 Gy in 25 fractions to CTV 50.4 (primary tumor + mesorectum). Response assessment was done after 6 weeks of completion of chemoradiotherapy using MRI pelvis. Patients who were operable after chemoradiotherapy underwent TME. The response was assessed by comparing pretreatment MRI with post radiation therapy MRI and pathological response. MSI status was assessed using immunohistochemistry (IHC) and was planned to correlate with the pathological response.

Magnetic resonance imaging pelvis protocol

The following MRI sequences were done using a 1.5T MRI scanner using a phased array coil without contrast or endorectal gel

High-resolution (HR) T2 in three planes using a small field of view (FOV) (oblique axial, oblique coronal, and sagittal planes)
T2-weighted coronal and axial planes using a large field of FOV
Short-tau inversion recovery (STIR) and T1-weighted axial plane using large FOV
Diffusion-weighted imaging in the axial plane with b-values of 0, 100, and 800 s/mm².

Magnetic resonance tumor regression grading

Modified Dworak et al.^[23] grading which was altered to a radiologic system.^[24]

Tumor regression grading 1 (TRG) – Complete radiologic response and no evidence of tumor.
TRG 2 – Good response: Dense fibrosis or “acellular” mucin, no obvious residual tumor, minimal residual disease, or no tumor
TRG 3 – Moderate response: >50% fibrosis or “acellular” mucin and visible intermediate signal
TRG 4 – Slight response: Little areas of fibrosis or “acellular” mucin, but mostly tumor
TRG 5 – No response: Intermediate signal intensity or same appearances as the original tumor.

Pathological tumor regression grading

In our center, we use the Mandard tumor response grading system to assess TRG.^[25]

Tumor response was graded as follows:

TRG 1 - Complete regression with the absence of residual cancer and fibrosis extending through the wall (complete response)
TRG 2 - Rare residual tumor cells scattered throughout the fibrosis (near complete response)
TRG 3 - Predominant fibrosis but increase in the number of cancer cells (moderate response)
TRG 4 - Residual cancer cells outgrowing the fibrosis (minimal response)
TRG 5 - The absence of regressive changes (no response).

Immunohistochemistry protocol to assess microsatellite instability status

Tissue sections embedded in paraffin were cut at a thickness of 4 μ and floated in poly L-lysine coated slides which were incubated at 37°C overnight. Then these slides were treated for 10 min with 4% milk powder solution. This will give a positive charge to the slides by eliminating the hydrophobic effect. The slides were loaded in Ventana Benchmark XT automated immunostainer.

Sample size

We calculated a sample size of 41 patients with a precision of around 15%, with a 95% confidence interval based on available literature.^[18]

Statistical analysis

Data were entered using EPI DATA software (Comprehensive Data Management and Basic Statistical Analysis System, Odense Denmark, EpiData Association). All categorical data were reported using frequency and percentage. Mean or median with range is used to represent the quantitative variables. The outcomes were calculated from the date of diagnosis. Kaplan − Meier curves were used to evaluate the survival outcomes. Statistical package for Social Sciences for Windows (SPSS Inc. Released 2007, version 23.0, Armonk, New York, USA) was used for statistical analysis.

Results

Twenty-two patients were recruited for the study. Eight patients were added prospectively and 14 patients were added retrospectively. Majority of our patients (59.1%, n = 13) were males. The median age group was 35 years (range –18–62 years). All patients had an Eastern Co-operative Oncology Group performance score of 1 [Table 1].

Table 1: Demographics, disease, and treatment characteristics (n=22)

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Tumor characteristics

Seventy-two percentage (n = 16) had T3 lesions and 63.6% (n = 14) had N2 disease at presentation. In 72.7% (n = 16), the tumor was located within 5 cm from the anal verge. CEA was normal in 59.1% (n = 13). Biopsy of all the patients had signet-ring cells. In 45.5% (n = 10) of patients, there were mucin components along with signet-ring cells [Table 1].

Chemoirradiation

Radiotherapy was delivered by 2D four-field technique in 77.3% (n = 17), 3DCRT in 13.6% (n = 3) and IMRT in 9.1% (n = 2). Most common dose prescription (95.5%, n = 21) was 50.4 Gy in 28 fractions. Out of 22 patients, 21 patients had concurrent Capecitabine (825 mg/m² twice daily on days of radiation therapy) and one patient did not receive concurrent Capecitabine due to his comorbid illness [Table 1].

Magnetic resonance imaging tumor regression grade

In the response assessment MRI, TRG 5 was seen in 5 patients. TRG 4 seen in 12, TRG 3 in 3, and TRG 2 in 1 patient [Figure 1] and [Table 2]. No patients had complete MRI response (TRG 1). Due to clinical progression, one patient did not have post radiotherapy response assessment MRI.

Table 2: Magnetic resonance imaging and pathological tumor regression grades

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Figure 1: MRI tumor regression grade of all patients. MRI: Magnetic resonance imaging

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Surgery

Inoperable lesion was found 31.82% (n = 7). Abdominoperineal excision was done for 31.82% (n = 7). Anterior resection (AR) was done for 22.73% (n = 5). Hartmann's operation was done for 9.09% (n = 2) and exenteration for 4.54% (n = 1) [Table 1]. The median time to surgery from the completion of radiotherapy was 58 days (Range: 40–97 days).

Pathological tumor regression grade

Fifteen patients underwent surgery and among them the postoperative histopathological examination revealed, TRG 5 in 26.7% (n = 4), TRG 4 in 6.6% (n = 1), TRG 2 in 26.7% (n = 4) and 40% (n = 6) with TRG 1 [Table 2]. Even patients who had MRI TRG 5 showed p CR. [Figure 2] shows the correlation between MRI TRG and pathological TRG. The median time to surgery from completion of radiotherapy in patients with a p CR was 55 days (Range: 45–68 days).

Figure 2: Correlation between MRI TRG and pathological TRG. MRI: Magnetic resonance imaging, TRG: Tumor regression grade

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On correlating MRI TRG with pathological TRG, two out of two MRI TRG 5 patients and three out of ten MRI TRG 4 patients had complete pathological response. A near-complete pathological response (TRG 2) was seen in three out of ten MRI TRG 4 patients. Among the 6 patients with the complete pathological response (TRG 1), one was MRI TRG 3, three were MRI TRG 4, and two were MRI TRG 5. [Table 3] shows the characteristics of patients with p CR.

Table 3: Characteristics of patients with complete response

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Microsatellite instability status

Only 17 patients underwent MSI IHC and MSI was stable for all of them. Hence, response of radiotherapy and MSI could not be correlated.

Survival Analysis

The median survival was 23 months. The 2-year and 3-year disease-free survival (DFS) was 46% and 38%, respectively [Figure 3].

Figure 3: Progression-free survival

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Discussion

The standard treatment for locally advanced rectal cancer is long-course chemoirradiation followed by TME. However, the same approach does not yield similar outcomes in the signet-ring cell carcinoma rectum. Few anecdotal reports suggest a poor response to radiotherapy in signet-ring cell carcinoma of the rectum. However, in our study, we found an unusual and very rarely reported outcome of around 40% (6/15) complete pathological response.

Blomqvist et al. divided rectal cancers into “good,” “bad,” and “ugly” rectal cancers. “Good” group were those with clear CRM, N0 disease and T1-T3b mid/upper rectum, T1-T2 low rectum, and T3a. “Bad” group were those with clear CRM, N1/N2 disease, and T3c/d mid/upper rectum, low rectum, and also T3b, T4 with peritoneal or vaginal involvement alone. The “Ugly” group comprises those with positive CRM and lateral pelvic lymph nodes and T4 with involvement of prostate, seminal vesicles, the base of the urinary bladder, pelvic sidewalls or floor, and sacrum. The majority of our patients were “Bad” and a few were “Ugly” rectal cancers.^[26]

The MERCURY trial, a landmark study that studied the MRI accuracy in predicting surgery with negative CRM and found a 92% specificity in achieving the same.^[27] A 5-year follow-up, the above-mentioned study showed that preoperative MRI with clear CRM correlated with decreased local recurrence and improvement in overall survival and DFS. When the CRM was involved in MRI, there was a significant increase in distant metastases.^[28]

Studies done by Elmashad et al. and Sathyakumar et al.^[16],[29] showed that there was both tumor and lymph node downstaging in the reassessment MRI after 6 weeks of neoadjuvant chemoiiradiation. However, in our study, the reassessment MRI showed that 57% had a minimal response and 23% had no response indicating no downstaging. Although there was only minimal response in reassessment MRI, many were operable based on surgeon's clinical judgment and underwent surgery.

The presence of mucin pools lacking neoplastic epithelium (“acellular” mucin) in resection specimens after neoadjuvant chemoradiotherapy is a well-described phenomenon. The current recommendation is to regard “acellular” mucin as treatment response and not as a residual tumor.^[30],[31]

On pretreatment T2-weighted MRI, mucinous tumors will be markedly hyperintense with intermediate signal areas which correspond to histologically seen malignant cells, cords, and vessels. After neoadjuvant chemoradiotherapy, necrosis will happen in the tumor and there will be the formation of “acellular” mucin which are nothing, but the mucin pools lacking tumor cells/neoplastic epithelium. “Acellular” mucin is seen on T2-weighted MRI as featureless high signal intensity fluid-like signal.^[30],[31] However, this distinction between “acellular” and “cellular” mucin is not fool-proof and is practically misleading in many instances which explains the failure in accurately understating the tumor response. This important limitation in MR-TRG should be kept in mind while planning or deferring further treatment after neoadjuvant treatment in mucinous rectal cancers. To overcome this limitation, Park et al. have proposed a further modified MRI-TRG. However, this modified system works under the assumption that all T2 hyperintense component seen in the post treatment MRI is nonviable, is therefore debatable, and needs wider validation before it can be adopted in routine practice.^[32] To date, no imaging technique or grading system is consistently or reproducibly able to predict complete response after chemoradiotherapy in the mucinous rectal tumor. Radiomics, a field in which quantitative texture analysis extracted from images can be correlated with tumor characteristics that cannot be assessed by the naked eye, is a promising tool in assessing treatment response in mucinous rectal cancers.^[33]

Many studies have shown that p CR leads to better long-term survival in rectal cancers. The 5-year disease-free and overall survival rates after p CR were 85% and 90%, respectively.^[34] The p CR also indicates that there will be fewer chances for local recurrences and distant metastasis and there was a trend toward improved survival.^[15] In our patients, 40% had a complete pathological response and 26.7% had a near-complete pathological response. Even patients with poor response in reassessment MRI had a histopathological complete response. Hence, the predictive role of MRI in response assessment after neoadjuvant chemoirradiation in signet-ring cell carcinoma rectum needs to be interpreted cautiously.

In our study, the 2-year and 3-year DFS was 46% and 38%, respectively. Wu et al. have shown a 2-year and 5-year survival of 63% and 48.1%, respectively, after preoperative radiation therapy.^[8] [Table 4] shows the comparison of our study with other studies in the literature.

Table 4: Studies showing median survival and disease-free survival in signet ring cell carcinoma rectum

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All the patients who had MSI IHC were MSI stable. Hence, we could not find out a correlation between MSI and radiation response. About one-third of signet-ring cell carcinomas can have MSI. However, it was not a significant predictor of survival.^[35]

The major limitation of our study was that we could not recruit the required sample size due to the rarity of this subtype of rectal cancer. Polymerase chain reaction (PCR) is a more sensitive and specific test for MSI. However, we used IHC to assess MSI status. The interesting observation from our study was that 40% had a complete histopathological response and many of them had their MRI predicted poor response. Hence, the predictive role of MRI in the downstaging of signet-ring cell carcinoma rectum needs more extensive research. In the future, these tumors with bad biology need a new paradigm which includes molecular and genetic markers and very aggressive treatment plans. These tumors may also benefit from total neoadjuvant therapy with FOLFOX, CAPEOX, or FOLFIRINOX.^[36]

Conclusion

The role of MRI in predicting downstaging in signet-ring cell carcinoma rectum after neoadjuvant chemoirradiation therapy is not always in concurrence with the histopathological downstaging. The pCR rate seen in our study matches with the response rates seen in non-signet ring cell types. Hence, long-course chemoirradiation plays a major significant role in managing these tumors. Although the pCR rate seen in this cohort appears to be encouraging and this needs to be estimated in a larger series. Assessment of MSI using IHC is suboptimal and should preferably use PCR techniques in future studies.

Research quality and ethics statement

This scientific study complies with standard reporting guidelines by the EQUATOR network. Prior approval was obtained from the institutional review board before the initiation of this study (IRB Number: 9777 dated 3/12/2015).

Acknowledgments

We are immensely thankful to the contributions made by the statistician, Ms. Dona Maria Philip.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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